A signal connecting method and apparatus is provided which can reduce noises and create natural synthesized voices. The signal connecting method (or apparatus) for connecting a plurality of waveform signals and creating a synthesized waveform signal, has: a step (or unit) for determining an upper limit frequency of a frequency spectrum of each of the plurality of waveform signals; and a step (or unit) for filtering at least a connection portion of each waveform signal by using predetermined filter characteristics having the determined upper limit frequency. The cut-off frequency of the filtering is the higher upper limit frequency in upper limit frequencies of spectra of adjacent two waveform signals before and after the connection portion of the waveform signals. Higher harmonics to be caused by discontinuity of the connection portion of waveform signals can be effectively removed and noises of synthesized waveform signals can be reduced considerably.

1. Field of the Invention
The present invention relates to a signal connecting method and apparatus for connecting waveform signals to create a synthesized waveform signal, and more particularly to a method and apparatus suitable for connecting a plurality of voice waveform signals.
2. Description of the Related Art
Voices synthesized by voice synthesizing technology are used widely nowadays. For example, voice synthesizing technology is used in various situations such as text reading software, telephone number guide, stock guide, traveller's guide, shop guide, and traffic information.
Voice synthesizing methods are classified mainly into a rule synthesizing method and a form editing method.
The rule synthesizing method performs morpheme analysis of a text from which voices are synthesized, and in accordance with the analysis results, performs a phonological process for the text to create voices. This rule synthesizing method has less constraints of the contents of a text from which voices are synthesized and can be used for voice synthesis of texts having a variety of contents. However, with the rule synthesizing method, the quality of output voices is inferior to that of the form editing method.
The form editing method records voices actually spoken by a person and coupling constituent elements obtained by dividing the recorded voices to create target voices. The form editing method is superior to the rule synthesizing method in terms of the voice quality. However, with this form editing method, it is not possible to synthesize voices which contain constituent elements unable to be derived from the recorded voices. Therefore, the larger the division unit of recorded voices, the more the constrains of voices to be synthesized. In this connection, a method capable of synthesizing voices of various types has been proposed by using the form editing method by finely dividing recorded voices to the level of vowel and consonant.
However, the waveform at the connection portion of constituent elements of recorded voices becomes discontinuous as shown in FIG. 6(a), resulting in the generation source of noises. If the division unit of recorded voices is small, noises become conspicuous because the connection portions are discontinuous and the quality of synthesized voices is lowered.
As one method of reducing such noises, it is considered, for example, to replace a discontinuous portion with a straight line as shown in FIG. 6(b) to reduce noises. However, this connection portion creates higher harmonics, also resulting in noises.
Another approach to reduce noises to be caused by discontinuous connection portions is a Minimum Distance Search (MDS) method. With this method, as shown in FIG. 6(c) when two waveforms are connected, a point having generally the same instantaneous value and tangent gradient is searched from a portion as near to the trailing edge of the forward waveform as possible and from a portion as near to the leading edge of the backward waveform, and these two points are connected together.
With the MDS method, however, the connection point of the two waveforms is generally a point different from the edge of each waveform. Parts of the waveforms to be connected are usually discarded so that synthesized waveforms become unnatural.

Advanced Telecommunications Research Institute International


An instruction dispatching apparatus in a multi threading microprocessor that concurrently executes N threads each in one of G groups each having one of P priorities. G round-robin vectors each have N bits corresponding to the threads, each being a 1-bit left-rotated and subsequently sign-extended version of an N-bit vector with a single bit true of the last thread selected for dispatching in the group. Each of N G-input muxes receive a corresponding one of the N bits of each of the round-robin vectors and selects for output one of the inputs specified by the corresponding thread's group. Selection logic selects for dispatching one of the N instructions corresponding to the thread whose dispatch value is greater than or equal to any of the N threads left thereof. Each dispatch value comprises a least-significant bit of the corresponding mux output, a most-significant dispatchable instruction bit, and middle thread group priority bits.

Microprocessor designers employ many techniques to increase microprocessor performance. Most microprocessors operate using a clock signal running at a fixed frequency. Each clock cycle the circuits of the microprocessor perform their respective functions. According to Hennessy and Patterson (see Computer Architecture: A Quantitative Approach, 3rd Edition), the true measure of a microprocessor's performance is the time required to execute a program or collection of programs. From this perspective, the performance of a microprocessor is a function of its clock frequency, the average number of clock cycles required to execute an instruction (or alternately stated, the average number of instructions executed per clock cycle), and the number of instructions executed in the program or collection of programs. Semiconductor scientists and engineers are continually making it possible for microprocessors to run at faster clock frequencies, chiefly by reducing transistor size, resulting in faster switching times. The number of instructions executed is largely fixed by the task to be performed by the program, although it is also affected by the instruction set architecture of the microprocessor. Large performance increases have been realized by architectural and organizational notions that improve the instructions per clock cycle, in particular by notions of parallelism.
One notion of parallelism that has improved the instructions per clock cycle, as well as the clock frequency, of microprocessors is pipelining, which overlaps execution of multiple instructions within pipeline stages of the microprocessor. In an ideal situation, each clock cycle one instruction moves down the pipeline to a new stage, which performs a different function on the instruction. Thus, although each individual instruction takes multiple clock cycles to complete, because the multiple cycles of the individual instructions overlap, the average clocks per instruction is reduced. The performance improvements of pipelining may be realized to the extent that the instructions in the program permit it, namely to the extent that an instruction does not depend upon its predecessors in order to execute and can therefore execute in parallel with its predecessors, which is commonly referred to as instruction-level parallelism. Another way in which instruction-level parallelism is exploited by contemporary microprocessors is the issuing of multiple instructions for execution per clock cycle. These microprocessors are commonly referred to as superscalar microprocessors.
What has been discussed above pertains to parallelism at the individual instruction-level. However, the performance improvement that may be achieved through exploitation of instruction-level parallelism is limited. Various constraints imposed by limited instruction-level parallelism and other performance-constraining issues have recently renewed an interest in exploiting parallelism at the level of blocks, or sequences, or streams of instructions, commonly referred to as thread-level parallelism. A thread is simply a sequence, or stream, of program instructions. A multithreaded microprocessor concurrently executes multiple threads according to some scheduling policy that dictates the fetching and issuing of instructions of the various threads, such as interleaved, blocked, or simultaneous multi threading. A multithreaded microprocessor typically allows the multiple threads to share the functional units of the microprocessor (e.g., instruction fetch and decode units, caches, branch prediction units, and load/store, integer, floating-point, SIMD, etc. execution units) in a concurrent fashion. However, multithreaded microprocessors include multiple sets of resources, or contexts, for storing the unique state of each thread, such as multiple program counters and general purpose register sets, to facilitate the ability to quickly switch between threads to fetch and issue instructions.
One example of a performance-constraining issue addressed by multi threading microprocessors is the fact that accesses to memory outside the microprocessor that must be performed due to a cache miss typically have a relatively long latency. It is common for the memory access time of a contemporary microprocessor-based computer system to be between one and two orders of magnitude greater than the cache hit access time. Instructions dependent upon the data missing in the cache are stalled in the pipeline waiting for the data to come from memory. Consequently, some or all of the pipeline stages of a single-threaded microprocessor may be idle performing no useful work for many clock cycles. Multithreaded microprocessors may solve this problem by issuing instructions from other threads during the memory fetch latency, thereby enabling the pipeline stages to make forward progress performing useful work, somewhat analogously to, but at a finer level of granularity than, an operating system performing a task switch on a page fault. Other examples of performance-constraining issues addressed by multi threading microprocessors are pipeline stalls and their accompanying idle cycles due to a data dependence; or due to a long latency instruction such as a divide instruction, floating-point instruction, or the like; or due to a limited hardware resource conflict. Again, the ability of a multithreaded microprocessor to issue instructions from other threads to pipeline stages that would otherwise be idle may significantly reduce the time required to execute the program or collection of programs comprising the threads.
As may be observed from the foregoing, a processor concurrently executing multiple threads may reduce the time required to execute a program or collection of programs comprising the multiple threads. However, the extent to which a multi threading processor may realize a performance increase over a single-threaded processor may be highly dependent upon the thread scheduling policy of the processor, i.e., how the processor schedules the various threads for issuing their instructions for execution. Furthermore, the appropriate thread scheduling policy may be highly dependent upon the particular application in which the processor is used. For example, multi threading processors may be employed in various applications, including real-time embedded systems like network switches and routers, RAID controllers, printers, scanners, hand-held devices, digital cameras, automobiles, set-top boxes, appliances, etc.; scientific computing; transaction processing; server computing; and general purpose computing. Each of these applications may require a different scheduling policy to optimize performance of the multi threading processor. Consequently, it is highly desirable to enable customers with various applications the ability to customize the thread scheduling policy to meet their particular requirements. A customizable thread scheduler is particularly desirable when attempting to design a multi threading microprocessor core that may be part of a microprocessor and/or system that is customizable to meet the needs of various customer applications. This makes the multi threading core reusable for various designs, which is highly desirable because it avoids having to redesign an entire processor for each application.
Because there are multiple threads in a multi threading processor competing for limited resources, such as instruction execution bandwidth, there is a need to fairly arbitrate among the threads for instruction issue bandwidth. It may be desirable to give higher priority to some threads and lower priority to others. However, having priorities may introduce certain problems, such as low priority threads being starved for bandwidth in favor of high priority threads. Another problem may be that if a single thread is at highest priority, the efficiency benefits of interleaving multiple threads for execution may be lost since for a significantly large number of clock cycles instructions from only the highest priority thread may be issued for execution.
Therefore, what is needed is a multi threading processor with a customizable thread scheduling architecture that allows threads to be prioritized and yet still fairly distributes the execution bandwidth and interleaves the multiple threads to enjoy the efficiency benefits of multi threading.

