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Pyridine Nucleotides in Erythrocyte Metabolism *
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1 Journal of Clinical Investigation Vol. 45, No. 2, 1966 Pyridine Nucleotides in Erythrocyte Metabolism * RUTH T. GROSS,t ELEANOR A. SCHROEDER, AND BEVERLY W. GABRIO (From the Department of Pediatrics, Albert Einstein College of Medicine, New York, N. Y., and the King County Central Blood Bank and the Department of Biochemistry, University of Washington, Seattle, Wash.) In recent years investigations of erythrocyte metabolism have been concerned with the interrelationships of enzymes, coenzymes, substrates, cofactors, and ph, inter alia (1-8). Although data have accumulated on these various aspects of the metabolism of normal and pathological erythrocytes, little direct information has been available heretofore on pyridine nucleotide coenzymes, despite their importance in both the glycolytic and the hexose monophosphate shunt pathways. The availability of these compounds in the blood of immature subjects is of particular interest because of certain abnormalities in the erythrocytes of fullterm newborn and prematurely born infants. These abnormalities, which include decreased survival of 51Cr-labeled erythrocytes in premature infants (9, 10) and increased susceptibility to Heinz body formation (11) and to methemoglobinemia (12-15) in both full-term and premature infants, as well as the occurrence of unexplained hemolytic anemias, suggest the possibility of transient biochemical defects in the erythrocytes of these young subjects. Although the activities of the pyridine nucleotide-dependent enzymes in both the glycolytic and shunt pathways are increased in the erythrocytes of young infants (16, 17), the levels of the oxidized and reduced coenzymes themselves have not been reported. In the present study the concentrations of DPN,l * Submitted for publication August 16, 1965; accepted November 4, This work was supported by grants HE-7890 and HD from the U. S. Public Health Service. t Address requests for reprints to Dr. Ruth T. Gross, Albert Einstein College of Medicine, Eastchester Rd. and Morris Park Ave., New York 61, N. Y. 1 The following abbreviations are used: DPN and TPN, di- and triphosphopyridine nucleotide; DPNH and TPNH, reduced DPN and TPN; DEAE, diethylaminoethyl; PGD, 3-phosphoglyceraldehyde dehydrogenase; LDH, lactic dehydrogenase; G-6-PD, glucose-6-phosphate dehydrogenase; 6-PGD, 6-phosphogluconic dehydrogenase; GR, glutathione reductase; Hb`R, TPNH- DPNH, TPN, and TPNH have been determined in the blood of healthy human adults and premature infants and in cord blood by the sensitive technique of enzymatic cycling (18). Reported also are correlative studies to evaluate the response of oxidized and reduced pyridine nucleotides as well as pyridine nucleotide-requiring enzymes (PGD, LDH, G-6-PD, 6-PGD, GR, and Hb3R) to in vitro preservation of blood at 40 C in ACD, ACDadenine, and ACD-inosine. Methods Subjects. The studies to be presented are a compilation of experiments performed independently in two laboratories, one in New York and the other in Seattle. Studies on adult human erythrocytes were undertaken in both laboratories. The samples from adult subjects both in New York and Seattle were obtained in each city from healthy hospital and laboratory personnel or blood donors and were studied only in the respective laboratories. Studies on erythrocytes from premature infants were performed in New York on infants selected from the premature nursery of the Bronx Municipal Hospital Center. Selection was made by exclusion of infants with recognized disease as well as infants receiving medications other than routine vitamin supplements. The premature infants ranged in age from 1 to 30 days and in weight from 1,240 to 2,260 g. The remainder of the studies were performed in Seattle. Umbilical cord blood was obtained at birth, immediately after the cord was cut, from full-term infants of known normal patients in the obstetrical service of Maynard Hospital in Seattle. Materials. Materials used were obtained commercially.2 methemoglobin reductase; and ACD, acid-citrate-dextrose (National Institutes of Health, formula A). 2DPN was obtained from Pabst Laboratories, Milwaukee, Wis., or from Sigma Chemical Co., St. Louis, Mo.; TPN, TPNH, DPNH, glucose-6-phosphate, adenine sulfate, and protamine sulfate from Sigma Chemical Co.; crystalline bovine serum albumin from Armour and Co., Kankakee, Ill.; a-ketoglutarate, 6-PGD, G-6-PD, glutamic dehydrogenase, and rabbit muscle LDH from Boehringer and Sons, Mannheim, Germany; calcium lactate from Calbiochem, Los Angeles, Calif.; beef heart LDH from Worthington Biochemical Corp., Freehold, N. J.; EDTA from J. T. Baker Chemical Co., Phillips- 249
2 250 R. T. GROSS, E. A. SCHROEDER, AND B. W. GABRIO Procedures. Blood was collected by venipuncture in ACD solution or heparin (2 mg per 100 ml of blood) or was used directly from a free flowing deep capillary puncture obtained from the finger tip in adults and from the heel in premature infants. Four aliquots of 10 /Al each was sufficient for the measurement of the oxidized and reduced pyridine nucleotides. For the studies on erythrocyte preservation, blood was collected in ACD and separated immediately into three aliquots of 100 ml each. The aliquots were supplemented with 10 ml of 0.9% NaCl (control), adenine sulfate (0.9 to 1.0 jumole adenine per ml of blood), or inosine (10.0 to 10.2 Amoles per ml). The blood was stored at 40 C in Cutter plastic bags. DPN, DPNH, TPN, and TPNH were determined in both laboratories by the enzymatic cycling method of Lowry, Passonneau, Schulz, and Rock (18). The nucleotides were used at concentrations well below their Michaelis constants during cycling. The reaction rates were proportional to nucleotide concentrations over the range of 2 X 10' to 1 X 10' mole per L for TPN and TPNH and 5 X 10' to 5 X 10-' mole per L for DPN and DPNH. In the Seattle laboratory the final fluorometric measurements, made in a volume of 2.30 ml for DPN and DPNH and 2.50 ml for TPN and TPNH, were performed with the use of the G. K. Turner fluorometer with primary Corning 5860, secondary Turner 3, and Turner 48 filters. In the New York laboratory the final volumes employed were 1.22 ml for DPN and DPNH and 1.11 ml for TPN and TPNH, and the fluorometric measurements were performed with a Farrand spectrophotofluorometer with primary Corning 737 and secondary Corning 372 filters. For measurements of DPN and DPNH the wavelength of the exciting monochromator was 370 m/ and of the analyzing monochromator 475 m/a. For TPN and TPNH the wavelengths were 350 and 480 m, for the two monochromators. Purification of 6-PGD from rat liver was performed according to the procedure of Lowry and associates (18) with the following modifications. Nucleic acids were precipitated directly from the homogenate by the addition of 0.3 vol of 2% protamine sulfate. After the first dialysis, the dialyzed preparation was added to 1 vol of DEAEcellulose suspension, prepared as described previously (2), and mixed for 15 minutes. About 40% of the G-6-PD remains on the adsorbent. The unadsorbed fraction, collected by centrifugation at 1,900 g in the International refrigerated centrifuge model PR-2, was treated with ammonium sulfate according to the original procedure. After the second dialysis, treatment of the dialyzed preparation with DEAE-cellulose was substituted for the series of calcium phosphate gel treatments, and the 6-PGD in the unadsorbed fraction was purified further as described originally. The above modifications burg, N. J.; DEAE-cellulose from Eastman Kodak Co., Rochester, N. Y.; dextran from Pharmacia Laboratories, Inc., New Market, N. J.; inosine from Schwarz Bio- Research, Mt. Vernon, N. Y.; and sodium heparin (10 mg per ml) from Invernex Corp., San Francisco, Calif. served to expedite the preparation and insure more consistent results. Leukocytes from 90 ml of pooled cord blood were isolated by a technique described previously (19). Hemoglobin was determined as cyanmethemoglobin (20) and plasma hemoglobin as pyridine hemochromogen (21). Assays for the following enzymes were performed on hemolysates (1, 2) essentially according to the published methods: G-6-PD (22), 6-PGD (23), LDH (24), PGD (1), GR (25), and Hb'R (26). Results Reliability of method. The stability of DPN, DPNH, TPN, and TPNH in blood samples collected in ACD and maintained thereafter at 40 C was tested at 5, 15, and 50 minutes and 1, 12, and 24 hours after collection. A variable decline, no greater than 15 %o, in one or more of the nucleotides was encountered in blood samples obtained from adults after 1 hour of storage. In samples from premature infants, there was no difference from adults in the stability of TPN and TPNH. However, 1 hour after collection there were losses as great as 40%o in both DPN and DPNH in samples from premature infants. Once the aliquots of blood were diluted in the appropriate acid or alkaline solutions, the levels of all four nucleotides from all subjects remained stable at 40 C in the refrigerator, but lost variable amounts of activity at the lower temperature of the laboratory ice bucket. These latter observations on the stability of dilute solutions of endogenous pyridine nucleotides are comparable to the findings of Lowry, Passonneau, and Rock (27) that dilute standard solutions of pyridine nucleotides are more stable at 40 C than at C. A comparison of values obtained from analyses of whole blood and from isolated, washed red cells showed that the process of washing the cells resulted in variable losses, up to 15% of the nucleotides. The recovery of pyridine nucleotides added to diluted blood samples just before heating was 97% for DPN and TPN, 98%o for DPNH, and 96%o for TPNH. Duplicate determinations of a particular blood sample agreed to within 5%o. Mean levels of pyridine nucleotides in the erythrqcytes of healthy adults obtained and determined independently in Seattle and New York differed at the most by 12%o for TPN and as little as 2%o for TPNH (cf. Table I).
3 ERYTHROCYTE PYRIDINE NUCLEOTIDES 251 TABLE I Levels of pyridine nucleotides in erythrocytes from adults, premature infants, and cord blood Source of DPN + TPN + DPN TPN DPN + DPNH blood DPN DPNH TPN TPNH DPNH TPNH DPNH TPNH TPN + TPNH Adult subjects m~umoles/g hemoglobin New York 188 A 6.1* 142 ± ± ( )t (97-204) (89-161) (54-129) (14)4 (13) (30) (13) Seattle 205 ± :1: ± ± ( ) (99-217) (73-133) (55-104) (19) (19) (19) (19) Combined 198 ± ± ± ±t ( ) (97-217) (73-161) (54-129) (33) (32) (49) (32) Cord : ± ± ( ) ( ) ( ) (65-118) Premature 205 ± ± infants ( ) (10-S221) ( ) (51-103) (17) (17) (30) (22) * Mean value 4 standard error of mean. t Range. * No. of subjects. Pyridine nucleotide levels of erythrocytes from ratio concomitant with increasing maturity from adults, premature infants, and cord blood. In premature to cord to adult blood, the significance Table I is presented a summary of the erythrocyte 'of the widest difference, between premature infants pyridine nucleotide levels from the. three groups and adults, is questionable (0.1 > p > 0.05, deof subjects. The mean values do not differ ap- termined by Student's t test). In Table II the preciably among the groups. No deficiency could levels encountered in adults in the present study be demonstrated in the erythrocytes of either the are further compared with results reported by full-term newborn (cord blood) or premature in- others using a variety of methods. fants. Within each group the ratio of oxidized to Leukocyte contribution to pyridine nucleotide reduced nucleotide was the same for both DPN levels in blood. Although the values for pyridine and TPN. Although there was a decrease in this nucleotides in the erythrocyte are derived from TABLE II Pyridine nucleotides in blood*: comparison of levels obtained from adults in the present study with those reported in the literature Reference Author no. Species DPN DPNH TPN TPNH moles X10' moles X10-' Present study* Man Szeinberg and Pipano 28 Man Barnett 29 Man Schrier, Kellermeyer, and Alving 30 Man Bishop, Rankine, and Talbott 31 Man Mills and Summers 32 Man Bonsignore, Fornaini, Segni, and Fantoni 33 Man Shafer and Bartlett 34 Man Oski and Diamond 35 Man Lohr and Waller 36 Man Brown and Clarke 37 Man JaffU and Neumann 38 Man Lowry, Passonneau, Schulz, and Rock 18 Rat Glock and McLean 39 Rat Rabbit * Calculated on basis of 15 g hemoglobin per 100 ml blood.
4 252 R. T. GROSS, E. A. SCHROEDER, AND B. W. GABRIO TABLE III Contribution of leukocytes to levels of pyridine nucleotides in whole blood* Adult blood Cord blood Erythro- Leuko- Erythro- Leuko- Nucleotide cytes cytes cytes cytes mjsmoles/ml whole blood DPN 25.8t 0.21$ 31.2t 0.24 DPNH TPN TPNH * Erythrocyte values in adult and cord blood and leukocyte values in cord blood are derived from data obtained in the present study using the technique of enzymatic cycling. The leukocyte values in adult blood are calculated from the data of Silber, Gabrio, and Huennekens (19) obtained by a different method. t Calculated by mean nucleotide levels given in Table I and mean hemoglobin levels of 12.6 and 13.0 g hemoglobin per 100 ml acid-citratedextrose (ACD) blood for adult and cord blood, respectively. t Calculated assuming an average leukocyte count in ACD blood of 6,500 per mm'. Leukocyte count in cord blood sample = 13,300 per mm3. analyses of whole blood, the data given in Table III indicate that this is justified. The figures given for erythrocytes are those obtained on whole blood, whereas those for leukocytes represent analyses made on isolated leukocyte preparations. It is apparent that the leukocyte contribution is negligible, i.e., less than 1% of the determined values for DPN, DPNH, and TPN and about 1.5%o of the values for TPNH in both adult and cord blood. If the total pyridine nucleotide in the formed elements of the blood is calculated on a cellular basis (cell count), the values are 190 mumoles per 1010 erythrocytes and 890 m~umoles per 1010 leukocytes. In vitro storage of blood: pyridine nucleotides. Three experiments were performed in which blood was collected in ACD, supplemented with adenine or inosine, and analyzed at weekly intervals for 48 days during storage at 40 C. The results were essentially the same in all three experiments. In Table IV are shown data from a representative experiment in which the additions at the beginning of storage were 0.9,umole of adenine or 10.0 jumoles of inosine per ml of ACD blood. Although there were no remarkable changes in pyridine nucleotide levels throughout the metabolic stress of storage, there was a small net loss of diphosphopyridine nucleotide in the control sample. The ratio of the oxidized to reduced forms decreased in all blood samples as a consequence of the slight fall in the levels of DPN and TPN and a compensatory slight increase in DPNH and TPNH. The significance of these small changes is not apparent. In vitro storage of blood: pyridine nucleotiderequiring enzymes. Concomitant with the relative stability of the pyridine nucleotides during storage of blood at 40 C are the essentially unchanged activities of several pyridine nucleotidedependent enzymes in the erythrocyte (cf. Table IV). Discussion The method of enzymatic cycling (18) has provided a precise and highly reproducible means of measuring each of the four pyridine nucleotide forms in micro quantities of blood. The concordance of data obtained from independent samples determined in two different laboratories is testimony to this fact. Comparative data obtained from isolated preparations of leukocytes indicate that the quantities measured in whole blood may be equated with erythrocyte pyridine nucleotide levels. The concentrations of both DPN and TPN in adult blood shown in Table II are similar to those TABLE IV Pyridine nucleotides and pyridine nucleotide-requiring enzymes in the erythrocyte during in vitro storage at 40 C* Pyridine nucleotides Enzymes Plasma hemo- Days stored DPN DPNH TPN TPNH PGD LDH G-6-PD 6-PGD GR Hb'R globin m.smoles/g hemoglobin.moles substrate converted/hour/g hemoglobin mg hemoglobin! 100 ml ACD plasma , Control , Adenine , Inosine , * PGD = 3-phosphoglyceraldehyde dehydrogenase, LDH lactic dehydrogenase, G-6-PD = glucose-6-phosphate dehydrogenase, 6-PGD = 6-phosphogluconic dehydrogenase, GR = glutathione reductase, and Hb'R = TPNH-methemoglobin reductase.
