Colicine Typing of Shigella sonnei

The colicine typing method of Shigella sonnei is described with experimental evidence supporting it as well as the manner of selection of eight indicator strains. The disparity of principle between this method and that of Abbott and Shannon's method is (1) selection of the indicators only from wild strains existing in this country, (2) employment of heart infusion broth for colicine production, (3) performance of the typing within 48 hours, and (4) determination of types and subtypes of test-strains by combining their colicinogenic activity against the indicators and their sensitivity to colicines produced by the indicators. A modification of the method is advocated which requires three days to extract colicine by cultivation and one day for sensitivity tests and which uses peptone as the sole nutriment in media. The efficiency of the technique of Abbott and Shannon, McGeachie and McCormick, and the authors' two methods was compared using the selected indicators. Only the technique of McGeachie and McCormick showed some discrepancies.     

Hyphomonas beringensis sp. nov. and Hyphomonas chukchiensis sp. nov., isolated from surface seawater of the Bering Sea and Chukchi Sea

Two Gram-negative, non-spore-forming, oval to pear shaped motile strains, designated 25B14_1^T and BH-BN04-4^T, isolated from surface seawater from the Bering Sea and Chukchi Sea, respectively, were subjected to polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strains 25B14_1^T and BH-BN04-4^T clustered together with Hyphomonas atlanticus 22II1-22F38^T and Hyphomonas oceanitis DSM 5155^T, respectively, within genus Hyphomonas. Based on whole genome sequence analysis, the calculated DDH and ANIm values between strain 25B14_1^T and BH-BN04-4^T are 18.8 and 83.19 % respectively. The calculated DDH values of strain 25B14_1^T and BH-BN04-4^T with seven type strains ranged from 18.2 to 19.9 % and from 18.4 to 40.4 %, respectively. The ANIm values of strain 25B14_1^T and BH-BN04-4^T with seven type strains ranged from 83.00 to 84.67 % and from 83.14 to 90.58 %, respectively. Both isolates were found to contain Q-11 as the predominant respiratory quinone. The major fatty acids of strain 25B14_1^T were identified as C_16:0, C_17:0, C_18:1 ω7c-methyl and Summed Feature 8 (C_18:1 ω6c/ω7c as defined by MIDI), while in the case of strain BH-BN04-4^T they were identified as C_16:0, C_18:1 ω7c-methyl and Summed Feature 8 (C_18:1 ω6c/ω7c). The G+C contents of 25B14_1^T and BH-BN04-4^T were determined to be 58.4 and 61.0 mol%, respectively. The combined phenotypic and genotypic data show that the two isolates each represent novel species of the genus Hyphomonas, for which the names Hyphomonas beringensis sp. nov. and Hyphomonas chukchiensis sp. nov. are proposed, with the type strain 25B14_1^T (=MCCC 1A07321^T = LMG 27914^T) and BH-BN04-4^T (=MCCC 1A07481^T = LMG 27915^T), respectively.     

Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (≤3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.     

Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera:Dytiscidae): I. Metabolism of androst-4-en-3,17-dione (AD)

Two Bacillus strains were isolated from the foregut of the water beetle Agabus affinis (Payk.) and tested for their steroid transforming ability. After incubation with androst-4-en-3,17-dione (AD), 13 different transformation products were detected. AD was hydroxylated at C₆, C₇, C₁₁ and C₁₄, resulting in formation of 6β-, 7α-, 11α- and 14α-hydroxy-AD. One strain also produced small amounts of 6β,14α-dihydroxy-AD. Partly, the 6β-hydroxy group was further oxidized to the corresponding 6-oxo steroids. In addition, a specific reduction of the Δ⁴-double bond was observed, leading to the formation of 5α-androstane derivatives. In minor yields the carbonyl functions at C₃ and C₁₇ were reduced leading to the formation of 3ξ-OH or 17β-OH steroids. EI mass spectra of the trimethylsilyl and O-methyloxime trimethylsilyl ether derivatives of some transformation products are presented for the first time.     