This patent is subject to a terminal disclaimer.

Multithreading instruction scheduler employing thread group priorities


According to one exemplary embodiment, a dielectric ceramic composition includes a main component group, where the main component group is represented by MgxCayZnzTiO2+x+y+z, where the sum of x, y, and z is less than or equal to 1.0 such that the dielectric ceramic composition has a wider sintering temperature range and reduced exaggerated grain growth. According to one embodiment, x can be greater than 0.0 and less than 1.0, y can be greater than 0.0 and less than 1.0, and z can be greater than 0.0 and less than 1.0. The dielectric ceramic composition can further include between 0.0 and 50.0 percent by weight of aluminum oxide. The dielectric ceramic composition can further include copper oxide. The dielectric ceramic composition can further include boron oxide.

1. Field of the Invention
The present invention is generally in the field of dielectric materials. More specifically, the invention is in the field of dielectric ceramic materials.
2. Background Art
Dielectric ceramic compositions, such as dielectric ceramic compositions that include magnesium ("Mg"), calcium ("Ca"), and titanium ("Ti"), are widely used in dielectric filters, dielectric resonators, and dielectric couplers in various types of radio frequency ("RF") and microwave systems. However, a conventional dielectric ceramic composition that comprises Mg, Ca, and Ti, typically has a narrow sintering temperature range and exaggerated grain growth. Since the narrow sintering temperature range is difficult to maintain in the sintering kiln, the conventional dielectric ceramic may be under-fired (i.e. sintered at a temperature below a desired temperature range) or over-fired (i.e. sintered at a temperature above the desired temperature range) in the sintering kiln. However, under-firing and over-firing can cause various problems in the resulting dielectric composition.
For example, under-firing can cause undesirable variations in the dielectric constant, low density, and reduced mechanical strength in the conventional dielectric ceramic. On the other hand, for example, over-firing can cause undesirable exaggerated grain growth, which can also reduce the mechanical strength of the conventional dielectric ceramic composition. Furthermore, under-firing or over-firing caused by a narrow sintering temperature range results in low manufacturing yield for the conventional dielectric ceramic composition.
Thus, there is a need in the art for a dielectric ceramic composition that has an increased sintering temperature range, reduced exaggerated grain growth, and increased manufacturing yield.

Dielectric ceramic composition having wide sintering temperature range and reduced exaggerated grain growth


A cam phaser for transferring rotational energy from a crankshaft (16) to a camshaft (12) of an internal combustion engine (2), having a stator (20) for torque-proof connection to one of the shafts (12, 16) and a rotor (22) received rotationally in the stator (20) for torque-proof connection to the other shaft (12, 16), with first and second chambers being formed in the stator (20), engaged by first and second vanes (26) of the rotor (22). A bore (42) is provided in the first vane (26), in which a locking element (40) can be received for blocking a rotary motion of the rotor (22) relative to the stator (20). Here in an impact position of the second vane (26) at a wall (30) of the second chamber, the first vane (26) is arranged spaced apart from a wall (30) of the first chamber (44).

Cam phasers are technical components to adjust the phasing between a crankshaft and a camshaft in an internal combustion engine.
A cam phaser with a locking mechanism at the rotor of the cam phaser is known from DE 199 03 622 A1, which prevents any rotary motion of the rotor in reference to the stator in case of certain operating states of the cam phaser.


An image projection apparatus such as a video projector or projection television is provided for decreasing light loss. The apparatus has three cathode ray tubes for respectively providing images of red, green and blue, two dichroic mirrors for combining the images onto the same axis, and a lens group for enlarging and projecting the combined image toward a screen, wherein the inclination of each dichroic mirror is adjusted such that the incident angle of light incoming from the cathode ray tube to the dichroic mirror is less than 45.degree.. Thus, the degree of stop-band shift due to refractive index variations of an incidence medium is decreased. Also, the reflectance for incoming light having a wavelength coincident with the stop band is increased.

The present invention relates to an image projection apparatus for projecting television images, and more particularly to an image projection apparatus for projecting color images by combining images generated from a plurality of cathode ray tubes.
In the technical field of image display, direct-viewing type cathode ray tubes (CRTs) for televisions have proven incapable of providing a relatively large image display, currently being limited to forty-inch screens. Thus, larger screen sizes generally bring about problems with respect to practicality, due to increased difficulty in manufacturing and diminishing resolution. On the other hand, since the image projection apparatus projects an image from image generating surfaces of small projection cathode ray tubes to a screen by means of an optical apparatus such as a lens, a large-sized image of better picture quality can be realized by improving performance of the optical apparatus.
The image projection apparatus generally comprises three projection-type CRTs each having an image generating surface with phosphors for illuminating red, green and blue, and projection lenses for enlarging and projecting images. Additionally, in order to combine the images of the three CRTs, a certain image projection apparatus comprises two dichroic mirrors which reflect one image of red, green and blue but allow other images to be passed (see U.S. Pat. Nos. 4,607,280 and 4,842,394).
Such an image projecting apparatus having two dichroic mirrors is constructed as shown in FIG. 1. Three CRTs 1, 2 and 3 are arranged in a T-configuration, and two dichroic mirrors 5 and 6 are arranged in an X-configuration such that respective dichroic mirrors are inclined by 45.degree. with respect to optical axes of the cathode ray tubes to intersect with each other at the point where the respective optical axes of the cathode ray tubes meet, and a projection lens group 7 is provided in front of the dichroic mirrors. In the image projection apparatus constructed as above, in order to enhance the performance of the optical system, an increase in the reflectance of the dichroic mirror is necessary along with the elimination of lens element aberration.
In U.S. Pat. No. 4,607,280, two dichroic mirrors are installed in a space filled with air. In such an arrangement, since coma aberration occurs due to the refractive index of the two dichroic mirrors, many lens elements are required for compensating the coma aberration.
In case of U.S. Pat. No. 4,842,394, the dichroic mirrors are installed in an oil-filled space having a predetermined refractive index, thereby contriving the improvement of axial performance. However, in such a structure, the refractive index of the oil is varied due to the heat generated in the cathode ray tubes, and the degree of shift in the so-called "stop band" according to the variance of incident angle of light emitted from the cathode ray tube toward the dichroic mirror is increased, resulting in a severe variation in color and a significant degradation of the reflectance.

Dichroic mirror and an image projection apparatus using the same


Disclosed are a diffusion plate and a display apparatus having the same. The diffusion plate includes a base and a plurality of convex portions. The base includes a front surface and a rear surface. Each convex portion has a crescent of an ellipse formed on a front surface of the base while extending in one direction. The display apparatus includes a display panel, a light source, and the diffusion plate.

1. Field of the Invention
The present disclosure is directed to a diffusion plate and a display apparatus having the same. More particularly, this disclosure is directed to a diffusion plate provided with crescent shape convex portions and a display apparatus having the same.
2. Description of the Related Art
With the development of digital technology, driving mechanisms of display apparatuses have changed from analog to digital. These changes occur naturally due to consumer demand for lightweight display apparatuses, and high definition and clear images.
In response to these demands, the liquid crystal display has developed as a slim image display apparatus having high resolution. However, to further expand the market for liquid crystal displays, many vendors have attempted to develop products having low power consumption and low manufacturing cost.
A liquid crystal display comprises a liquid crystal panel and a backlight unit. The liquid crystal panel serves as a light receiving element. The backlight unit serves as a light source to provide light to the liquid crystal panel. In particular, the backlight unit typically includes a light source, which is provided in the form of a linear light source or a point light source, and an optical sheet, which allows light emitted from the light source to pass therethrough. The optical sheet converts light from linear or point light sources into surface light and increases light brightness.
Currently, slim display apparatuses with low power consumption and a low manufacturing cost are desired. However, there are challenges to making a liquid crystal display slimmer due to the geometrical structure of the backlight unit. In addition, for a low power consumption display apparatus, a more appealing external appearance may be obtained by using fewer lamps.

Diffusion plate and display apparatus having the same


A satellite constellation optimized for stationkeeping fuel consumption is provided. The satellite constellation includes a plurality of satellites, each satellite having a corresponding inclined elliptical orbit, each orbit having an initial right ascension of ascending node (“RAAN”) value, a RAAN drift rate, a semi-major axis, an eccentricity, an argument of perigee and an inclination. Each satellite has a fuel consumption value required to maintain the RAAN drift rate, the semi-major axis, the eccentricity, the argument of perigee and the inclination of the corresponding orbit. The initial RAAN value and the RAAN drift rate for each orbit correspond to a minimized fuel consumption value for the satellite having the highest fuel consumption value. The initial RAAN value and RAAN drift rate may be determined by calculating, for each possible data combination of an initial RAAN value for each orbit and a RAAN drift rate for the constellation, a fuel consumption value for each satellite in the constellation, and selecting, from the fuel consumption values thus determined, the data combination corresponding to a lowest fuel consumption for a highest fuel-consuming satellite.

