Population genetic studies in the Balkans. II. DNA-STR-systems

Within a study of the genetics of Southeastern European populations four DNA-STR-systems (D21S11, FGA, TH01, VWA) were examined in seven samples (samples of three Aromuns and four other Balkan populations). The results have been compared to data from four samples from literature (Austrians, Germans, Hungarians, Slovenians). The results show three clusters: a) the Aromuns from Albania (Andon Poci) and Macedonia (Stip region), b) the Romanian Aromuns (Kogalniceanu), Romanians (Constanta, Ploiesti) and Albanians (Tirana) und c) the data from literature. A sample of Northeastern Greece clearly differs from these three clusters. Including seven serum protein polymorphisms (without the populations from literature) results in two clusters: a) the three Aromun populations and b) Albanians and Romanians. Again the sample of Northeastern Greece clearly differs from these clusters.     

Investigations into the mechanisms controlling prostaglandin production by the guinea-pig placenta: roles of calcium and gonadotrophin-releasing hormone

The outputs of PGF(2 alpha), PGE(2) and 6-keto-PGF(1 alpha) were higher from the day 29 guinea-pig placenta than from the sub-placenta in culture, with PGF(2 alpha)being the major prostaglandin produced by the placenta. Lack of extracellular calcium reduced the production of all three prostaglandins by the sub-placenta and 6-keto-PGF(1 alpha) production by the placenta, but had no effect on the production of PGF(2 alpha) and PGE(2) by the placenta. EGTA (a calcium chelator) and a low concentration (30 microM) of TMB-8 (an intracellular calcium antagonist) generally inhibited prostaglandin output from the placenta and sub-placenta at various time points during culture, although EGTA had no effect on PGE(2) output from the placenta. Trifluoperazine and W-7 (calmodulin inhibitors) had no inhibitory effect on the outputs of PGF(2 alpha) and PGE(2) from the placenta, nor on the outputs of any prostaglandin from the sub-placenta. However, these two compounds inhibited the output of 6-keto-PGF(1 alpha) from the placenta. Nifedipine and verapamil (calcium channel blocking drugs) generally reduced the outputs of prostaglandins from the placenta and sub-placenta, except verapamil had no inhibitory effect on PGF(2 alpha) output from the sub-placenta. Gonadotrophin-releasing hormone (GnRH) did not stimulate the output of prostaglandins from the placenta, and tended to have a weak inhibitory action on this tissue. On the sub-placenta, GnRH had an initial inhibitory action on the outputs of PGF(2alpha) and 6-keto-PGF(1 alpha), which was then followed by a stimulation of the outputs of PGF(2 alpha) and, to a lesser extent, of PGE(2).     

In vitro developmental competence of buffalo oocytes collected at various stages of the estrous cycle

The objective was to determine the in vitro developmental competence of buffalo oocytes collected from abattoir-derived ovaries at various stages of the estrous cycle and follicular status. In Experiment 1, ovaries (n=476 pairs) were collected and divided into the following five groups: (a) ovaries with a corpus hemorragicum and no dominant follicle (CH-NO-DF); (b) ovaries with a mature functional corpus luteum (CL) and a dominant follicle (CL-DF); (c) ovaries with a mature functional CL and no dominant follicle (CL-NO-DF); (d) ovaries with a regressing CL and a dominant follicle (RCL-DF); and (e) ovaries without any luteal structures and only small follicles (ANEST). In Experiment 2, 144 pairs of ovaries with a CL (or regressing CL) and a dominant follicle were collected and follicles were classified as dominant, largest subordinate, and subordinate. In both experiments, the dominant follicle was defined as any follicle >10mm in diameter that exceeded the diameter of all other (subordinate) follicles. Although oocytes were collected from each group of ovaries, only Grades A or B oocytes were used for in vitro embryo production. Cleavage rates were higher (P<0.05) from oocytes collected from ovaries in the CH-NO-DF (59.6%) and CL-NO-DF (59.2%) groups than those collected from CL-DF (52.2%) and ANEST (43.6%) groups. The yield of transferable embryos was higher (P<0.05) from oocytes collected from CH-NO-DF (27.4%) and CL-NO-DF (24.0%) ovaries than from CL-DF (16.2%), RCL-DF (15.4%), and lowest (P<0.05) from ANEST (8.8%). In Experiment 2, oocytes from the dominant follicle had a higher (P<0.05) cleavage rate (65.2 %) and transferable embryo yield (30.2%) than those collected from the largest subordinate and subordinate follicles. In conclusion, oocyte competence depended on the morphofunctional state of ovaries. Oocyte development was maximal in pairs of ovaries with a corpus hemorragicum or CL and no dominant follicle; in paired ovaries with a CL and a dominant follicle, development was maximal in oocytes derived from the dominant follicle.     