野生鸟类分离的多重耐药肺炎克雷伯氏菌(Klebsiella pneumoniae)耐药基因分子进化特征研究

【目的】调查野生鸟类携带菌的耐药状况,探索其在细菌耐药性传播过程中的作用。【方法】从野生鸟类石鸡、绯胸鹦鹉、太阳锥尾鹦鹉和黑领椋鸟的新鲜粪便分离4株Klebsiella pneumoniae,采用微量肉汤稀释法评估其多重耐药表型,并利用全基因组测序技术和细菌全因组关联分析、比较基因组学方法对分离株进行分子溯源,系统解析其携带的多重耐药质粒或基因与其宿主、同源质粒间的关联。【结果】4株肺炎克雷伯菌的耐药谱各不相同,来自石鸡样本的分离株S90-2对9种药物耐受,绯胸鹦鹉样本分离株S141对3种药物耐受,太阳锥尾鹦鹉分离株M911-1仅耐受氨苄西林,黑领椋鸟的样本分离株S130-1对所使用的14种药物完全敏感。S90-2属于ST629型,携带bla_CTX-M-14、fosA6、aac(3)-Iid和bla_SHV-11为主的30个耐药基因和携带1个耐药性质粒pS90-2.3 (IncR型)。S141属于ST1662型,携带fosA5、bla_SHV-217等27个耐药基因,1个质粒pS141.1 [IncFIB(K)(pCAV1099-114)/repB型]仅携带耐药基因adeF。M911-1为新ST类型,携带bla_SHV-1、fosA6等共计27个耐药基因,其质粒pM911-1.1携带了3个耐药基因。S130-1属于ST3753型,携带bla_SHV-11、fosA6等27个耐药基因,pS130-1 [IncFIB(K)型]则仅携带一个耐药基因tet(A)。质粒比对表明,质粒pS90-2.3携带的耐药基因片段源自不同的肠杆菌科菌株染色体或质粒。pS90-2.3的同源质粒主要来自人类宿主菌,且主要在中国分布,这些质粒主要细菌宿主为K. pneumoniae和Escherichia coli,且ST11型K. pneumoniae分离株为重要宿主菌。【结论】本研究中来自野生鸟类的多重耐药K. pneumoniae,其耐药基因主要来自质粒,质粒耐药基因主要由转座子、插入序列、整合子和前噬菌体等可移动元件介导,这些多重耐药质粒与人类的宿主菌密切相关。     

Oceanobacillus aidingensis sp. nov., a moderately halophilic bacterium

Two Gram-positive, rod-shaped moderately halophilic bacterial strains, designated AD7-25^T and AB-11, were isolated from Aiding and Manasi salt lakes in Xinjiang of China, respectively. The strains were found to be able to grow at NaCl concentrations of 0–21 % (w/v), with optimum growth occurring at 6–8 % (w/v) NaCl. The optimal temperature and pH for growth were determined to be 33–37 °C and pH 7.0–7.5. Cells of the strains are motile by means of polar flagella. Both strains can produce ellipsoidal spores. The major cellular fatty acids were identified as anteiso-C_15:0, iso-C_15:0, iso-C_14:0, anteiso-C_17:0 and iso-C_16:0. The diamino acid in the peptidoglycan and the major quinone system were determined to be meso-diaminopimelic acid (meso-DAP) and MK-7, respectively. The DNA G+C contents of stains AD7-25^T and AB-11 were 39.8 and 40.0 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that these two novel strains are closely related to the genus Oceanobacillus showing 90–99.5 % similarity with respect to type strains. These two novel strains were most closely related to Oceanobacillus oncorhynchi subsp. incaldanensis DSM 16557^T (99.1 and 99.5 %), followed by O. oncorhynchi subsp. oncorhynchi JCM 12661^T (99.1 and 99.4 %), Oceanobacillus neutriphilus CGMCC 1.7693^T (97.0 and 97.5 %), Oceanobacillus sojae JCM 15792^T (97.6 and 98.0 %) and Oceanobacillus locisalsi KCTC 13253^T (96.5 and 96.9 %). The DNA–DNA hybridization data indicated that DNA relatedness between strains AD7-25^T and AB-11 was 91.0 %, and the genomic homology of representative strain AD7-25^T with O. oncorhynchi subsp. incaldanensis DSM 16557^T, O. oncorhynchi subsp. oncorhynchi JCM 12661^T, O. neutriphilus CGMCC 1.7693^T, O. sojae JCM 15792^T and O. locisalsi KCTC 13253^T were 41, 39, 20, 23 and 17 %, respectively. On the basis of phenotypic and phylogenetic distinctiveness, strains AD7-25^T and AB-11 should be assigned to the genus Oceanobacillus as a new species, for which the name Oceanobacillus aidingensis sp. nov. was proposed. The type strain is AD7-25^T (=CGMCC 1.9106 ^T = NBRC 105904^T).     