Constellations of satellites having inclined, elliptical orbits are useful for providing communication coverage to Earth. One approach to providing continuous coverage uses two or more satellites having the same ground track. To ensure that the satellites have the same ground track, a fixed angular separation is maintained between the ascending node of each orbit (the intersection of the orbit with the equatorial plane of Earth where the satellite crosses from the southern hemisphere into the northern hemisphere). The ascending nodes of the orbits may be equally or unequally spaced, depending upon the mission requirements. Because each satellite orbit therefore has a different position with respect to the Earth, the sun and the moon, gravitational effects of these bodies perturb the orbit of each satellite to a differing degree, causing each orbit to tend to precess around the Earth at a different rate. This precession is measured as a drift rate in the right ascension of the ascending node ("RAAN") of each orbit. These perturbations also affect the other orbital elements, such as the argument of perigee, the semi-major axis, the eccentricity, the argument of perigee and the inclination.
Because the RAAN drift rate for each orbit in a constellation is prone to varying at a different rate, the orbits, if left uncorrected, would tend to lose their fixed angular separation. Accordingly, stationkeeping fuel stored onboard each satellite must be expended in controlled engine burns to maintain a single RAAN drift rate for all of the orbits, as well as to maintain the other orbital elements of the satellite orbits. If the effects of these orbital perturbations could be minimized for the constellation, the satellites would require less stationkeeping fuel onboard, permitting a greater payload mass for a given launch vehicle.
Accordingly, there is a need to optimize inclined, elliptical satellite constellations with a shared ground track to experience less orbital perturbations and to require less stationkeeping fuel. The present invention satisfies these needs and provides other advantages as well.

Stationkeeping optimization for inclined elliptical satellite orbit constellations


The present invention relates to an apparatus and method for use with a mass spectrometer. The ion enhancement system of the present invention is used to direct a heated gas toward ions produced by a matrix based ion source and detected by a detector. The ion enhancement system is interposed between the ion source and the detector. The analyte ions that contact the heated gas are enhanced and an increased number of ions are more easily detected by a detector. The method of the invention comprises producing analyte ions from a matrix based ion source, enhancing the analyte ions with an ion enhancement system and detecting the enhanced analyte ions with a detector.

Most complex biological and chemical targets require the application of complementary multidimensional analysis tools and methods to compensate for target and matrix interferences. Correct analysis and separation is important to obtain reliable quantitative and qualitative information about a target. In this regard, mass spectrometers have been used extensively as detectors for various separation methods. However, until recently most spectral methods provided fragmentation patterns that were too complicated for quick and efficient analysis. The introduction of atmospheric pressure ionization (API) and matrix assisted laser desorption ionization (MALDI) has improved results substantially. For instance, these methods provide significantly reduced fragmentation patterns and high sensitivity for analysis of a wide variety of volatile and non-volatile compounds. The techniques have also had success on a broad based level of compounds including peptides, proteins, carbohydrates, oligosaccharides, natural products, cationic drugs, organoarsenic compounds, cyclic glucans, taxol, taxol derivatives, metalloporphyrins, porphyrins, kerogens, cyclic siloxanes, aromatic polyester dendrimers, oligodeoxynucleotides, polyaromatic hydrocarbons, polymers and lipids.
According to the MALDI method of ionization, the analyte and matrix is applied to a metal probe or target substrate. As the solvent evaporates, the analyte and matrix co-precipitate out of solution to form a solid solution of the analyte in the matrix on the target substrate. The co-precipitate is then irradiated with a short laser pulse inducing the accumulation of a large amount of energy in the co-precipitate through electronic excitation or molecular vibration of the matrix molecules. The matrix dissipates the energy by desorption, carrying along the analyte into the gaseous phase. During this desorption process, ions are formed by charge transfer between the photo-excited matrix and analyte.
Conventionally, the MALDI technique of ionization is performed using a time-of-flight analyzer, although other mass analyzers such as an ion trap, an ion cyclotron resonance mass spectrometer and quadrupole time-of-flight are also used. These analyzers, however, must operate under high vacuum, which among other things may limit the target throughput, reduce resolution, capture efficiency, and make testing targets more difficult and expensive to perform.
To overcome the above mentioned disadvantages in MALDI, a technique referred to as AP-MALDI has been developed. This technique employs the MALDI technique of ionization, but at atmospheric pressure. The MALDI and the AP-MALDI ionization techniques have much in common. For instance, both techniques are based on the process of pulsed laser beam desorption/ionization of a solid-state target material resulting in production of gas phase analyte molecular ions. However, the AP-MALDI ionization technique does not rely on a pressure differential between the ionization chamber and the mass spectrometer to direct the flow of ions into the inlet orifice of the mass spectrometer.
AP-MALDI can provide detection of a molecular mass up to I06 Da from a target size in the attamole range. In addition, as large groups of proteins, peptides or other compounds are being processed and analyzed by these instruments, levels of sensitivity become increasingly important. Various structural and instrument changes have been made to MALDI mass spectrometers in an effort to improve sensitivity. Additions of parts and components, however, provides for increased instrument cost. In addition, attempts have been made to improve sensitivity by altering the analyte matrix mixed with the target. These additions and changes, however, have provided limited improvements in sensitivity with added cost. More recently, the qualitative and quantitative effects of heat on performance of AP-MALDI has been studied and assessed. In particular, it is believed that the performance of an unheated (room temperature) AP-MALDI source is quite poor due to the large and varying clusters produced in the analyte ions. These large clusters are formed and stabilized by collisions at atmospheric pressure. The results of different AP-MALDI matrixes to different levels of heat have been studied. In particular, studies have focused on heating the transfer capillary near the source. These studies show some limited improvement in overall instrument sensitivity. A drawback of this technique is that heating and thermal conductivity of the system is limited by the materials used in the capillary. Furthermore, sensitivity of the AP MALDI source has been limited by a number of factors including the geometry of the target as well as its position relative to the capillary, the laser beam energy density on the target surface, and the general flow dynamics of the system.
Thus, there is a need to improve the sensitivity and results of AP-MALDI mass spectrometers for increased and efficient ion enhancement.

Apparatus and method for ion production enhancement


The invention relates to transgenic non-human mammals characterized in that the genome of said mammals contain at least one heterologous gene encoding for the production of heterologous catalytic entity selected from the group consisting of enzymes and antibodies, and wherein said catalytic entity produces a second heterologous product in the milk of said mammal. Especially useful in the practice of the invention are human glycosyltransferases and transgenic sheep, goats and cows. The heterologous product includes oligosaccharides and glycoconjugates. Specifically exemplified, is the production of 2'-fucosyl-lactose in the milk of transgenic mice which contain and express a transgene encoding .alpha.-1,2-fucosyltransferase operatively linked to a mammary gland specific promoter.