5 ERYTHROCYTE PYRIDINE NUCLEOTIDES 253 reported by a number of investigators using different analytical techniques. Few comparative data are available for reduced pyridine nucleotides. In the present study, DPNH content is about five times greater and the TPNH level is only 80%o of the respective values reported by Lbhr and Waller (36), who employed a different method for analysis. With the use of a modification of the present method of enzymatic cycling for the TPNH assay, Brown and Clarke (37) have reported a mean concentration of TPNH that is 2j times greater than the present value. However, their data were obtained on 23 clinic patients without an attempt to correlate clinical diagnoses, and the authors state that the wide range of values might indicate the presence of abnormal specimens. This fact, coupled with the methodological difference whereby the depletion of glucose-6-phosphate in the enzymatic cycling mixture was measured, may contribute to the variance of data as compared with the present study. There is almost twice as much total DPN as TPN in the human erythrocyte. This ratio is similar to the distribution in a number of other tissues reported by Lowry and his associates (18) and by Burch, Lowry, and Von Dippe (40). In the steady state condition the quantity of the oxidized forms of both DPN and TPN exceeds the reduced forms. Furthermore, the quantity of oxidized TPN appears adequate for the dehydrogenase reactions of the hexose monophosphate shunt pathway. The TPN level of 10-5 mole per L is an order of magnitude higher than the Km for TPN in the G-6-PD reaction in human erythrocytes (41, 42). Thus additional investigation appears to be indicated to reveal why glycolysis predominates in the erythrocyte. The present studies provide no evidence for pyridine nucleotide deficiency in the erythrocytes of full-term newborn or premature infants. This observation is of interest in view of abnormalities characteristic of the erythrocytes of young infants. Limited availability of TPN in particular has been considered a possible explanation for the increased susceptibility to Heinz body formation and drug-induced hemolysis in newborn infants (43). It should be emphasized, therefore, that measurements in this study have been obtained in the absence of oxidative stress to the erythrocytes. Since the rate of erythrocyte glycolysis is greatly increased in newborn infants, particularly in the presence of an oxidizing agent (44, 45), the occurrence under stress of a relative deficiency of one or more of the pyridine nucleotides has not been ruled out. The further possibility that oxidants may have a direct effect upon erythrocyte pyridine nucleotide levels is considered in detail elsewhere (46). Stability studies demonstrating losses of DPN and DPNH as great as 40% when whole blood from premature infants is maintained at 40 C for 1 hour suggest that the erythrocytes of these subjects have increased activity of the enzyme that hydrolyzes the DPN. Although this hydrolytic activity ceases upon dilution of the blood in acid or base, it is possible that unappreciated losses may be sustained during the 1 to 2 minutes often involved in collecting blood from premature infants. Thus the in vivo levels of the DPN in the erythrocytes of premature infants may be somewhat higher than those reported here. The finding of slightly higher levels of DPN and DPNH in cord blood from full-term infants and the stability of these compounds in cord blood at 40 C indicates that the presumed increased activity of DPNase in the premature is no longer evident in the erythrocytes of full-term newborn infants. During the in vitro preservation of blood at 40 C in ACD, numerous physical and chemical changes occur in the erythrocyte. Supplementation of the blood with adenine (47), nucleosides (inosine or adenosine) (48-50), or both (51) allays many of the deteriorative changes. The present evidence indicates, however, that pyridine nucleotides and pyridine nucleotide-dependent enzymes are maintained at approximately their steady state levels throughout storage with or without additives. These findings are of interest in light of the evidence of Jaffe and Neumann that stored erythrocytes have a diminished capacity to synthesize DPN from nicotinic acid as well as incorporate nicotinic acid or nicotinamide into DPN and TPN in comparison with fresh cells (38). Further, Jaffe and Neumann found that although inosine is inhibitory to these processes in fresh blood, the nucleoside could effect, in part, a restoration of the ability of stored cells to carry out these reactions (38). Since the pyridine nucleotide levels in the present study remained stable throughout storage despite the decline in the cell's
6 254 R. T. GROSS, E. A. SCHROEDER, AND B. W. GABRIO pertinent synthetic capacity, it might be concluded that the enzymes that hydrolyze pyridine nucleotides in erythrocytes of adults are inactive during storage at 40 C. Quite different results in the levels of these nucleotides and enzymes can be obtained if the temperature of storage is increased to 200 C. LUhr and Waller (4) found that DPN and TPN declined to about 20% of their original levels and PGD, LDH, G-6-PD, and Hb8R to about 10 to 40%o of their original activities after 7 days of storage at 20 C. The latter data serve to emphasize that experiments performed on blood stored at elevated temperatures are not comparable to those carried out at the standard 40 C. Summary The concentrations of oxidized and reduced diphosphopyridine nucleotide and triphosphopyridine nucleotide have been determined and are essentially the same in erythrocytes of adults, premature infants, and cord blood. There is almost twice as much total DPN as total TPN, and in the steady state both nucleotides exist predominantly in the oxidized form. Comparative data from adult and cord leukocytes indicate that measurements of whole blood are valid indexes of the pyridine nucleotide content of erythrocytes. During the in vitro preservation of blood at 40 C in acid-citrate-dextrose solution or with added adenine or inosine, the levels of oxidized and reduced pyridine nucleotide remain fairly constant as do the activities of pyridine nucleotide-requiring enzymes, phosphoglyceraldehyde dehydrogenase, lactic dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, glutathione reductase, and TPNH-methemoglobin reductase. Acknowledgments The authors are indebted to Mrs. Aline Alenty and Mrs. Ann Waltersdorph for expert technical assistance. References 1. Chapman, R. G., M. A. Hennessey, A. M. Waltersdorph, F. M. Huennekens, and B. W. Gabrio. Erythrocyte metabolism. V. Levels of glycolytic enzymes and regulation of glycolysis. J. clin. Invest. 1962, 41, Hennessey, M. A., A. M. Waltersdorph, F. M. Huennekens, and B. W. Gabrio. Erythrocyte metabolism. VI. Separation of erythrocyte enzymes from hemoglobin. J. clin. Invest. 1962, 41, Bartlett, G. R. Human red cell glycolytic intermediates. J. biol. Chem. 1959, 234, L6hr, G. W., and H. D. Waller. Zellstoffwechsel und Zellalterung. Klin. Wschr. 1959, 37, Murphy, J. R. Erythrocyte metabolism. II. Glucose metabolism and pathways. J. Lab. clin. Med. 1960, 55, London, I. M. The metabolism of the erythrocyte. Harvey Lect. 1961, 56, Szeinberg, A., and P. A. Marks. Substances stimulating glucose catabolism by the oxidative reactions of the pentose phosphate pathway in human erythrocytes. J. clin. Invest. 1961, 40, Fornaini, G., G. Leoncini, L. Luzzatto, and G. Segni. Glucose metabolism in human erythrocytes from normal and fava bean-sensitive subjects. J. clin. Invest. 1962, 41, Vest, M. F., and H-R. Grieder. Erythrocyte survival in the newborn infant, as measured by chromium' and its relation to the postnatal serum bilirubin level. J. Pediat. 1961, 59, Kaplan, E., and K. S. Hsu. Determination of erythrocyte survival in newborn infants as measured by Cr'-labeled erythrocytes. Pediatrics 1961, 27, Brown, A. K. Peculiarities of erythrocytes of the newborn in Kernicterus, A. Sass-Kortsak, Ed. Toronto, University of Toronto Press, 1961, p Cornblath, M., and A. F. Hartmann. Methemoglobinemia in young infants. J. Pediat. 1948, 33, Kravitz, H., L. D. Elegant, E. Kaiser, and B. M. Kagan. Methemoglobin values in premature and mature infants and children. Amer. J. Dis. Child. 1956, 91, McDonald, C. D., Jr., and T. H. J. Huisman. A comparative study of enzymic activities in normal adult and cord blood erythrocytes as related to the reduction of methemoglobin. Clin. chim. Acta 1962, 7, Ross, J. D. Deficient activity of DPNH-dependent methemoglobin diaphorase in cord blood erythrocytes. Blood 1963, 21, Gross, R. T., and R. E. Hurwitz. The pentose phosphate pathway in human erythrocytes. Relationship between the age of the subject and enzyme activity. Pediatrics 1958, 22, Gross, R. T., E. A. R. Schroeder, and S. A. Brounstein. Energy metabolism in the erythrocytes of premature infants compared to full term newborn infants and adults. Blood 1963, 21, Lowry, 0. H., J. V. Passonneau, D. W. Schulz, and M. R. Rock. The measurement of pyridine nucleotides by enzymatic cycling. J. biol. Chem. 1961, 236, Silber, R., B. W. Gabrio, and F. M. Huennekens. Studies on normal and leukemic leukocytes. III. Pyridine nucleotides. J. clin. Invest. 1962, 41, 230.
7 ERYTHROCYTE PYRIDINE NUCLEOTIDES Crosby, W. H., J. L. Munn, and F. W. Furth. Standardizing a method for clinical hemoglobinometry. U. S. armed Forces med. J. 1954, 5, Flink, E. B., and C. J. Watson. A method for the quantitative determination of hemoglobin and related heme pigments in feces, urine, and blood plasma. J. biol. Chem. 1942, 146, Kornberg, A., and B. L. Horecker. Glucose-6-phosphate dehydrogenase in Methods in Enzymology, S. P. Colowick and N. 0. Kaplan, Eds. New York, Academic Press, 1955, vol. 1, p Horecker, B. L., and P. Z. Smyrniotis. Phosphogluconic acid dehydrogenase from yeast. J. biol. Chem. 1951, 193, Kornberg, A. Lactic dehydrogenase of muscle in Methods in Enzymology, S. P. Colowick and N. 0. Kaplan, Eds. New York, Academic Press, 1955, vol. 1, p Racker, E. Glutathione reductase (liver and yeast) in Methods in Enzymology, S. P. Colowick and N. 0. Kaplan, Eds. New York, Academic Press, 1955, vol. 2, p Huennekens, F. M., R. W. Caffrey, R. E. Basford, and B. W. Gabrio. Erythrocyte metabolism. IV. Isolation and properties of methemoglobin reductase. J. biol. Chem. 1957, 227, Lowry, 0. H., J. V. Passonneau, and M. K. Rock. The stability of pyridine nucleotides. J. biol. Chem. 1961, 236, Szeinberg, A., and S. Pipano. Oxidized pyridine nucleotides in normal and glucose-6-phosphate dehydrogenase deficient red cells. Proc. Tel Hashomer Hosp. 1964, 3, Barnett, H. N. Erythrocyte pyridine nucleotides. Fed. Proc. 1957, 16, Schrier, S. L., R. W. Kellermeyer, and A. S. Alving. Coenzyme studies in primaquine-sensitive erythrocytes. Proc. Soc. exp. Biol. (N. Y.) 1958, 99, Bishop, C., D. M. Rankine, and J. H. Talbott. The nucleotides in normal human blood. J. biol. Chem. 1959, 234, Mills, G. C., and L. B. Summers. The metabolism of nucleotides and other phosphate esters in erythrocytes during in vitro incubation at 37. Arch. Biochem. 1959, 84, Bonsignore, A., G. Fornaini, G. Segni, and A. Fantoni. Biosynthesis of pyridine coenzymes in erythrocytes of subjects with a case history of favism. Ital. J. Biochem. 1961, 10, Shafer, A. W., and G. R. Bartlett. Phosphorylated carbohydrate intermediates of the human erythrocyte during storage in acid citrate dextrose. III. Effect of incubation at 370 C with inosine, inosine plus adenine, and adenosine after storage for 6, 10, 14, and 18 weeks. J. clin. Invest. 1962, 41, Oski, F. A., and L. K. Diamond. Erythrocyte pyruvate kinase deficiency resulting in congenital nonspherocytic hemolytic anemia. New Engl. J. Med. 1963, 269, L6hr, G. W., and H. D. Waller. The enzyme-deficiency haemolytic anaemias. Germ. med. Mth. 1961, 6, Brown, E., and D. L. Clarke. Simplified method for the determination of reduced triphosphopyridine nucleotide by means of enzymatic cycling. J. Lab. dlin. Med. 1963, 61, Jaffe, E., and G. Neumann. The synthesis of pyridine nucleotides in fresh and stored human erythrocytes. Transfusion (Philad.) 1965, 5, Glock, G. E., and P. McLean. Levels of oxidized and reduced diphosphopyridine nucleotide and triphosphopyridine nucleotide in animal tissues. Biochem. J. 1955, 61, Burch, H. B., 0. H. Lowry, and P. Von Dippe. The stability of the triphosphopyridine nucleotide and its reduced form in rat liver. J. biol. Chem. 1963, 238, Marks, P. A., A. Szeinberg, and J. Banks. Erythrocyte glucose-6-phosphate dehydrogenase of normal and mutant human subjects. Properties of the purified enzymes. J. biol. Chem. 1961, 236, Kirkman, H. N. Glucose 6-phosphate dehydrogenase from human erythrocytes. I. Further purification and characterization. J. biol. Chem. 1962, 237, j Gross, R. T., and E. A. R. Schroeder. The relationship of triphosphopyridine nucleotide content to abnormalities in the erythrocytes of premature infants (abstract). J. Pediat. 1963, 63, Gross, R. T. Unpublished data. 45. Zinkman, W. H. An in-vitro abnormality of glutathione metabolism in erythrocytes from normal newborns: mechanism and clinical significance. Pediatrics 1959, 23, Gabrio, B. W., E. A. Schroeder, and R. T. Gross. The response of erythrocyte pyridine nucleotides to ascorbic acid and methylene blue. In preparation. 47. Simon, E. R., R. G. Chapman, and C. A. Finch. Adenine in red cell preservation. J. clin. Invest. 1962, 41, Donohue, D. M., C. A. Finch, and B. W. Gabrio. Erythrocyte preservation. VI. The storage of blood with purine nucleosides. J. clin. Invest. 1956, 35, Mollison, P. L., and M. A. Robinson. Observations on the effects of purine nucleosides on red-cell preservation. Brit. J. Haemat. 1959, 5, Bartlett, G. R., and A. W. Shafer. Phosphorylated carbohydrate intermediates of the human erythrocyte during storage in acid citrate dextrose. II. Effect of the addition of inosine late in storage. J. clin. Invest. 1961, 40, Wada, T., F. Takaku, K. Nakao, M. Nakao, T. Nakao, and H. Yoshikawa. Post-transfusion survival of the red blood cells stored in a medium containing adenine and inosine. Proc. Jap. Acad. 1960, 36, 618.
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|Mark Thomas <ma...@apache.org>
|Re: [pool] [POOL-208] Support Java 1.5 Generics in version 1.x.
|Fri, 23 Dec 2011 13:29:23 GMT
On 23/12/2011 11:33, Christian Grobmeier wrote: > So far i have see the reason: Tomcat 5.5 runs with jdk1.4.2. Is this > actually the reason we don't go forward? Because another project needs > the 1.5 series? > If this is the case, then the EOL of Tomcat 5.5 is the earliest EOL of Pool 1.5. > > This does mean pool can earliest go forward in September 2012: > http://tomcat.apache.org/tomcat-55-eol.html Pool is already going forward in the 1.6.x branch and in 2.0.x > On 1.6, question is if this series will be so time consuming as > expected. After all, 1.5 dev is at a minimum as understood it. It > should be easy for Gary to move the changes to 1.6 version, if he is > willing to care on that (nasty tongues tell me Git would help in this > case). It is certainly more work but how much more is the critical question. As long as the code bases stay broadly similar it should be easy to patch 1.6.x to fix a bug and svn merge the change to 1.5.x. We do this in Tomcat all the time and merging from trunk (8.0.x) to 7.0.x and 6.0.x is generally fairly simple. Merging to 5.5.x requires more work as the source layout is completely different. Releases would be more work but there are folks that look to be willing to release 1.5.x and 1.6.x The other bit I can think of is that the website may need some tweaks to handle multiple versions. I'm not familiar with the Commons web site generation process to judge if that is easy or hard. > If 1.6 is coming and this project does not support it, how can we > handle it? After all we are not working for the Tomcat project, we are > working for ourselves, and Gary needs a generic version. I would not > like to see him going and make up a github fork. People outside of > this project are already saying we are like dinosaurs (collections vs > guava). We should probably discuss how we can do this - probably Gary > could make some kind of "half official" release, a bit of an > "experimental" tagged one. One people do not rely upon, except they > know what they are doing. Probably a 1.5-extended version, like in > Lord of the Rings. All that is required for a release is someone willing to tag and release and three PMC members to vote +1. As long as 1.5.x and 1.6.x remain broadly similar (i.e. 1.6.x = 1.5.x + generics) then the additional work of porting fixes between the two should be minimal. We want to avoid "unofficial" releases. That raises all sorts of red flags at ASF board level. Mark --------------------------------------------------------------------- To unsubscribe, e-mail: firstname.lastname@example.com For additional commands, e-mail: email@example.com
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March 21, 2012
Last Thursday on Day One of Funkshion, students at the University Of Miami got a chance to show the best that they had on the runway. The event was held at The Moore Building 4040 N.E 2nd Avenue Miami FL. Click below to see all the photos, shot courtesy of S. Louis Photography.
Follow S. Louis Photography at www.facebook.com/S.LOUISPHOTOGRAPHY
CategoriesMiami Fashion