准噶尔盆地北缘哈拉玛盖组中的Anchitherium

发现于新疆准噶尔盆地北缘中新世哈拉玛盖组中的安琪马过去都被归入Anchitheriumaurelianense种。近年来在该地区新发现的Anchitherium的材料表明新疆准噶尔盆地北缘中新世哈拉玛盖组中的安琪马具有Anchitheriumgobiense的特征:上臼齿的原小尖弱但可辨认,次附尖较发育并与后部齿带共同封闭成一三角形窝,前齿带发育但内齿带弱;下颊齿相对较宽,下颊齿齿叶呈V形,外端显得较为尖锐;下臼齿列由前向后明显变窄;距骨较高窄。这些特征均与内蒙古通古尔的AnchitheriumgobienseColbert,1939一致,而与欧洲的A.a.aurelianense、A.a.steinheimense及A.a.hippoides者不同。因此哈拉玛盖组中的安琪马应归属Anchitheriumgobiense。我国湖北房县的安琪马也应是A.gobiense种。A.gobiense的前臼齿列的宽度由后向前,尤其是臼齿列的宽度由前向后减小的程度,大于欧洲的A.aurelianense种,这一特征与下颊齿相对较宽、齿叶外端较尖锐、上颊齿的内齿带弱等特征一起组成了中国Anchitherium支系的特征。中国的三趾马化石层位中的大型安琪马Sinohippuszitteli具有明显不同于欧洲安琪马类的特征:1)齿冠很大,上颊齿外脊的外壁呈宽V形;2)其前臼齿列及臼齿列的宽度分别向前和向后明显变小,特别是下前臼齿列向前变窄的程度远超过安琪马属。因此应保留Sinohippus属,Sinohippuszitteli是亚洲大陆上特有的安琪马支系的晚期代表。     

Newcastle disease virus in double-crested cormorants in Alabama, Florida, and Mississippi.

In order to understand the epidemiology of Newcastle disease (ND) outbreaks in double-crested cormorants (Phalacrocorax auritus), a study was conducted on wintering migratory cormorants (P. a. auritus) in Alabama and Mississippi (USA) and non-migratory cormorants (P. a. floridanus) that breed in Florida (USA). Antibodies against ND virus were detected by the hemagglutination-inhibition method in sera from 86 of 183 (47%) migratory cormorants over-wintering in eight roosting sites in Alabama and Mississippi between November, 1997 and April, 1999. Titers ranged from 5 to 40. Antibody prevalences in sera collected from females in early winter (November and December) (26%) and late winter (February and March) (56%) were significantly different (P = 0.0007). None of 45 serum samples from 1- to 7-wk-old nestlings from 11 colonies in Florida during the 1997-98 and 1998-99 breeding seasons was positive. However, antibodies were detected in yolk samples from 98 of 126 (78%) eggs collected in these same colonies. Titers ranged from 4 to 256. The prevalence of antibodies in eggs collected from fresh-water colonies (63% prevalence, n = 30) and salt-water colonies (82% prevalence, n = 96) was significantly different (P = 0.041). ND virus was not isolated from tissues of 18 cormorants and cloacal and tracheal swabs from 202 cormorants collected in Alabama and Mississippi; virus was also not isolated from cloacal and tracheal swabs from 51 nestlings from Florida.     