Carbohydrates are an important class of biological compounds. The term "saccharides" encompasses a wide variety of carbohydrate-containing compounds. These include polysaccharides, oligosaccharides, glycoproteins and glycosides with non-carbohydrate aglycones. Biological macromolecules composed of protein or lipids containing oligosaccharide moieties are collectively known as glycoconjugates. The carbohydrate moiety provides many biological functions.
In cells, carbohydrates function as structural components where they regulate viscosity, store energy, or are key components of the cell surface. The complex oligosaccharide chains of various glycoconjugates (especially glycoproteins and glycolipids) mediate or modulate a variety of biological processes. For a general review of the bioactivity of carbohydrates see: (a) Biology of Carbohydrates, Volume 2, Ginsburg et al, Wiley, N. Y. (1984); and (b) P. W. Rademacher et al, Annual Review of Biochemistry, Volume 57, page 785, 1988). Among other things, it is known that:
(a) carbohydrate structures are important to the stability, activity, localization and degradation of glycoproteins;
(b) certain oligosaccharide structures activate plant secretion of antimicrobial substances;
(c) glycoconjugates are frequently found on the surfaces of various cells and are important, inter alia, to cell interactions with the surroundings since they function as receptors or regulators when bonded to cell surfaces of, for example, peptides, hormones, toxins, viruses, bacteria and during cell-cell interaction;
(d) carbohydrate structures are antigenic determinants (for example, blood group antigens);
(e) carbohydrates function as cell differentiating antigens during normal tissue development;
(f) carbohydrates are important in oncogenesis since specific oligosaccharides have been found to be cancer-associated antigenic determinants; and
(g) oligosaccharides are important to sperm/egg interaction and to fertilization.
Isolated oligosaccharides are known to inhibit the agglutination of uropathogenic coliform bacteria with erythrocytes. Other oligosaccharides have been shown to possess potent antithrombic activity by increasing the levels of plasminogen activator. This same biological activity has been used, by covalently attaching these oligosaccharides to the surface of medical instruments, to produce surfaces which have anticoagulation effects. These surfaces are useful in the collection, processing, storage and use of blood. Still other oligosaccharides have found utility as gram positive antibiotics and disinfectants. Further, certain free oligosaccharides have been used in the diagnosis and identification of specific bacteria. A considerable future market is envisaged for fine chemicals based on biologically active carbohydrates.
Universities and industry are at present working intensely on developing the additional uses of biologically active oligosaccharides. These efforts include, but are not limited to:
(a) the development of novel diagnostics and blood typing reagents;
(b) the development of a novel type of therapy as an alternative to antibiotics, based on the prevention of the adhesion of bacteria and viruses to cell surfaces by means of specific oligosaccharides; and
(c) the use of oligosaccharides to stimulate plant growth and provide protection against certain plant pathogens.
A large number of oligosaccharide structures have been identified and characterized. The smallest building block or unit of an oligosaccharide is a monosaccharide. The major monosaccharides found in mammalian glycoconjugates are: D-glucose (Glc), D-galactose (Gal), D-mannose (Man), L-fucose (Fuc), N-acetyl-D-galactose amine (GalNAc), N-acetyl-D-glucose amine (GlcNAc) and N-acetyl-D-neuraminic acid (NeuAc). The abbreviations in parentheses are the standard abridged terminology for monosaccharides according to the recommendations of the International Union of Physics, Chemistry and Biology Council; Journal Biological Chemistry, Volume 257, pages 3347-3351, (1982). These abbreviations will be used hereinafter. Despite the relatively small number of fundamental building blocks, the number of possible combinations is very great because both the anomeric configuration (alpha- or .beta.-glycosidic linkage) as well as the position of the O-glycosidic bond can be varied.
Thus, a large variety of oligosaccharide structures can exist. The bioactivity of oligosaccharides is known to be specific in terms of both sugar conformation and composition. Individual monosaccharides provide one element of bioactivity but they also contribute to the overall conformation of the oligosaccharide thereby providing another level of specificity and bioactivity. It is the diversity of glycoconjugates and oligosaccharides that produces biological specificity of certain oligosaccharide structures. However, this diversity also causes a particular problem for the practical utility of these compounds. Glycoconjugates are typically potent immunogens and the biospecificity, as noted above, is determined not only by the particular monosaccharide sequence but also by the nature of the glycosidic bond. Consequently it is often not possible to use oligosaccharide structures found in one animal species in another species. Similar restrictions on use may also apply on an individual basis. For example, since certain blood group antigens are known to be formed from specific oligosaccharides, it is necessary to be especially careful when conjugating a blood group oligosaccharide to a protein and then using that glycoprotein therapeutically. Careful consideration of the potential immunogenicity concerns must be made.
Despite these potential difficulties, it is well accepted that there is a need to produce large quantities of human oligosaccharides and/or glycoconjugates bearing those oligosaccharides. Numerous methods have been contemplated as suitable means for achieving this goal. Such methods include synthesis of oligosaccharides by conventional organic chemistry or the use of enzymes in vitro. Immobilized enzymes are presently the preferred mode for large scale in vitro oligosaccharide production. This is because of an enzyme's high regio- and stereoselectivity, as well as a high catalytic efficiency under mild reaction conditions. The literature discloses a number of enzyme-catalyzed oligosaccharide syntheses. For example, see the scientific review articles by Y. Ichikawa et al, "Enzyme-catalyzed Oligosaccharide Synthesis" in Analytical Biochemistry, Volume 202, pages 215-238, (1992); and K. G. I. Nillson, "Enzymatic Synthesis of Oligosaccharides" Trends in Biotechnology, Volume 6, pages 256-264, (1988). Both hydrolases and transferases have been used to faciltate production of oligosaccharides. The glycosidase enzymes, a subclass of the hydrolases, are especially useful in the synthesis of oligosaccharides by a process of reversing the degradative cycle. In general, however, enzymatic oligosaccharide synthesis is based on the biosynthetic pathway. While the biosynthetic pathway of oligosaccharide synthesis is principally regulated by the gene encoding the production of each glycosyltransferase, the actual oligosaccharide structures are determined by the substrate and acceptor specificity of the individual glycosyltransferases. Oligosaccharides are synthesized by transferring monosaccharides from sugar nucleotide donors to acceptor molecules. These acceptor molecules may be other free oligosaccharides, monosaccharides, or oligosaccharides bound to proteins or lipids.
Enzymatic oligosaccharide synthesis has generally been conducted only on a small scale because the enzymes, particularly the glycosyltransferases from natural sources, have been difficult to isolate. Also, the sugar nucleotide donors are very difficult to obtain from natural sources and are very expensive when derived from organic chemistry synthesis. More recently however, a recycling and reutilization strategy has been developed for synthesizing large quantities of oligosaccharides. U.S. Pat. No. 5,180,674, incorporated herein by reference, discloses a novel affinity chromatography method in which the reaction products are repetitively recycled over the matrix or resin bound glycosyltransferases. Furthermore, recent progress in gene cloning techniques have made several glycosyltransferases available in sufficient quality and quantity to make enzymatic synthesis of oligosaccharides more practical.
The literature is replete with descriptions of recombinant or transgenic expression of a heterologous glycosyltransferase. However, before continuing a discussion of the literature, it is necessary to clarify the meaning of various terms as used herein and in the claims:
(a) Host, host cell or host animal: These terms are used to refer to the cell or mammal which is responsible for the biosynthesis of biological material.
(b) Homologous: This word means that the entity thus characterized is normally present or produced by the host.
(c) Heterologous: This word means that the entity thus characterized is not normally present or produced by the host. In other words, the entity thus characterized is foreign to the host.
(d) Catalytic activity: This term is used to refer to the inherent property of certain biological compounds to facilitate chemical change in other substances.
(e) Catalytic entity: This term is used to refer to biological compounds which inherently possess catalytic activity which results in the production of new, different or altered compounds. Examples hereof are enzymes and antibodies. An enzyme is a biochemical catalyst of a specific biochemical reaction. An enzyme product is formed as a result of the enzyme's catalytic activity on a substrate material.
(f) Genome: This word is used to refer to the complete genetic material found in the host. This material is arranged in chromosomes.
(g) Gene: This word refers to a functional portion of the genome which is responsible for the biosynthesis of a specific biological entity.
(h) Insertion: This word is used to refer to the process whereby a portion of heterologous DNA or a heterologous gene that is introduced into the genome of a host. The DNA which is inserted is referred to as an "insert".
(i) Transgene: This refers to heterologous genetic material which is transferred by insertion from the genome of one animal species to the genome of another animal species. More simply, a transgene is a gene which is heterologous to the host. The transgene encodes a specific biological material.
(j) Transgenic mammal or transgenic host: These terms are used to refer to a mammal or cell which has had a transgene inserted into its genome. As a result of this insertion, the transgenic host produces heterologous biological material that it would not normally synthesize. Heterologous entities are present or are produced by a transgenic host as a result of the insertion of foreign genetic material into the host cell genome.
(k) Primary gene product: This refers to a biological entity which is formed directly as a result of the transcription and translation of a homologous or heterologous gene. Examples thereof include proteins, antibodies, enzymes and the like.
(l) Secondary gene product: This refers to a product which is formed as a result of the biological activity of a primary gene product. An example thereof, is an oligosaccharide which is formed as a result of the catalytic activity of an enzyme.
(m) Biological products: This term is used to refer to products produced or synthesized by a transgenic host as a result of the insertion of a transgene into the genome of the mammal. More specifically, the term means biological products which are secondary gene products. One example hereof, as described below, is human oligosaccharides produced by transgenic cows. Human oligosaccharides are produced as a result of the catalytic activity of human glycosyltransferases. As discovered herein, when the gene encoding human glycosyltransferases is inserted into the murine genome, the resultant transgenic mouse produces a heterologous human glycosyltransferase as the primary gene product. The human glycosyltransferase, using homologous substrate materials, produces oligosaccharides and glycosylated proteins. The oligosaccharide, formed as a result of enzyme activity of the primary gene product, is also properly called a secondary gene product. Glycoconjugates are another example of the class of compounds referred to herein and in the claims referred as "biological products".
(n) Product: This word is used to refer to the secondary gene products of the instant invention and is used as an alternative to "biological product".
(o) Humanized milk: This refers to milk obtained from a non-human mammal which, through alteration of the host genome, is made to produce milk which more closely resembles human milk. One example of humanized milk is cow's milk containing products found in human milk but not normally found in the cow's milk. Human oligosaccharides are produced in cow's milk as a result of the insertion of the gene encoding human glycosyltransferases into the bovine genome. Humanized milk also contains proteins glycosylated with human oligosaccharides.
As noted above, there is a considerable body of literature which describes the recombinant or transgenic expression of heterologous glycosyltransferases. However, the literature does not disclose or in any other manner suggest production of secondary gene products in the milk of non-human transgenic mammals as claimed in the instant invention. Examples of the literature are:
1) U.S. Pat. No. 5,032,519 to Paulson teaches a method for genetically engineering cells so that they produce soluble and secretable Golgi processing enzymes instead of the naturally occuring membrane-bound enzymes.
2) U.S. Pat. No. 5,047,335 to Paulson teaches the alteration by genetic engineering of the genome of Chinese Hamster Ovary Cells (CHO) so that the CHO cells produce a sialytransferase.
3) International Patent Application No. PCT/US91/08216 teaches a transgene capable of producing hetrologous recombinant proteins in the milk of transgenic bovine species. This published patent application teaches a method for obtaining the primary gene product only. This published patent application also discloses methods of producing and using the altered milk obtained from these transgenic animals.
4) International Patent Application No. PCT/US91/05917 teaches methods for intracellularly producing DNA segments by homologous recombination of smaller overlapping DNA fragments. This published patent application teaches a method for obtaining the primary gene product only.
5) International Patent Application No. PCT/GB87/00458 teaches methods of producing a peptide, said method involving incorporating a DNA sequence coding for the peptide into the gene of a mammal coding for a milk whey protein in such a way that that the DNA sequence is expressed in the mammary gland of the adult female mammal. This published patent application teaches a method for obtaining only the primary gene product, the peptide, in the milk of the transgenic mammal, and also discloses methods of producing and using the altered milk obtained from these transgenic animals.
6) International Patent Application No. PCT/GB89/01343 teaches methods for producing proteinaceous materials in transgenic animals that have genetic constructs integrated into their genomes. The construct comprises a 5'-flanking sequence from a mammalian milk protein gene and DNA coding for a heterologous protein other than a milk protein. This published patent application teaches a method for obtaining only the primary gene product, the heterologous protein, in the milk of the transgenic mammal.
7) European Patent Application No. 88301112.4 teaches methods for targetting specific genes to the mammary gland which results in the efficient synthesis and secretion of biologically important molecules into the milk of these transgenic animals. This published patent application also teaches methods of producing and using the altered milk obtained from these transgenic animals, and a method for obtaining only the primary gene product in the milk of the transgenic mammal.
8) International Patent Application No. PCT/DK93/00024 teaches methods for producing human kappa-casein in the milk of transgenic animals. The genetic construct comprises a 5'-flanking sequence from a mammalian milk protein gene, such as casein or whey acid protein, and DNA coding for human kappa casein. The DNA sequence contains at least one intron. This published patent application teaches a method for obtaining only the primary gene product, the heterologous human kappa casein, in the milk of the transgenic mammal.