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Entire home/apt
Payallar Belediyesi, Antalya, Turkey
Alanya, Antalya, Turkey
Beautiful, bright and wonderful apartment. The house has everything you need for a comfortable stay. The apartments are in an excellent complex with friendly neighbors, mostly Scandinavians. Conveniently located within walking distance from the center of Konakli, but also to Alanya by car can be reached in just 7 minutes. Nearby there are all the necessary infrastructure, particularly recommend coming in the summer, an ideal place for family and youth recreation. Mantas and his mother, who greeted me on arrival, very nice and helpful people, always ready to help.
We had a very nice vacation in This apartment. A very nice and clean place, the pool and sourrandings is allso very nice. The houst Mantas is allways quick to respond on any quastion
Konaklı, Antalya Province, Turkey
Konaklı Belediyesi, Antalya, Turkey
The host canceled this reservation 2 days before arrival. This is an automated posting.
Private room
Конаклы, Анталья, Turkey


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Pirate suspects caught in Spanish legal no-man's land
Seven suspected Somali pirates detained earlier this week by a Spanish navy ship were stuck in legal no-man's land Sunday after Madrid rejected a Spanish judge's order to release them.MADRID - Seven suspected Somali pirates detained earlier this week by a Spanish navy ship were stuck in legal no-man's land Sunday after Madrid rejected a Spanish judge's order to release them.
Spain's Defence Minister Carme Chacon announced Sunday that the Madrid government would "appeal," while Foreign Minister Miguel Angel Moratinos said it was in contact with Kenyan authorities to hand the seven over to Nairobi.
National Court judge Fernando Andreu, who opened a preliminary investigation on Thursday, argued that sending the suspects to Kenya to be prosecuted would violate their rights since an inquiry was already underway in Spain.
Andreu had ordered the defence ministry Thursday to remand the seven suspects in custody and bring them to Spain so they could be questioned under a new piracy law adopted last year after a Spanish trawler and its crew were held hostage for six days by Somali pirates.
But public prosecutors said Friday the suspects should be taken to Kenya which signed an agreement with the European Union in March to take suspected pirates detained by EU navies patrolling Somalia's waters and prosecute them in Kenyan courts.
The group were captured in international waters in the Indian Ocean on Wednesday after their boat capsized when they were allegedly trying to board a Panamanian-flagged vessel.
The seven are currently stuck aboard the Marques de la Ensenada supply vessel, which also captured another seven suspects on Thursday as they appeared to be attempting to board a Maltese-flagged merchant ship.
AFP / Expatica