Characterization of electrostatic binding sites of extracellular polymers by linear programming analysis of titration data

Electrostatic binding sites of extracellular polymeric substances (EPS) were characterized from titration data using linear programming analysis. Test results for three synthetic solutions of given solutes comprising amino, carboxyl, and phenolic groups indicated that this method was able to identify the electrostatic binding sites. For the six sites with pK(a) between 3 and 10, the estimated pK(a) deviated 0.11 +/- 0.09 from the theoretical values, and the estimated concentrations deviated 3.0% +/- 0.9% from the actual concentrations. Two EPS samples were then extracted from a hydrogen-producing sludge (HPS) and a sulfate-reducing biofilm (SRB). Analysis of charge excess data in titration from pH 3 to 11 indicated that the EPS of HPS comprised of five electrostatic binding sites with pK(a) ranging from 3 to 11. The pK(a) values of these binding sites and the possible corresponding functional groups were pK(a) 4.8 (carboxyl), pK(a) 6.0 (carboxyl/phosphoric), pK(a) 7.0 (phosphoric), pK(a) 9.8 (amine/phenolic), and pK(a) 11.0 (hydroxyl). EPS of the SRB comprised five of similar binding sites (with corresponding pK(a) values of 4.4, 6.0, 7.4, 9.4, and 11.0), plus one extra site at pK(a) 8.2, which was likely corresponding to the sulfhydryl group. The total electrostatic binding site concentration of EPS extracted from HPS were 10.88 mmol/g-EPS, of which the highest concentration was from the site of pK(a) 11.0. The corresponding values for the EPS extracted from SRB were 16.44 mmol/g-EPS and pK(a) 4.4. The total concentrations of electrostatic binding sites found in this study were 20- to 30-fold of those reported for bacterial cell surface, implying that EPS might be more crucial in biosorption of metals than bacterial cell surface in wastewater treatment and in bioremediation.     

Single-beat estimation of right ventricular end-systolic pressure-volume relationship

Assessment of right ventricular (RV) contractility from end-systolic pressure-volume relationships (ESPVR) is difficult due to problems in measuring RV instantaneous volume and to effects of changes in RV preload or afterload. We therefore investigated in anesthetized dogs whether RV ESPVR and contractility can be determined without measuring RV volume and without changing RV preload or afterload. The maximal RV pressure of isovolumic beats (P(max)) was predicted from isovolumic portions of RV pressure during ejecting beats and compared with P(max) measured during the first beat after pulmonary artery clamping. In RV pressure-volume loops obtained from RV pressure and integrated pulmonary arterial flow, end-systolic elastance (E(es)) was assessed as the slope of P(max)-derived ESPVR, pulmonary artery effective elastance (E(a)) as the slope of end-diastolic to end-systolic relation, and coupling efficiency as the E(es)-to-E(a) ratio (E(es)/E(a)). Predicted P(max) correlated with observed P(max) (r = 0.98 +/- 0.02). Dobutamine increased E(es) from 1.07 to 2.00 mmHg/ml and E(es)/E(a) from 1.64 to 2.49, and propranolol decreased E(es)/E(a) from 1.64 to 0.91 (all P < 0.05). After adrenergic blockade, preload reduction did not affect E(es), whereas hypoxia and arterial constriction markedly increased E(a) and somewhat increased E(es) due to the Anrep effect. Low preload did not affect E(es)/E(a) and high afterload decreased E(es)/E(a). In conclusion, in the right ventricle 1) P(max) can be calculated from normal beats, 2) P(max) can be used to determine ESPVR without change in load, and 3) P(max)-derived ESPVR can be used to assess ventricular contractility and ventricular-arterial coupling efficiency.     