9) International Patent Application No. PCT/US87/02069 teaches a method for producing mammals capable of expressing recombinant proteins in their milk.
These publications each teach, in one manner or another, a means for obtaining the primary gene product of the transgene, that gene product being the active protein or enzyme which is encoded by the transgene. This literature discloses transgenic means for obtaining glycosyltransferases in non-human milk. However, none of the aforementioned publications teaches or suggests the use of transgenic animals as a means of obtaining a desired secondary gene product which is the product of the active enzyme. More particularly, however, none of the aforementioned publications teaches or suggests or in any other manner discloses the use of transgenic human glycosyltransferases in non-human milk to produce human oligosaccharides or glycoconjugates bearing those oligosaccharides. These oligosaccharides, which are the product of active glycosyltransferases, are hereinafter referred to as the "secondary gene product". Thus, the various oligosaccharides found in human milk are formed as a direct result of the genetically regulated expression of certain specific glycosyltransferases. In this regard, oligosaccharides may be properly considered to be "secondary gene products" since they are synthesized as a result of the biochemical activity of the primary gene product, the heterologous glycosyltransferase enzymes.
Human milk contains a variety of oligosaccharides and proteins. Free, soluble oligosaccharides are not normally produced by animal cells and tissues with the exception of the highly differentiated lactating mammary glands. Oligosaccharides constitute the major portion of the total carbohydrate content of human and bovine milk. The major carbohydrate constituent of mammalian milk is the disaccharide lactose. Lactose is typically found at a concentration greater than 10 mg/ml and is synthesized by the attachment of galactose to glucose. This reaction is catalyzed by the enzyme, .beta.-1,4 galactosyltransferase. The milk of most mammals, including cows, contains only very small quantities of a few additional oligosaccharides. In contrast, human milk contains substantial amounts of a number of additional soluble oligosaccharides that are larger than lactose. All human oligosaccharides are synthesized by the sequential addition of monosaccharides to lactose. Representative oligosaccharides found in human milk are shown in Table 1.
TABLE 1 __________________________________________________________________________ OLIGOSACCHARIDES PRESENT IN HUMAN MILK Structure Common Name Concentration (mg/liter) __________________________________________________________________________ Gal.beta.-1,4-Glc Lactose 50,000 Fuc-a-1,2-Gal-.beta.-1,4-Glc 2-fucosyllactose 200 Gal-.beta.-1,3-GlcNAc-.beta.-1,3-Gal-.beta.-1,4-Glc Lacto-N-tetraose 400 Gal-.beta.-1,4-GlcNAc-.beta.-1,3-Gal-.beta.-1,4-Glc Lacto-N-neotetraose 60 Fuc-a-1,2-Gal-.beta.-1,3-GlcNAc-.beta.- Lacto-N-fucopentaose I 200 1,3-Gal-.beta.-1,4-Glc Gal-.beta.-1,3Fuc-a-1,4!GlcNAc-.beta.- Lacto-N-fucopentaose II 20 1,3-Gal-.beta.-1,4-Glc Gal-.beta.-1,4Fuc-a-1,3GlcNAc-.beta.- Lacto-N-fucopentoase III 50 1,3-Gla-.beta.-1,4-Glc Fuc-a-1,2-Gal-.beta.-1,3Fuc-a-1,4!- Lacto-N-difucopentaose I 25 GlcNAc-.beta.-1,3-Gal-.beta.-1,4-Glc NeuAc-a-2,6-Gal-a-1,4-Glc 6-sialyllactose 25 10. NeuAc-a-2,3-Gal-.beta.-1,4-Glc 3-sialyllactose 10 NeuAc-a-2,3-Gal-.beta.-1,3-R Sialyltetrasaccharide a 10 Gal-.beta.-1,3NeuAc-a-2,6!GlcNAc- Sialyltetrasaccharide b 35 .beta.-1,3-R NeuAc-a-2,6-Gal-.beta.-1,4-GlcNAc- Sialyltetrasaccharide c 50 .beta.-1,3-R NeuAc-a-2,3-Gal-.beta.-1,3NeuAc-a-2,6!- Disialyltetrasaccharide 60 GlcNAc-.beta.-1,3-Gal-.beta.-1,4-Glc NeuAc-a-2,3-Gal-.beta.-1,3Fuc-a-1,4!- Sialyl Lacto-N-fucopentaose 50 GlcNAc-.beta.-1,3-Gal-.beta.-1,4-Glc __________________________________________________________________________ -a-: denotes an alpha glycosidic linkage R: Gal.beta.-1,4-Glc
The oligosaccharides in human milk are present as a result of the activity of certain specific glycosyltransferases found in human mammary tissue. For example, the alpha 1,2 linked fucose residues in structures 2,5, and 8 are produced by a unique human fucosyltransferase and characterize a phenotype known in the field of immunohematology as "secretors". These individuals are thus characterized because they synthesize human blood group substances in their salivary and other mucus secretions where the oligosaccharides are covalently attached to various proteins.
The alpha 1,4 linked fucose residues in structures 6,8, and 15 are formed as a result of the enzymatic action of a different fucosyltransferase. These oligosaccharides represent a phenotype present in individuals characterized as having a "Lewis positive" blood type. Such individuals use this fucosyltransferase to synthesize an oligosaccharide structure which corresponds to a human blood group antigen. This oligosaccharide is also found in the saliva, and other mucus secretions, and covalently attached to lipids found on the membrane of red blood cells of "Lewis positive" individuals. Structure 5 is related to the H-antigen of the ABO blood group; structure 6 is the "Lewis a" blood group antigen; structure 8 is the "Lewis b" blood group antigen.
At least fifteen human milk proteins have been identified. Some of these proteins are generally recognized to be glycosylated, i.e. they are covalently attached to certain specific oligosaccharides. The particular oligosaccharides which are covalently attached to the protein are the same as, or similar to, the oligosaccharides described above, and their formation is the result of normal genetically regulated expression of certain specific glycosyltransferase genes. The presence of a heterologous glycosyltransferase would also affect the post-translational modification of proteins. The proteins glycosylated by a heterologous glycosyltransferase are also properly known as "secondary gene products". Both homologous as well as heterologous proteins would be modified by the glycosyltransferase in a manner different from that resulting from the activity of homologous glycosyltransferases.
It has long been known that these oligosaccharides and glycosylated proteins, promote the growth of desirable bacteria in the human intestinal tract. It is also believed that the oligosaccharides in human milk inhibit the attachment of harmful microorganisms to the mouth and throat. These human oligosaccharides and specifically glycosylated proteins are absent from, or present in markedly different amounts in, bovine milk. Further, as noted previously, bovine milk contains predominantly lactose only. Human milk contains not only lactose but also numerous other oligosaccharides. Also, the amino acid composition of human milk proteins is significantly different from the amino acid composition of the corresponding cow's milk proteins. As a consequence, infants fed infant formula which comprises cow's milk may be more susceptible to intestinal disturbances such as diarrhea, or their blood plasma amino acid ratios and levels may differ from breast fed infants. For the same reasons, elderly, immunocompromised and critically ill patients also have an urgent need for the availability of a nutritional product which biochemically closely resembles the composition of human milk.
The complicated chemistry of human milk proteins and oligosaccharides has made their large scale synthesis extremely difficult. Before they can be incorporated into commercial nutritional product, a practical method for obtaining large amounts of glycosylated human milk proteins and oligosaccharides must be devised. One potential solution to this problem is the use of transgenic animals, more particularly transgenic cows which express genes or cDNAs encoding enzymes which catalyse the formation of oligosaccharides and/or proteins glycosylated with the same human oligosaccharides. Transgenic milk-bearing domestic animals, such as rabbits, pigs, sheep, goats and cows, are herein proposed as a means of producing milk containing human oligosaccharides and proteins glycosylated with human oligosaccharides. More particularly, transgenic cows are highly suitable for the production of oligosaccharides and recombinant proteins, because a single cow can produce more than 10,000 liters of milk containing as much as 300 kilograms of protein (mainly casein) per year at a very minimal cost. Thus, transgenic cows appear to be a less costly production route than other recombinant protein production methods since investment in fermentation facilities would not be required. Also, cow mammary glands are more cost effective than cultured cells, are likely to produce continuously and since milk is collected several times a day, the time between the actual synthesis and harvest can be as short as a few hours. The cow's genetic stability is greater than microbial or cell based production systems. Also, cows are relatively easy to reproduce using artificial insemination, embryo transfer and embryo cloning techniques. Further, downstream processing of cow's milk containing human transgenic proteins may require little or no purification. Publications teaching such methods are referred to below. However, none of these publications teaches, discloses or in any other manner suggests production of secondary gene products in the milk of non-human transgenic mammals as claimed in the instant invention.
"Molecular Farming: Transgenic Animals as Bioreactors" by J. Van Brunt, Biotechnology, Volume 6, page 1149-1154, 1988, describes the alteration of the genome of various large domestic milk bearing animals yielding transgenic animals capable of producing various heterologous entities. This publication suggests methods for obtaining the primary gene product only.
International Patent Application No. PCT/US91/08216 describes a transgene capable of producing heterologous recombinant proteins in the milk of transgenic bovine species. This published patent application teaches a method for obtaining the primary gene product only. This application also discloses methods of producing and using the altered milk obtained from these transgenic animals.
International Patent Application No. PCT/GB87/00458 describes methods for producing a peptide, said method involving incorporating a DNA sequence coding for the peptide into the gene of a mammal coding for a milk whey protein in such a way that that the DNA sequence is expressed in the mammary gland of the adult female mammal. This published patent application teaches a method for obtaining only the primary gene product, the peptide, in the milk of the transgenic mammal. This application also discloses methods of producing and using the altered milk obtained from these transgenic animals.
International Patent Application No. PCT/GB89/01343 teaches methods of producing proteinaceous materials in transgenic animals that have genetic constructs integrated into their genome. The construct comprises a 5'-flanking sequence from a mammalian milk protein gene and DNA coding for a heterologous protein other than a milk protein. This published patent application teaches a method for obtaining only the primary gene product, the heterologous protein, in the milk of the transgenic mammal.
European Patent Application No. 88301112.4 describes methods for targeting specific genes to the mammary glands which results in the efficient synthesis and secretion of biologically important molecules into the milk of these transgenic animals. This published application also discloses methods of producing and using the altered milk obtained from these transgenic animals and teaches a method for obtaining only the primary gene product in the milk of the transgenic mammal.
International Patent Application No. PCT/US87/02069 teaches a method for producing mammals capable of expressing recombinant proteins in the milk of lactating animals. This patent application does not disclose or in any other manner suggest production of secondary gene products in the milk of non-human transgenic mammals as claimed in the instant invention.
While transgenic animals can be used for the production of large quantities of human proteins, they have not been used for the production of secondary gene products, such as human oligosaccharides or proteins and lipids glycosylated with certain specific oligosaccharides, or human milk proteins and lipids glycosylated with certain specific oligosaccharides. None of the aforementioned publications discloses or suggests a method for producing human oligosaccharides and glycoconjugates in non-human mammalian milk. The aforementioned publications also do not disclose or suggest a method for obtaining glycoconjugates in non-human mammalian milk wherein the glycosylation is with the desired oligosaccharides. Achieving this result requires that the genome of non-human milk-bearing mammals be altered so as to ensure, that the mammary tissue which selectively expresses a desired human glycosyltransferase which would then glycosylate certain proteins with the desired oligosaccharide. This approach requires the DNA encoding the desired human glycosyltransferase be incorporated into said genome. The literature also does not disclose or suggest a method for obtaining glycosylated human proteins in non-human mammalian milk wherein the glycosylation is with the desired oligosaccharides. The literature also does not disclose or suggest a method for obtaining glycosylated human milk proteins in non-human mammalian milk wherein the glycosylation is with the desired oligosaccharide moieties. Achieving this result would require that the genome of non-human milk-bearing mammals to be altered so as to ensure that its mammary tissue selectively expresses both the human glycosyltransferase as well as the desired human proteins which are then appropriately glycosylated with the desired oligosaccharides by the active human glycosyltransferase. This approach requires not only that the DNA encoding the desired glycosyltransferase be inserted into said genome but also that the DNA encoding the desired human proteins also be incorporated into said genome.
Accordingly, it is an aspect of the present invention to provide methods for detecting succesful transgenesis of fertilized oocytes prior to implantation, such that the transplanted oocytes contain the genetic constructs required to achieve the desired glycosylation and oligosaccharide production.
It is also an aspect of the present invention to provide transgenic non-human milk bearing mammalian species which are capable of producing human glycosyltransferases that are secreted extracellularly by the mammary tissue of said mammalian species.
Further, it is also an aspect of the present invention to provide transgenic non-human milk bearing mammalian species which are capable of producing human glycosyltransferases that are secreted extracellularly by the mammary tissue into the milk produced by said mammalian species.
In addition, it is an aspect of the present invention to provide transgenic non-human milk bearing mammalian species which are capable of producing glycosylated human proteins and oligosaccharides that are secreted extracellularly by the mammary tissue into the milk produced by said mammalian species.
The present invention also relates to transgenic non-human milk bearing mammalian species which are capable of producing glycosylated human milk proteins and lipids in the milk of such transgenic animals.
It is also an aspect of the present invention to provide transgenic non-human milk bearing mammalian species which are capable of producing human oligosaccharides in the milk of such transgenic animals.
The present invention also relates to food formulations containing glycosylated human proteins, lipids and oligosaccharides from such transgenic milk.
The present invention also relates to pharmaceutical, medical diagnostic and agricultural formulations containing glycosylated proteins, lipids and oligosaccharides obtained from the milk of transgenic animals.
It is also an aspect of the present invention to provide transgenic bovine species that are capable of producing glycosylated proteins such as glycosylated human milk proteins and lipids in their mammary glands.
It is a further aspect of the present invention to provide transgenic bovine species that are capable of producing human oligosaccharides in the milk of such transgenic cows.
The present invention also relates to food formulations containing glycosylated proteins, lipids and oligosaccharides from such transgenic bovine milk.
The present invention also relates to pharmaceutical, medical diagnostic and agricultural formulations containing glycosylated proteins, lipids and oligosaccharides obtained from the milk of transgenic cows.