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Valine helps prevent the breakdown of muscle by supplying the muscles with extra glucose for energy production during intense physical activity. Valine also helps remove potentially toxic excess nitrogen from the liver, and is able to transport nitrogen to other tissues in the body as needed.
Valine is an essential amino acid, which means that it cannot be manufactured in the body and must be obtained through dietary sources. Natural sources of valine include meats, dairy products, mushrooms, peanuts, and soy protein. Although most people get enough valine from their diet, there have been recorded cases of valine deficiency.
People that exercise a lot, have a low-protein diet, or are seriously trying to build muscle mass should consider valine supplementation. Valine should always be taken together with the other two branched-chain amino acids, isoleucine and valine The ideal balance is 2 milligrams of leucine and valine for each 1 milligram of isoleucine.
An excessively high intake of valine may cause a skin crawling sensation and even hallucinations. Too much valine in the diet can also disrupt liver and kidney function and increase the amount of ammonia in the body. People with impaired liver or kidney function should not take isoleucine without first consulting a physician, as large doses of amino acids may aggravate these conditions.
Additional Information
|Supplement Direct
|Serving Size:
|1/4 tsp (1g)
|Per Container:
|Ingredients (Amount Per Serving)
|1g (1000 mg) Valine
|As a dietary supplement, take 1/4 level teaspoon one or more times daily as needed, preferably on an empty stomach or use as directed by a health care professionals.
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Premier, Saskatchewan Party Candidate Kick Off By-election Campaign
By-election Presents Voters With Clear Choice on Carbon Tax Issue Premier Brad Wall today said Saskatchewan Party candidate Brent Penner and his team will run a strong campaign in...
Brent Penner Nominated as Candidate for Saskatoon Meewasin By-election
Saskatoon— Brent Penner was nominated last night to become the Saskatchewan Party candidate in Saskatoon Meewasin for the upcoming by-election.
New Poll Shows Strong Opposition In Saskatchewan to Federal Carbon Tax
A new province-wide Insightrix poll conducted last week shows a strong majority of Saskatchewan people oppose Justin Trudeau's plan to impose a federal carbon tax in Saskatchewan.
Remembering Roger Parent 1953-2016
The Saskatchewan Party is mourning the loss of our good friend and colleague Roger Parent, MLA for Saskatoon Meewasin. Parent passed away last night at Royal University Hospital in Saskatoon...
NDP Convention to Debate Israel Boycott, Adopting LEAP Manifesto, Shutting Down Coal and Drug Decriminalization
NDP Convention to Debate Israel Boycott, Adopting LEAP Manifesto, Shutting Down Coal and Drug Decriminalization
The NDP's provincial convention is this weekend. Once again, they will be debating policies that should how increasingly out of touch they are with Saskatchewan people. These include boycotting and...
NDP Spend Over 72% of Election Budget on Out-of-Province Consultants
Once again, the NDP is caught saying one thing but doing another.
70 Per Cent in Saskatchewan Support Wall's Position on Carbon Tax
A new Insightrix poll commissioned by the Saskatchewan Party shows that seven out of 10 Saskatchewan people support Brad Wall's position over Justin Trudeau's position on a national carbon tax....
Don't Miss Our Fall Premier's Events
We will be having two new Premier's events this fall.
Premier's Golf Ball 2016
Last Sunday we hosted the 6th annual Premier's Golf Ball Charity Event. Proceeds this year from the event went to SOFIA House here in Regina. SOFIA House is a second...
2016-2017 Budget
Photo:CTV Saskatoon Last week we released the 2016-2017 budget. Throughout the last election our party campaigned on a responsible, affordable plan to keep Saskatchewan strong. The 2016-17 Saskatchewan budget...