New and already known acanthocephalans mostly from mammals in Vietnam, with descriptions of two new genera and species in Archiacanthocephala

Adults of 2 new species and 2 new genera of acanthocephalans in class Archiacanthocephala, collected between 1998 and 2004 in Vietnam from the intestines of mammals, are described, i.e., Cucullanorhynchus constrictruncatus n. gen., n. sp. (Oligacanthorhynchidae) from a leopard Panthera pardus (Linnaeus) (Mammalia: Felidae) and Paraprosthenorchis ornatus n. gen. n. sp. (Oligacanthorhynchidae) from the Chinese pangolin Manis pentadactyla (Linnaeus) (Mammalia: Manidae). Adult Sphaerechinorhynchus macropisthospinus Amin, Wongsawad, Marayong, Saehoong, Suwattanacoupt, and Sey, 1998 (Plagiorhynchidae) are described for the first time from 2 females collected from a tiger Panthera tigris (Linnaeus) (Mammalia: Felidae) and from 1 male from a water monitor Varanus salvator Laurenti (Reptilia: Varanidae). Characteristic features distinguishing the new species or genera from related taxa are as follows. The trunk of C. constrictruncatus has an anterior hood in both sexes and a posterior constriction in females. The anterior trunk of P. ornatus has many small festoons and proboscis hooks are inserted in elevated papillae separated by beady, near hexagonal, ornate grids.     

Measurement of dopamine D2-like receptors in postmortem CNS and pituitary: differential regional changes in schizophrenia

In situ radioligand binding with autoradiography and anti-human dopamine D(2) receptor antibodies with Western blots have been used to measure the density of dopamine D(2)-like receptors in the caudate-putamen and pituitary from schizophrenic subjects who did or did not have residual antipsychotic drugs in their tissue at death. There was a significant decrease in the Ki for haloperidol displaceable [(125)I]iodosulpride binding in the pituitary (p < 0.01) and caudate-putamen (p < 0.05) from subjects with schizophrenia with residual drugs in their tissue. There was a significant decrease in the density of [(125)I]iodosulpride in the pituitary (p < 0.001) and a strong trend to a decrease in binding in the caudate-putamen (p = 0.055) from subjects with schizophrenia. By contrast, [(3)H]spiperone binding was decreased in the caudate-putamen (p < 0.05) with a trend to decreased binding in the pituitary (p = 0.07) from subjects with schizophrenia. There was no difference in the density of dopamine D(2) receptors in the caudate-putamen from subjects with schizophrenia (p = 0.31). All the findings on receptor densities were independent of drug status. [(125)I]iodosulpride binds to the dopamine D(2&3) receptors. We have shown that there is no change in the dopamine D(2) receptor in the caudate-putamen from subjects with schizophrenia and therefore, these data would be consistent with there being a decrease in the dopamine D(3) in the caudate-putamen from subjects with schizophrenia. Since dopamine D(3) receptors are absent or present at low concentrations in the pituitary, our data would suggest the dopamine D(2) receptor is decreased in that tissue from schizophrenic subjects.     

Yersinia enterocolitica and related species isolated from wildlife in New York State.

Fecal specimens for Yersinia screening were obtained from a variety of wild mammals, birds, reptiles, fish, and invertebrates throughout New York State. One specimen from each of 1,426 animals was examined. A total of 148 isolates of Yersinia enterocolitica and related species were obtained from 133 (9.3%) of the animals. Y. enterocolitica was isolated from 100 (7%) of the animals tested, including 81 (10%) of 812 mammals and 19 (3.3%) of 573 birds. Y. intermedia, Y. frederiksenii, and Y. kristensenii were isolated from 39 (2.7%), 5 (0.35%), and 4 (0.28%) animals, respectively. The 81 Y. enterocolitica isolates from mammals belonged to 15 serogroups and included three pathogens: two isolates of typical serogroup 0:8, the "American strain," one from a gray fox (Urocyon cinereoargenteus) and one from a porcupine (Erethizon dorsatum); and one isolate of serogroup 0:3, bacteriophage type IXb, the "Canadian strain," from a gray fox. The most prevalent serogroups recovered from mammals were 0:6,31 (16 isolates) and 0:5,27 (6 isolates). The 19 isolates of Y. enterocolitica from birds belonged to nine serogroups and included one serogroup 0:6,31 isolate from a common grackle (Quiscalus quiscula) and two serogroup 0:5,27 isolates from great horned owls (Bubo virginianus).     