Transgenic non-human mammals producing oligosaccharides and glycoconjugates


A method of using a triple-effect absorption system to recover methane from landfill gas contaminated with CO2 and trace contaminates such as chlorinated hydrocarbons and aromatics involves processing the landfill gas with three absorbers and a flash system. One absorber uses a solvent to absorb the trace contaminants from the landfill gas, the second absorber in conjunction with the flash system extracts CO2 from the gas, and just a first portion of that CO2 is used for stripping the trace contaminates from the solvent in the third absorber. The rest of the extracted CO2 is vented to atmosphere to prevent dampening the combustion of the trace contaminants absorbed by the first portion of CO2.

1. Field of the Invention
The subject invention generally pertains to processing landfill gas and more specifically to an absorption system for recovering methane gas.
2. Description of Related Art
Decomposing garbage buried in a landfill can generate landfill gas that can be extracted and processed to provide relatively clean methane gas. Processing plants have been developed for extracting the methane and disposing of its contaminants in a responsible manner. In order to attain the full value of the methane, processing plants need to remove the contaminants efficiently and thoroughly. Otherwise, unacceptably high concentrations of the contaminants can remain in the methane, thereby decreasing its value. In some cases, certain contaminants may accumulate in the processing plant itself, which can reduce the plant's operating efficiency.
Current gas processing plants are more efficient at removing some impurities than others, so there is still a need for a better method of recovering clean methane gas from landfills.

Triple-effect absorption system for recovering methane gas


A multilayer ceramic capacitor exhibits superior life characteristics in a high temperature load test despite the use of very thin dielectric layers. As a dielectric ceramic constituting a dielectric layer of the multilayer ceramic capacitor, a perovskite-type compound is used and contains Ba and Ti (a portion of Ba can be replaced with at least one of Ca and Sr, and a portion of Ti can be replaced with Zr) as a main component, and including La within the range of 2-6 parts by mole, Mg within the range of 3-5 parts by mole, and Mn within the range of 1.5-3 parts by mole in a case where a total content of Ti and Zr is 100 parts by mole.

1. Field of the Invention
The present invention relates to a multilayer ceramic capacitor. Further, the present invention relates to a method for manufacturing a multilayer ceramic capacitor.
2. Description of the Related Art
A multilayer ceramic capacitor as a typical ceramic electronic component generally includes a multilayer body having a plurality of laminated dielectric layers and a plurality of internal electrodes arranged along interfaces between the dielectric layers and a plurality of external electrodes formed on an outer surface of the multilayer body and electrically connected to the internal electrodes.
With the progress of electronics technology in recent years, miniaturization and capacity enlargement in a multilayer ceramic capacitor are required. To meet these requirements, thinning of a dielectric layer in a multilayer ceramic capacitor has been promoted. However, thinning of a dielectric layer causes a field intensity applied to each layer to be relatively high. Therefore, improvement in reliability during voltage application and particularly in life characteristics presented by a high temperature load test is required with respect to a dielectric ceramic included in a dielectric layer.
An example of a dielectric ceramic constituting a dielectric layer is known from the disclosure of Japanese Patent Publication No. 4-169003. Japanese Patent Publication No. 4-169003 discloses a dielectric ceramic. The dielectric ceramic contains 100 parts of main components including 95.0-98.0 molar % of BaTiO3 having an unreacted BaO content of less than or equal to 0.7 weight % and a Ba/Ti molar ratio of 1.005-1.025, and 2.0-5.0 molar % of rare-earth oxide of at least one type selected from the group consisting of La, Nd, Sm, Dy and Er. The dielectric ceramic contains subcomponents including 0.3-1.5 parts by weight of MnO and 0.5-2.5 parts by weight of oxide glass having BaO—SrO—Li2O—SiO2 as a main component, with respect to the main components.
Japanese Patent Publication No. 4-169003 does not describe the case where the dielectric layers are formed to be thinner. Therefore, when the dielectric ceramic disclosed in Japanese Patent Publication No. 4-169003 is employed as a dielectric layer for a multilayer ceramic capacitor exhibiting the progress of thinning, high reliability during voltage application is not assured.