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When Saviour showed up minster seemed to hap true here. It did not entity if He was at the local activity hollow and a female next to a bad laurels amalgamated Him; church happened. Astonishingly clerical happened minus a program, short a building, without a 3-point sermon, and without a respect team musical performance the up-to-the-minute CD thatability is the 'next fastest item to truly individual in heaven' (Remember the Memorex commercial?-Is it region or is it Hillsong?).
Like it or not we are the church: you and me. As guileless as thatability sounds, as odd as it may seem, and as unlovely as it looks we are named to be the minster. The 'church' is not a building, a program, or a day but a people: you and me. Once we establish up christian church should surface and no amount of scheduling and selling and mystic stalk and jivingability is going to occurrence thatability.
Is location ever a time in recorded ancient times where on earth Saviour welcome human to clerical to acquire the truth, show for sightless eyes, or for any healing? I don't deliberate so (feel release to let me cognize otherwise). He was the original animal house of worship of world thatability all of us are designed to be present but we strain so rough to calendar and system of rules what a living, vital, close relation with Powerful God looks and feels similar to.Post ads:
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(Sometimes it all retributory feels approaching compromise to me; resembling I have listed in an mindboggling spirited human relationship purchasable 24/7/365 for a scheduled time period engagement next to Being I fondness).
And material possession me once I say it does not facial expression suchlike present house of worship on Sundays! Self-conceited doubling is unloved by God because it breeds triteness. God is unadulterated and viable and desires for us to cognise him relationallyability in the mundane efficiency of energy.
I reflect it was thatability sick Faith contributor Donald Alton Glenn Miller who said (and I abhor to damage what he aforementioned so infernal me if I get thisability false) thatability if we have a bond near God than it is active to be uncomfortable and muddled and always dynamic.Post ads:
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And, I'd suchlike to add, fitting because we have a connotation thatability we know Him now does not be set to thatability after one-minuteability in His existence or one-minuteability in debate near Him won't sort my herald spin, my bosom go pitter spiel (Or is thatability Santa's feet on the protection top?), and breed me surface close to I don't even know thisability astonishing Begetter thatability is so impressive thatability He can solitary dispense me least small bits and glimpses of Who He is.
God is grand. He cannot be restrained in tens of thousands of Rest day morning services or even in jillions of Christiansability. His musing is seen in every keen accomplishment thatability was through with today and His paw is seen as the sun rise, the moon shines, and the twist blows.
God is so massive thatability He is even seen in the irreligious (another history nudeness).
God is massive so for The Nazarene interest get Him out of the basilica.