Arginine kinase from the Tardigrade, Macrobiotus occidentalis: molecular cloning, phylogenetic analysis and enzymatic properties

Arginine kinase (AK), which catalyzes the reversible transfer of phosphate from ATP to arginine to yield phosphoarginine and ADP, is widely distributed throughout the invertebrates. We determined the cDNA sequence of AK from the tardigrade (water bear) Macrobiotus occidentalis, cloned the sequence into pET30b plasmid, and expressed it in Escherichia coli as a 6x His-tag—fused protein. The cDNA is 1377 bp, has an open reading frame of 1080 bp, and has 5′- and 3′-untranslated regions of 116 and 297 bp, respectively. The open reading frame encodes a 359-amino acid protein containing the 12 residues considered necessary for substrate binding in Limulus AK. This is the first AK sequence from a tardigrade. From fragmented and non-annotated sequences available from DNA databases, we assembled 46 complete AK sequences: 26 from arthropods (including 19 from Insecta), 11 from nematodes, 4 from mollusks, 2 from cnidarians and 2 from onychophorans. No onychophoran sequences have been reported previously. The phylogenetic trees of 104 AKs indicated clearly that Macrobiotus AK (from the phylum Tardigrada) shows close affinity with Epiperipatus and Euperipatoides AKs (from the phylum Onychophora), and therefore forms a sister group with the arthropod AKs. Recombinant 6x His-tagged Macrobiotus AK was successfully expressed as a soluble protein, and the kinetic constants (K(m), K(d), V(ma) and k(cat)) were determined for the forward reaction. Comparison of these kinetic constants with those of AKs from other sources (arthropods, mollusks and nematodes) indicated that Macrobiotus AK is unique in that it has the highest values for k(cat) and K(d)K(m) (indicative of synergistic substrate binding) of all characterized AKs.     

Genetic Diversity of Algal Viruses Which Lyse the Photosynthetic Picoflagellate Micromonas pusilla (Prasinophyceae)

The genetic similarity among eight clones of Micromonas pusilla virus (MpV) isolated from five geographic locations was measured by DNA hybridization. Our objective was to explore the existence of genetically distinct populations of MpV by comparing the similarity among MpVs isolated from a single water sample to the similarity among viruses isolated from geographically distant locations. The highest and lowest similarities we observed were 70% (plusmn) 1.1% (mean (plusmn) standard error [SE], n = 3) for virus strains SP1 and SP2 isolated from a California coastal water sample and 13% (plusmn) 1.9% for strains SP2 and PB6; the latter was isolated from New York estuarine water. However, the similarity between MpV isolated from a single water sample was not always greater than the similarity between viruses isolated from different locations. Viruses PB7 and PB8 were isolated from a single New York estuarine sample but were only 16% (plusmn) 0.5% similar, whereas PB7 was quite similar (43% (plusmn) 2.9%) to PL1, a virus from Texas coastal water. Overall, the similarity among MpVs isolated from a single geographic location, 34% (plusmn) 12.6% (mean (plusmn) SE, n = 4), was not significantly different from the similarity among MpVs isolated from geographically distant locations, 26.6% (plusmn) 2.7% (mean (plusmn) SE, n = 24) (P = 0.92, Mann-Whitney U test). Clones of MpV were more similar to each other than they were to the related algal virus PBCV-1, and three groups of MpVs consisting of (i) PL1, SG1, PB6, and PB7, (ii) PB8, and (iii) GM1, SP1, and SP2 were resolved. The genetic variation among MpVs isolated from a single water sample was as large as the variation between viruses isolated from different oceans. If MpVs within a geographic location share genetic characteristics not shared with MpVs from geographically distant locations, this was not reflected in the overall similarity of their genomes.     