Multilayer ceramic capacitor and method for manufacturing multilayer ceramic capacitor


A method for producing a horizontal magnetic recording medium that has as its magnetic film a granular film with grains of a chemically-ordered FePt or FePtX (or CoPt or CoPtX) alloy in the tetragonal L1.sub.0 structure uses an etched seed layer beneath the granular film. The granular magnetic film reveals a very high magnetocrystalline anisotropy within the individual grains. The film is produced by sputtering from a single alloy target or cosputtering from several targets. The granular structure and the chemical ordering are controlled by means of sputter parameters, e.g., temperature and deposition rate, and by the use of the etched seed layer that provides a structure for the subsequently sputter-deposited granular magnetic film. The structure of the seed layer is obtained by sputter etching, plasma etching, ion irradiation, or laser irradiation. The magnetic properties, i.e., H.sub.c and areal moment density M.sub.r t, are controlled by the granularity (grain size and grain distribution), the degree of chemical ordering, and the addition of one or more nonmagnetic materials, such as Cr, Ag, Cu, Ta, or B. The resulting granular magnetic film has magnetic properties suitable for application in high-density, horizontal magnetic recording media.

Conventional magnetic recording media for horizontal recording, such as hexagonal-close-packed (HCP) cobalt-platinum (CoPt) alloys, are unable to achieve recording densities above approximately 2-5 Gbit/in.sup.2. An historic trend has been to require reduced magnetic areal moment density (M.sub.r t) and increased coercivity (H.sub.c) to achieve recording at higher recording densities. High-density recording media also need to have exchange decoupled particles or grains. Smaller grains are required at higher densities for reduced intrinsic media noise to obtain a higher signal-to-noise ratio in the readback data. Thus, high-density recording media require both a reduced M.sub.r t and a reduced grain size.
Bulk tetragonal L1.sub.0 -ordered phase materials (also called CuAu materials), such as CoPt and FePt, are known for their high magnetocrystalline anisotropy and magnetic moment, properties that are also desirable for high-density magnetic recording media. The C-axis of the L1.sub.0 phase is similar to the C-axis of HCP CoPt alloys in that both are the easy axis of magnetization. Thus, while the disordered face-centered-cubic (FCC) solid solution of Co and Pt has cubic symmetry and low magnetic anisotropy, the ordered L1.sub.0 phase has uniaxial anisotropy similar to, but greater in magnitude than, HCP CoPt alloys.
Previous studies on the L1.sub.0 phase of FePt have concentrated mainly on the epitaxial growth of highly chemically-ordered films grown by molecular beam epitaxy. Several studies dedicated to the application of sputter-deposited, chemically-ordered alloys of FePt and CoPt as thin films for horizontal magnetic recording media describe the requirement for a relatively high-temperature post-deposition annealing to achieve the chemical ordering. These are described in Coffey et al., "High Anisotropy L1.sub.0 Thin Films for Longitudinal Recording", IEEE Transactions on Magnetics, Vol. 31, No. 6, November 1995, pp. 2737-2739; and Watanabe et al., "Microstructure and Magnetic Properties of High-Coercive Fe-Pt Alloy Thin Films", Materials Transactions, JIM, Vol. 37, No. 3, 1996, pp. 489-493. This post-deposition annealing process results in an increase of the grain size from about 10 nm in the as-deposited films to about 30 nm in the annealed films. Due to this larger grain size, these films have shown rather poor recording properties, in particular a low signal-to-noise ratio. In addition, the high-temperature annealing process is not compatible with existing magnetic recording media fabrication processes and materials.
What is needed is a method for making chemically-ordered, high magnetocrystalline anisotropy FePt and CoPt thin films with a small grain size and without post-deposition annealing.

Method for making horizontal magnetic recording media having grains of chemically-ordered FePt or CoPt

International Business Machines Corporation


A method is described for processing a signal at a relay node in a wireless communication system. More specifically, in one embodiment, the relay node receives a codeword broadcast from a source node. The codeword is partitioned into a plurality of sub-blocks. Then, the relay node generates two network encoded codewords by combining linearly the plurality of sub-blocks, and transmits the two network encoded codewords to a destination node. The destination node detects the codeword by using a zero-forcing receiver.

1. Field of the Invention
The present invention relates to a network coding for cooperative relay network. More specifically, the invention integrates the network coding with the clustered cooperative communication at the physical layer.
2.Discussion of the Related Art
In the present invention, the network coding for the cooperative relay network is suggested. Hereafter, a D2D (device-to-device) communication is explained. FIG. 1 illustrates a concept of the D2D communication.
Referring to FIG. 1, UE1 and UE2 are performing the D2D communication, and UE3 and UE4 are also performing the the D2D communication. An eNB may perform controls for the D2D communication, such as a location of time/frequency resources or a transmission power, using a suitable control signals. But, if the UE exists out of coverage of the eNB, the D2D communication can be performed without the controls for the D2D communication of the eNB.

Method for network coding for cooperative relay network in wireless communication system

RESEARCH & BUSINESS FOUNDATION SUNGKYUNKWAN UNIVERSITY


A particulate material production apparatus is disclosed. The particulate material production apparatus includes a droplet ejector to eject droplets of a particulate material composition liquid or a melted particulate material composition in a droplet ejection direction from nozzles; a solidifying device to solidify the droplets; a first airflow forming device to form a first airflow to feed the ejected droplets to the solidifying device with the first airflow; and a second airflow forming device to form a second airflow to apply the second airflow the droplets before the droplets are fed by the first airflow. The second airflow forming device forms the second airflow by supplying a pressed gas from a slit, and the traveling direction of the first airflow is substantially perpendicular to the droplet ejection direction.

Uniformly-shaped particulate resins have been used for various purposes such as electrophotographic toner particles, spacer particles for use in liquid crystal panels, colored particles for use in electronic papers, and carriers of medical agents for use in drugs and medicines.
Specific examples of the method for producing such uniformly-shaped particulate resins include methods in which a particulate resin having a uniform particle diameter is produced by making a reaction in a liquid, such as soap-free polymerization methods. Soap-free polymerization methods generally have advantages such that a particulate resin having a relatively small particle diameter can be easily produced; the particle diameter distribution is sharp; and the particle form is nearly spherical. However, on the other side, soap-free polymerization methods have problems to be solved such that the production efficiency is bad because a solvent is removed from resin particles in a medium, which is typically water; it takes a long time for the polymerization process; and a long time and large amounts of water and energy are necessary for producing a particulate resin because it is necessary for the methods to perform various processes after the end of a solidification process, such as a process for separating the resultant particulate resin from the solvent used, and processes for repeatedly washing and drying the particulate resin, to produce the particulate resin.
In attempting to solve the problems mentioned above, one of the present inventors and other inventors propose a toner production method using an ejection granulation method in JP-2008-292976-A. Specifically, according to the toner production method, a toner is produced as follows. Namely, in a droplet ejecting unit to eject droplets of a toner composition liquid, which is a raw material of toner, a thin film, on which multiple nozzles are formed, is vibrated by an electromechanical converter serving as a vibration generator to periodically vibrate the thin film up and down. Thereby, the pressure in a chamber, which contains the toner composition liquid and which includes the thin film having the multiple nozzles as a constitutional member, is changed, resulting in ejection of droplets of the toner composition liquid from the nozzles to an air space below the nozzles in response to the periodical change of pressure. The thus ejected droplets of the toner composition liquid naturally fall through the air space and proceed in the same direction, thereby forming lines of droplets of the toner composition liquid. In this regard, the ejected droplets are reshaped so as to be spherical due to the difference in surface tension between the toner component liquid and air in the air space. The reshaped droplets are then dried, resulting in formation of a particulate toner.
In addition, one of the present inventors and other inventors propose a toner production method in JP-2008-286947-A in attempting to prevent uniting of ejected droplets. Specifically, in this second toner production method, droplets of a toner composition liquid are ejected from multiple nozzles to continuously form droplets of the toner composition liquid. In addition, an airflow forming device is provided to supply an airflow in the droplet ejection direction through a narrow passage provided on a downstream side relative to the droplet ejection direction, so that the droplets are fed by the airflow and thereby uniting of the droplets is prevented.

Particulate material production apparatus, and particulate material production method


Proppants used to prop open subterranean formation fractions. Proppant formulations using one or more proppants. Methods to prop open subterranean formation fractions and other uses for the proppants, as well as methods of making the proppants.