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Kaymu, an online marketplace that allows buyers and sellers to come together and get the best deals on a variety of products, has great deals available for the upcoming New Year. Products on Kaymu range from electronics and fashion, to home appliances and an array of other items, and the New Year collection includes deals on these and many more. We have exclusive deals on men and women's clothing and accessories, must-have gadgets, and a wide range of cosmetics and beauty products. What's more is that this collection comprises of the best deals from the top sellers on Kaymu, most of whom offer free delivery! You can simply click on 'New Year 2015' in the 'Categories' tab on the website: kaymu.com.np to see the amazing products featured in the collection. Kaymu also has an updated Android App for a smarter, swifter and smoother shopping experience! So be sure download from the Google Play Store, and check out the Kaymu website to make the most of the deals available.
Kaymu is the largest and safest online marketplace for emerging markets. It connects buyers and sellers from all over and let them take advantage of the best deals on an extensive range of products, including electronics, books, fashion items, gadgets and cosmetics. Thanks to a fixed price system, Kaymu guarantees that users will always find the lowest prices on the market, and that the sellers are serious about online retail. Kaymu is part of the Asia Pacific Internet Group (APACIG).
The Asia Pacific Internet Group (APACIG), a joint venture of Rocket Internet and Ooredoo, is the leading internet platform in the Middle East, supporting some of the top internet companies in the region. Founded by Rocket Internet in 2014, the APACIG's mission is to promote innovation and entrepreneurship throughout Asia Pacific, as well as to support the development of a vibrant online culture. The APACIG's network currently consists of 10 companies, operating in 15 countries.


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GEI Headquarters First to Carry LEED and GREENGUARD Certifications
GREENGUARD Environmental Institute (GEI) is relocating its Atlanta headquarters to new offices that will be a model of sustainable construction and indoor air quality management. Scheduled for completion in December 2007, the project is registered with USGBC's LEED
for Commercial Interiors rating system. GEI will also certify the space under its GREENGUARD for Building Construction ProgramSM, which establishes best practices for reducing moisture and mold risk. When completed, the office will be the first commercial space to carry both certifications. For more information on GEI programs and indoor air quality, visit


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We live in a wonderful world that is full of beauty, charm and adventure. There is no end to the experience we can have if only we seek them with our eyes open
After my Bachelor in Business studies i started working in Tourism. I am almost 2 years with Royal Mountain Travel now in my 5 year Travel experience working as a Sales & Operations Officer.
Favorite Places in Nepal are Pokhara, Nagarkot


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Toke Fearnim is a stableowner in Jurador.
Master Fearnim is a wiry man who has a fringe of gray hair.
When Matrim Cauthon is traveling with Valan Luca's Traveling Show across Altara, Mat goes into Jurador to buy a horse for Tuon Athaem Kore Paendrag. Master Fearnim has his stablehands, Adela and Hurd, show Mat several horses. After much hard barganing, Mat buys a razor from Master Fearnim.