Anaerobic degradation of benzoate: batch studies

Response of benzoate along with phenol to different anaerobic inocula has been investigated in batch reactors. In Phase I, the anaerobic biodegradability of benzoate and phenol were evaluated using (a) washed acclimatized granular sludge (WAGS) collected from a passive phenol fed bench-scale up-flow anaerobic sludge blanket reactor (UASBR) and (b) unacclimatized flocculent sludge (UFS) from a UASB based sewage treatment plant (STP). The effect of varying concentrations of benzoate has been investigated in Phase II using acclimatized granular sludge (AGS) from a bench-scale UASBR. Extent of degradation of benzoate was more than the phenol. Increasing benzoate COD from 2500 to 11,700mg/L, resulted in decrease in (i) rate constant, k from 0.79 to 0.11/d and (ii) ultimate biochemical methane potential (microb, g CH4-COD formed/g benzoate COD) from 84% to 60%. Temporal trend conforming to logistic S-curve indicated stressed conditions at higher benzoate concentration. Benzoate degradation was found to be sensitive to nature as well as quantity i.e. food to microorganism (F/M) of inocula used.     

Comparison of an avian osteopetrosis virus with an avian lymphomatosis virus by RNA-DNA hybridization.

Myeloblastosis-associated virus (MAV)-2(0), a virus which was derived from avian myeloblastosis virus and induced a high incidence of osteopetrosis, was compared with avian lymphomatosis virus 5938, a recent field isolate which induced a high incidence of lymphomatosis. The following information was obtained. (i) MAV-2(0) induced osteopetrosis, nephroblastoma, and a very low incidence of hepatocellular carcinoma. No difference was seen in the oncogenic spectrum of end point and plaque-purified MAV-2(0). (ii) 125I-labeled RNA sequences from MAV-2(0) formed hybrids with DNA extracted from osteopetrotic bone at a rate suggesting five proviral copies per haploid cell genome. The extent of hybridization of MAV-2(0) RNA with DNA from osteopetrotic tissue was more extensive (87%) than was observed in reactions with DNA from uninfected chicken embryos (52%). (iii) Competition of unlabeled viral RNA in hybridization reactions between the radioactive RNA from the two viruses and their respective proviral sequences present in tumor tissues showed that 15 to 20% of the viral sequences detected in these reactions were unshared. In contrast, no differences were detected in competition analyses of RNA sequences from the two viruses detected in DNA of normal chicken cells. (iv) MAV-2(0) 35S RNA was indistinguishable in size from avian lymphomatosis virus 5938 35S RNA by polyacrylamide gel electrophoresis.     

Synthesis of a shortened pro-alpha 2(I) chain and decreased synthesis of pro-alpha 2(I) chains in a proband with osteogenesis imperfecta

Synthesis of type I procollagen was examined in skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta. The fibroblasts synthesized shortened pro-alpha 2(I) chains and these shortened chains accounted for all the pro-alpha 2(I) chains synthesized by the cells. In addition, there was a decrease in the relative rate of synthesis of pro-alpha 2(I) chains. Fragmentation of the shortened pro-alpha 2(I) chains with vertebrate collagenase and cyanogen bromide demonstrated that the shortening was in alpha 2(I)-CB3,5A, a fragment from about the middle of the chain containing amino acid residues 361 to 775. Based on the relative mobility in electrophoretic gels, the shortening was about 20 amino acid residues. The decreased synthesis of pro-alpha 2(I) chains was demonstrated by an increase in the ratio for the rates of synthesis of pro-alpha 1(I):pro-alpha 2(I) chains. It was associated with an increase in the ratio of mRNAs for pro-alpha 1(I):pro-alpha 2(I) in the cells. Fibroblasts from the father also demonstrated a decreased synthesis of pro-alpha 2(I) chains as reflected by an increase in the ratio of newly synthesized pro-alpha 1(I):pro-alpha 2(I) chains. No shortened pro-alpha 2(I) chains were seen in fibroblasts from either the father or the mother. The observations suggested that the proband inherited a nonfunctioning pro-alpha 2(I) gene from her father and that the gene for the shortened pro-alpha 2(I) chain probably arose from a sporadic mutation.     