The present invention relates to proppant materials and other uses for the proppants. The present invention further relates to methods to make a proppant.
Previously, some proppants were made by Oxane Materials, Inc., so that the proppant had a template, such as a template sphere, with a shell wherein the template sphere or core typically provided a lightweight or low density material that had a void or voids, such as a cenosphere. The shell that was present preferably was a strength-bearing shell that lead to the overall crush resistance of the proppant once the shell encapsulated or coated the template sphere. This proppant holds much promise for addressing many of the disadvantages with previous proppant materials. However, if a proppant could be prepared having the same or similar lightweight qualities, as well as strength-bearing qualities, as the core/shell proppant, but without the need to form a shell on the template sphere, this would have numerous advantages with respect to costs, the amount of material used, and the like. Through a reaction of the proppant, such as on the surface, a separate shell is not needed, since the reaction forms a phase that can replace the need of a shell.
Ceramic proppants are widely used as propping agents to maintain permeability in oil and gas formations. Conventional proppants offered for sale exhibit exceptional crush strength but also extreme density. Typical densities of ceramic proppants exceed 100 pounds per cubic foot. Proppants are materials pumped into oil or gas wells at extreme pressure in a carrier solution (typically brine) during the fracturing process. Once the pumping-induced pressure is removed, proppants "prop" open fractures in the rock formation and thus preclude the fracture from closing. As a result, the amount of formation surface area exposed to the well bore is increased, enhancing recovery rates. Proppants also add mechanical strength to the formation and thus help maintain flow rates over time. Three grades of proppants are typically employed: sand, resin-coated sand and ceramic proppants. Proppants are principally used in gas wells, but do find application in oil wells.
Relevant quality parameters include: particle density (low density is desirable), crush strength and hardness, particle size (value depends on formation type), particle size distribution (tight distributions are desirable), particle shape (spherical shape is desired), pore size distribution (tight distributions are desirable), surface smoothness, corrosion resistance, temperature stability, and hydrophilicity (hydro-neutral to phobic is desired).
Ceramic proppants dominate sand and resin-coated sand on the critical dimensions of crush strength and hardness. They offer some benefit in terms of maximum achievable particle size, corrosion and temperature capability. Extensive theoretical modeling and practical case experience suggest that conventional ceramic proppants offer compelling benefits relative to sand or resin-coated sand for most formations. Ceramic-driven flow rate and recovery improvements of 20% or more relative to conventional sand solutions are not uncommon.
Ceramic proppants were initially developed for use in deep wells (e.g., those deeper than 7,500 feet) where sand's crush strength is inadequate. In an attempt to expand their addressable market, ceramic proppant manufacturers have introduced products focused on wells of intermediate depth.
Resin-coated sands offer a number of advantages relative to conventional sands. First, resin coated sands exhibit higher crush strength than uncoated sand given that resin-coating disperses load stresses over a wider area. Second, resin-coated sands are "tacky" and thus exhibit reduced "proppant flow-back" relative to conventional sand proppants (e.g. the proppant stays in the formation better). Third, resin coatings typically increase sphericity and roundness thereby reducing flow resistance through the proppant pack.
Ceramics are typically employed in wells of intermediate to deep depth. Shallow wells typically employ sand or no proppant. As will be described in later sections, shallow "water fracs'" represent a potential market roughly equivalent to the current ceramic market in terms of ceramic market size.
The family of non oxide based ceramic materials, specifically the carbides and nitrides of metallic materials, display exceptional mechanical, thermal and chemical properties all of which in combination would be ideal candidates for a proppant system. Although, they display very high intrinsic failure strengths, hardnesses, and fracture toughnesses, their apparent properties are highly dependent upon the microstructure of the ceramic material that develops during the sintering stage. Significant research has been conducted in the sintering of the carbide and nitride class of materials, the most important of which is the use of a glass forming liquid phase sintering aid to assist with the densification of the system. Although, the liquid phase sintering approach assists with the densification, the properties of such a system are less than optimal and fail to reach the intrinsic properties that these materials are capable of, due primarily to the effects of a relatively weak phase existing at the grain boundaries of the ceramic material. In addition, with the liquid phase sintering approach, a high level of shrinkage occurs during sintering. The degree of shrinkage is dependent upon a number of parameters, the most critical of which is particle size. Typically the degree of shrinkage can approach 20% or higher.
Another approach to improve the sintering and consequently the properties of such ceramic systems has been with a reaction mechanism that forms the appropriate carbide and/or nitride phase directly from the metallic phase. In this method, a preform of the appropriate metal is produced, with approximately 25-30% percent residual porosity. The component is then subjected to thermal treatments under the appropriate atmosphere to induce the formation of the carbide or nitride phase. During the formation of the carbide or nitride phase, a volume increase occurs, which serves to close the residual porosity and yield a highly dense ceramic body that is more or less pore free. By carefully controlling the initial porosity of the preform, the volume expansion associated with the formation of the carbide or nitride phase will completely fill all internal porosity and the outer volume of the preform will remain unchanged. This process is termed net shape forming.

Proppants with carbide and/or nitride phases


An electrical cable or an optical cable, which has a core with a cladding. An imprint is on the cladding and has the imprint formed by a plastic powder which contains copolymers of ethylene with unpolar comonomers.

The present invention is directed to a cable which can be used either as an electrical cable or optical cable and has a cladding that comprises imprints produced from a plastic powder.
British Patent No. 1,257,769, which claims priority from German Application 19 51 597, discloses a cable which has imprints on a cable cladding or sheath. The raised imprints are formed by sintering a plastic powder on the cable cladding, and this powder should be selected as close as possible to or the same material as the cable cladding. White or chromatically pigmented low-density polyethylene (LDPE) powders are usually used for the black LDPE claddings, which are resistant to stress tears and are usually employed as a cladding. For example, plastic powders that are known under the tradenames xe2x80x9cPECMA 200xe2x80x9d or xe2x80x9cCEVAMxe2x80x9d, which is an acrylic acid copolymer sold by Interorgana of Stuttgart, Germany, are used as products for the imprinting.
In addition to having a good workability and adequate adhesion, the plastic powders used for the imprinting must be of such a nature that they do not lead to any stress cracks. Mechanically, the imprinting of the respective identifier to be applied on the cable cladding or sheath represent a certain intervention that intrinsically harbors the risk of forming stress cracks.
The present invention is based on the object of creating a plastic powder suitable for the imprinting, wherein the protection of the cable cladding against the formation of stress cracks is also assured to the farthest-reaching extent possible in addition to providing a good workability.
To accomplish these objects, the invention is directed to an improvement in a cable having a core with a cable cladding that comprises imprints manufactured of a plastic powder. The improvement is achieved in that the plastic powder contains copolymers of ethylene with unpolar comonomers.
The plastic powder, which is constituted according to the present invention, can be easily worked, yields good adhesion and, as investigations have shown, harbors far less of a risk of forming stress cracks.
Suitable copolymers of ethylene with unpolar comonomers for the inventive plastic powder are preferably commercially available as linear low density polyethylene (LLDPE) as well as very low density polyethylene (VLDPE). For example, powders offered under the tradenames xe2x80x9cATTANAxe2x80x9d and xe2x80x9cENGAGE POPxe2x80x9d as well as xe2x80x9cENGAGE POExe2x80x9d of Dow Chemical as well as xe2x80x9cCLEARFLEXxe2x80x9d of ENI-Chemie are particularly suitable. Altogether good results with respect to the resistance to stress cracks were capable of being achieved in tests with the aforementioned materials.
Linear polyethylenes that, in particular, contain alpha olefins, preferably alpha-olefin propene, butene, hexene, octene and mixtures thereof as comonomers are preferably employed.
The proportion of comonomers within the plastic powder should be advantageously selected so that the reduced density of the ethylene copolymer caused by the comonomers is less than or equal to 0.92.
Various additives can also be added to the plastic powder within the scope of the invention, especially those for optimizing the adhesion, such as, for example, ethylene copolymers with polar comonomers, such as ethylene vinyl acetate (EVA) copolymers.
Additional stabilizers against heat and/or light as well as potentially anti-oxidants can also be advantageously added to the plastic powder of the invention. Including chromatic pigments into the plastic powder for improving the visibility or the legibility of the imprint is also expedient.
The plastic powder of the invention should expediently have a grain fineness with a grain diameter in a range of 50 xcexcm through 200 xcexcm.
Other advantages and features of the invention will be readily apparent from the following description of the preferred embodiments, the drawing and claims.

Electrical or optical cable with an imprint on the cable cladding


An electrical connector including an insulative housing (10) comprising a longitudinal base (110), a pair of shoulder portions (130) respectively projecting forwards from two opposite end sections of the longitudinal base, and a tongue plate (120) projecting forwards from a middle section of the longitudinal base and located between the pair of shoulder portions; a plurality of terminals (30) arranged on the tongue plate; and a shell (20) provided with a rectangle frame surrounding the tongue plate and defining a mating opening therein for accommodating a mating portion of a mating connector, the rectangle frame fitly set between the shoulder portions.

1. Field of the Invention
The present invention relates to an electrical connector, and more particularly to an electrical connector having a shielding shell.
2. Description of Related Art
U.S. Pat. No. D471,872 discloses a conventional connector comprising an insulative housing, a plurality of electrical terminals, and a metal shell. The housing includes a tongue plate on which the electrical terminals are arranged and a circuit of side walls surrounding the tongue plate and defining a rectangle mating opening for receiving a mating portion of a mating connector. The shell is of a two-double structure respectively attached to inner surfaces and outer surfaces of the side walls. However, as the mating opening is provided by the side walls of the housing together with the shell attached thereon, it increases the size of connector and thus does not meet the minimizing request for some kind of connectors. Otherwise, the complicated structure of the two-double shell increases manufacturing difficulty of the connectors and thus increases manufacturing cost of the connectors.
Therefore, a new connector is desired to overcome above-mentioned problems.

Electrical connector with shielding shell