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Hibiscus On The Beach
Select your stay dates to check availability at Hibiscus On The Beach
Hibiscus On The Beach
About the Hotel
Property Location
With a stay at Hibiscus On The Beach in Main Beach, you'll be on the waterfront and convenient to Mariner's Cove Marina and Sea World. This beach apartment is within close proximity of Marina Mirage Shopping Centre and King Tutts Putt Putt.
Make yourself at home in one of the 15 guestrooms, featuring kitchens with refrigerators and stovetops. Wireless Internet access (surcharge) keeps you connected, and cable programming is available for your entertainment. Conveniences include microwaves and coffee/tea makers, and you can also request cribs/infant beds (surcharge).
Enjoy recreational amenities such as an outdoor pool and a spa tub. Additional amenities at this apartment include wireless Internet access (surcharge), tour/ticket assistance, and barbecue grills.
Business, Other Amenities
Featured amenities include dry cleaning/laundry services, luggage storage, and laundry facilities. Free self parking is available onsite.
Hotel Facilities
- Luggage storage
- Spa tub
- Garden
- Total number of rooms - 15
- Laundry facilities
- Elevator/lift
- Front desk (limited hours)
- Tours/ticket assistance
- Year Built 1980
- Barbecue grill(s)
- Outdoor pool
- WiFi (surcharge)
- Parking onsite
- Free self parking
- Dry cleaning/laundry service
Room Facilities
- Stovetop
- Cribs/infant beds (surcharge)
- Cable TV service
- Hair dryer
- Coffee/tea maker
- Bathtub or shower
- Washer/dryer
- Cookware, dishware, and utensils
- Separate bedroom
- Rollaway/extra beds (surcharge)
- Iron/ironing board
- Private bathroom (not in room)
- WiFi (surcharge)
- Separate living room
- Microwave
- Refrigerator
- Television
- Kitchen
Hotel Policy
The following fees and deposits are charged by the property at time of service, check-in, or check-out.
- Crib (infant bed) fee: AUD 45 per stay
- Rollaway bed fee: AUD 45 per stay
The above list may not be comprehensive. Fees and deposits may not include tax and are subject to change.Show all hotel policy Show less hotel policy


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Boat That Capsized Was One Crew Member ShortOct 4, 2005 | AP
The owner of the boat that capsized on Lake George, killing 20 elderly tourists, could face a fine as low as $25 for failing to have enough crew members on board, police said Tuesday.
As divers searched for evidence and the victims' belongings, State Police Maj. Gerald Meyer told reporters that a state inspector determined in May the 38-foot Ethan Allen needed one crew member besides the captain because it carried up to 48 passengers.
The captain, 74-year-old Richard Paris, was the only crew member aboard when the boat, with 47 passengers, overturned Sunday afternoon during what was supposed to be a relaxing, one-hour fall foliage tour for a group of senior citizens. He was among the 28 survivors.
The state late Monday suspended the operating certificates for all five tour boats run by Shoreline Cruises after the determination that one crew member was aboard, said Wendy Gibson, spokeswoman for the state Office of Parks, Recreation and Historic Preservation.
Meyer said the fine for violating state navigation regulations is between $25 and $100.
The Ethan Allen was hauled out of the lake late Monday night and taken to a county airport, where National Transportation Safety Board investigators were to examine the vessel to determine why it capsized.
"The inspector made the determination that there was in fact an additional crew required for that vessel and did list one crew in addition to the pilot on that permit," Meyer said.
The Ethan Allen has a maximum capacity of 50 people - 48 passengers and two crew, Gibson said earlier. Commercial boats in New York that carry between 21 and 48 passengers must have two crew members.
Police said a wave from a passing boat and a sudden shift of passengers' weight on the boat's long benches may have factored into the capsizing. An investigation is ongoing.
"The bottom line is, any one of these little factors could not have upset the boat," said Warren County Sheriff Larry Cleveland. "If four or five of these came together, it's possible."
State officials originally suspended the certificates for two small boats similar to the Ethan Allen, but Gibson said they had expanded the suspension to include the Adirondac and the Horicon, larger cruise ships carry 400 and 200 guests, respectively.
Shoreline Cruises did not return messages left at its office Sunday and Monday.
Cleveland cautioned not to draw conclusions from the suspended certificates. "I do not believe there is any criminal culpability on any of the parties we have spoken with," he said.
The captain of the glass-enclosed boat told authorities it was hit by waves from at least one other vessel and turned over as he tried to steer out of them, authorities said.
The boat flipped so fast that no passenger could put on a life jacket. Forty-six passengers were from Michigan, with one from Ohio. Eight people were hospitalized with shortness of breath, broken bones and other injuries.
State Police Superintendent Wayne Bennett said passengers either slid or were thrown to one side of the boat after it began lurching. "And that, of course, would automatically mean an even bigger shift of weight," Bennett said.
The Ethan Allen was rated for 50 passengers based on the Coast Guard standard of 150 pounds per person. Cleveland said that standard may have to be revised in light of data showing Americans growing fatter.
On Monday afternoon, crews using inflatable bags raised the sunken boat 70 feet to the surface. Orange life vests could be seen floating inside. The search continued for other items.
"We know there are articles on the bottom," Cleveland said. "We're trying to get all those things put back together so we can get them returned."
Only Colorado, Indiana and New Hampshire require adults to wear life preservers when a boat is motion, said Melissa Savage of the National Conference of State Legislatures. No state has special laws governing boaters who are elderly or infirm.
The Lake George Park Association and the sheriff's department are responsible for enforcing safety on the lake, and they may reconsider the rules governing crew size and life jacket use, particularly when elderly or infirm passengers are involved, said James Hood, a spokesman for the association.
"It seems like a logical question or at least something to review," Hood said.
The captain was not tested for drug or alcohol after the accident. The sheriff said he had no legal grounds for administering such a test. The sheriff also said Paris had a state license, rather than a Coast Guard one, which would have required a test for drugs or alcohol.
The boat was last inspected in May and no problems were found, officials said.


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This is BOLDY.
A perfect sphere on which your phone or tablet sticks. Easy to attach.
Convenient don't you think? For in your home, car, caravan, boat and… where else?
You can attach the BOLDY-mainholder on any suitable surface with a 3M VHB self adhesive backing. For those who prefer to drill holes – the BOLDY-mainholder can also be secured with two stainless steel screws. Your phone or tablet equipped with the BOLDY-Connector or BOLDY case, will automatically connect once you move it towards BOLDY, et voilà… it will stay in the desired position.


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Toll Free: (888) 223-2545
Customer ServicePress 3
Hardware SpecialistPress 2
Hours:9:00 am - 8:30 pmM-F Eastern Time
The Bamboo chandelier offers a fresh blend of outdoor influences and richly detailed elements. Each piece is made of hand worked wrought-iron, outfitted in natural-slate and distressed bronze finishes. Resembling bamboo, the cylinder shades feature a striated pattern with an amber hue for a warm, diffused light. Dimensions: 20" H x 18" W. Height is adjustable using the included 48" chain. Fixture has 3 medium base sockets rated for 60W each.
How to install this product in a typical situation.
All Site Contents ©2015, House of Antique Hardware, Inc. Antique-By-Hand® is a registered trademark of House of Antique Hardware, Inc.