Influence of the scale function on wavelet transformation of the surface electromyographic signal

The scale function in wavelet transformation (WT) determines wavelet dilation and optimises the processing of a given signal. Here, the objective was to determine the influence of the scale function on the WT of 160 surface electromyograms using second-degree polynomial (WT(poly)) and exponential (WT(exp)) scale functions. For each WT, a mean frequency (MNF) was calculated from the original wavelet spectrum and from the cubic spline interpolated wavelet spectrum, and these were compared with the MNF obtained from a fast Fourier transform (FFT). The total intensity (Tp) for each WT was compared with the root mean square (RMS). The MNFs computed from the original wavelet spectra were significantly (P < 0.05) lower and higher when computed from the reconstructed wavelet spectra than those from the FFT. The Tp computed from WT(poly) showed significantly higher agreement with the RMS than the Tp from WT(exp). Finally, the WT(poly) may serve as a reference in electromyography.     

A PCR primer-specific to Cylindrocarpon heteronema for detection of the pathogen in apple wood

Abstract An oligonucleotide primer (ChInt) was synthesised from the variable internally transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) of Cylindrocarpon heteronema . PCR with primers ChInt and ITS4 (from a conserved sequence of the rDNA) amplified a 470-bp fragment from several isolates of C. heteronema but not from various apple wood saprophytes. Amplification of this fragment was achieved from 1–2 pg of fungal DNA. These primers amplified a fragment of the same size from DNA extracted from cankered wood but only after impurities were removed from the DNA on a Qiagen tip-5 column. Southern hybridization analysis confirmed the 470-bp fragment from C. heteronema DNA and cankered wood to be identical.     

Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.     

A real-time electrochemical technique for measurement of cellular hydrogen peroxide generation and consumption: evaluation in human polymorphonuclear leukocytes

There has been a long-standing need for sensitive and specific techniques for hydrogen peroxide (H(2)O(2)) measurement. We describe the development and application of a highly sensitive electrochemical sensor, utilizing a membrane-coated platinum microelectrode, suitable for real-time measurement of hydrogen peroxide generation and consumption in biochemical or cellular systems. This sensor provides high sensitivity enabling measurement of hydrogen peroxide down to 5-10 nM concentrations. We demonstrate that it can be used to measure the magnitude and time course of H(2)O(2) generation from the NADPH oxidase in leukocytes as well as the rate of H(2)O(2) degradation. After human polymorphonuclear leukocytes (PMNs) were activated by phorbol 12-myristate acetate, H(2)O(2) concentration increased with time and reached a peak concentration, from 5 to 15 microM in PMNs prepared from different individuals, within 3 to 8 min, then decreased slowly. The H(2)O(2) concentration in the solution is less than the total H(2)O(2) generation from the activated PMNs because a part of H(2)O(2) generated is decomposed. H(2)O(2) in solution, generated from the PMNs, was rapidly consumed after the activated PMNs were treated with 10 microM diphenylene iodonium (DPI). The rate of H(2)O(2) consumption was measured following the addition of exogenous H(2)O(2). The total production of H(2)O(2) from the activated PMNs was calculated from the measured H(2)O(2) concentration and the rate of H(2)O(2) consumption. This technique enables sensitive and continuous real-time measurement of H(2)O(2) concentration and total H(2)O(2) generation in cellular or enzyme systems without addition of any detection reagents.