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Taking Care in Winter Weather
The onset of winter does not signal the end of exercise and outdoors activities. There's plenty of skiing and snowboarding available in the Northeast, the American Rockies, across Canada, and even ...View Article
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| Good Nutrition & Lots of Exercise for Healthy Kids
We're in the middle of a raging epidemic affecting America's youth - the obesity epidemic. The number of American adolescents who are obese increased 300% in the last 40 years.


text: Description:A smooth and rusticated, bulldog from Erik Nørding's Valhalla series. Can vary in colour from brown to brown/black.Specifications: Length: 12,8 cm. Weight: 34 grams Height: 3,8 cm. Chamber, inside, diameter: 18 mm. Chamber outside: 3,8 cm.Chamber depth: 29 mmFilter: No Stem: Acrylic Produced in: Denmark TAX FREE: Please note that if you are an outside EU citizen the VAT (20%) will be deducted the listed prices when/if you finish your order.


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Turning Out The Lights: ProQuo
Art by Mike Lucas of Dow Jones
(Note: We're keeping an ongoing tally of venture-backed company shutdowns this year as VentureWire reports on them. See the list below. Look for these postings under the title, "Turning Out The Lights.")
ProQuo Inc., a start-up that offered to filter out people's junk mail, quietly went belly up in July, joining our list of this year's failed venture-backed start-ups.
The company had raised $15 million over two rounds from Draper Fisher Jurvetson and Mission Ventures.
Founded in 2006, ProQuo provided a service designed to help consumers remove their names from thousands of marketing lists, data brokers and other organizations that send unsolicited postal mail, while continuing to receive the offers they want.


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Winston-Salem, North Carolina – (The Hosting News) – May 22, 2006 – SilkRoad Equity, LLC has expanded its capital investment in web-based collaborative software technology firm, SolidSpace LLC. The additional funds will be used to further expand the company and support the creation of a more dynamic product line.
Jim Capps, President of SolidSpace LLC explained, "SolidSpace's evolution has always been based on the changing needs of our customers. By meeting their demands, we have been able to grow and diversify our offerings. Our success has been a direct result of that and we're excited to be in an even greater position to provide the most powerful collaborative tools to small and mid-sized companies."
The additional funds will bolster the continued development of the company's SolidSuite platform, a suite of software as a service (SAAS) collaboration tools, including podcast management, enterprise chat and IM and survey and poll systems.
Additionally, SolidSpace will also provide back-end capabilities including expense reporting, web conferencing, project management and sales lead tracking. This summer, SolidSpace will also expand its core business of managed services and data center hosting with the debut of SolidWaves, a business-class wireless broadband service for both corporate and residential customers.
All of these enhanced offerings help to further the company's stance to provide complete, cost-effective, fully-hosted business solutions for small to medium businesses (SMBs).
Andrew J. "Flip" Filipowski, Executive Chairman and CEO of SilkRoad offered, "SilkRoad Equity has always looked to support the most promising and energetic young companies. SolidSpace is no exception. We are looking forward to being a part of the company's success as it rises to meet its potential and continues its unprecedented growth."
SolidSpace LLC provides web-based collaborative software technologies, along with a hosting infrastructure that includes website, application and database hosting, and managed services. SolidSpace LLC is headquartered in Winston-Salem, N.C., and is a portfolio company of SilkRoad Equity LLC. SolidSpace divisions include Pendulab, the developer of the AffiliateShop affiliate management products, ChatBlazer Enterprise real-time communications tools and InstantEngage live customers service chat products.
SilkRoad Equity, LLC is a private investment firm that acquires businesses in a broad range of industries and manages them for growth and profitability. SilkRoad Equity is located in Winston-Salem, NC and manages investments in the life sciences, media and entertainment, technology and telecommunications sectors.
To learn more about SilkRoad Equity, please visit: www.silkroadequity.com.
For more information about SolidSpace, please visit: www.solidspace.com.


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Gakenke: Men urged to help women in acquiring loans
After many complaints from women about their husbands not supporting them when they want to acquire loans from banks, men were advised to help their wives so that they can get such and be able to develop.
This was a call from the coordinator of the National women's council in Gakenke district Immaculee Mukagacinya, who said that many men do not take this as a very important issue, hence called them on to give a hand to their women.
She said this while celebrating the International women's day on the 08/03/2013, were she advised men across the country to give their women the necessary items that can help them get loans from banks and therefore be able to work and develop.
She added on saying that since family property is for both parties, she did not see any reason as to why men should not give their women part of it to help them get money and be able to work.
The Gakenke vice mayor in charge of finance and economic development Odette Uwitonze also called on women in the district to get together in cooperatives which will help them develop.
"About 29% of women are the only ones formed under cooperative and therefore I think more women should join cooperative to be able to acquire loans and therefore work and develop", said Uwitonze.
She then asked the women to open up accounts with Sacco which can easily give them loans that can help them develop.
The number of women working with financial institutions is still very low compared with the men who are at a 50%, and therefore this is why women were called upon to join such institutions.


text: Kids' Bling2o™ sea monster swim goggles Item G7005 Color: BLUE MULTI Size: ONE SIZE ONE SIZE Size Charts Quantity: 1 2 3 4 5 6 7 8 9 Add to Bag Wishlist Add to Bag Add To Wishlist share: Find In Store Please select a size above. size & fit Product Details Makers of the awesome-est swim goggles ever, Bling2o was started as an in-home craft project with the four founders and their kids, quickly becoming an international business in 2013. All goggles are latex- and lead-free, have antifog properties and are a must for the ultimate underwater exploring adventure this summer. Import. Online only. Item G7005.


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Spire Group NY
20 W 23rd St. Fl. 5
New York, NY
Posted: August 12 2016
Always be on the alert for potentially fraudulent job postings online - never send money to a potential employer.
DescriptionWith nearly 10,000 units sold and rented since 1996, Spire (a Kurland Holdings Company) is one of Manhattan's most successful real estate companies. Whether you are in the market for a new property, selling or renting, Spire has the innovative real estate solutions you are looking for to transform your life.
Spire makes it easy. We ensure you get every listing, every opportunity, and all the tools you need wherever you are – at home, in our office, or on the go – with our industry-leading virtual office technology, enhanced database technology (including all of New York City's 20,000+ listings), Spire exclusives, open houses, and innovative mobile tools. We work with the city's best landlords and vendors, and boast one of Manhattan's most experienced sales forces. Unlike traditional firms, Spire empowers its agents with autonomy, eliminating the red tape that often hinders our competitors – to provide you with the highest level of personalized service to match you with a property you'll love.
Start living better.
Our Mission
Provide breakthrough real estate solutions to help clients live better lives. Lead through innovation, employing a progressive platform of streamlined operations, advanced technologies, and greater rewards. Expand as both clients and agents see the value of our industry-best offerings, our commitment to better living, and the positive impact we have on their lives.
Responsibilities- Create signage, newsletters, email campaigns, online promotions, etc.
- Provide support to social media efforts to target new audiences.
- Assist with trade shows and special events to promote company and increase attendance.
- Research and analyze competitor marketing and sales materials.
- Collaborate on team presentations for management and clients.
Requirements- Pursuing a Bachelors Degree in Marketing
- Possess excellent written and verbal communication and customer service skills.
- Possess creativity, flexibility, variety, and growth potential.
- Should be open-minded, a fast learner, enthusiastic, and adaptable.
SkillsRequired - Social Media Marketing
Preferred - Graphic Design


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