The metabolism of pyrimidines by proteolytic clostridia

Uracil was used by growing cultures of Clostridium sporogenes, and by proteolytic strains of C. botulinum types A and B. Uracil was not used by C. bifermentans; C. botulinum, type B (non-proteolytic); C. botulinum, type F (non-proteolytic); C. botulinum, type E; C. butyricum; C. cochlearium; C. difficile; C. histolyticum; C. oedematiens, type A; C. paraputrificum; C. scatologenes; C. septicum; C. sordellii; C. sticklandii; C. tertium; C. tetani; C. tetanomorphum; C. welchii, types A, B, C, E and 4 untyped strains. The growth of C. sporogenes was not increased by uracil; it was reduced to dihydrouracil. Experiments with washed cells of C. sporogenes showed that the uracil-reducing system was inducible. Washed cell suspensions incubated under hydrogen with uracil, thymine, iso-barbituric acid, 5-amino uracil and cytosine consumed 1 mole H₂/mole pyrimidine. The reduction product of cytosine was dihydrouracil indicating that it was deaminated before reduction. The reduction products of the remaining pyrimidines were the corresponding dihydro derivatives. Extracts of C. sporogenes reduced uracil in the presence of NADPH₂ but not NADH₂.     

The Phylogeny of Acetic Acid Bacteria Based on the Partial Sequences of 16S Ribosomal RNA: The Elevation of the Subgenus Gluconoacetobacter to the Generic Level

Thirty-six strains of acetic acid bacteria classified in the genera Acetobacter, Gluconobacter, and Acidomonas were examined for their partial base sequences in positions 1220 through 1375, 156 bases, of 16S rRNA. The strains of the Q₁₀-equipped Gluconobacter species examined were divided into two subgroups, which included the type strains of Gluconobacter oxydans, the type species of the genus Gluconobacter, and of a second species, Gluconobacter cerinus, respectively. The base differences numbered four between the two type strains. The strains of the Q₉-equipped species examined classified in the type subgenus Acetobacter of the genus Acetobacter were not very distant phylogenetically from those of the genus Gluconobacter. The calculated number of base differences was 9–6 between the type strains of G. oxydans and G. cerinus and the type strains of Acetobacter aceti and Acetobacter pasteurianus. In contrast, the strains of the Q₁₀-equipped species examined classified in the subgenus Gluconoacetobacter of the genus Acetobacter were very distant phylogenetically from those of the Acetobacter and Gluconobacter species mentioned above. The number of base differences was calculated to be 14-8. Furthermore, the strains of the methanol-assimilating, Q₁₀-equipped species of the genus Acidomonas examined were located in phylogenetically isolated positions. The type strain of Acidomonas methanolica (≡ Acetobacter methanolicus), the type species of the genus Acidomonas, had 16–9 base differences. The data obtained here indicated that the members of the subgenus Gluconoacetobacter of the genus Acetobacter can be distinguished at the generic level. The new genus Gluconoacetobacter was proposed with the type species, Gluconoacetobacter liquefaciens, in recognition of the genus Acidomonas along with the genera Acetobacter and Gluconobacter in the classification of the acetic acid bacteria.     

Cohesin and recombination proteins influence the G1-to-S transition in azygotic meiosis in Schizosaccharomyces pombe

To determine whether recombination and/or sister-chromatid cohesion affect the timing of meiotic prophase events, the horsetail stage and S phase were analyzed in Schizosaccharomyces pombe strains carrying mutations in the cohesin genes rec8 or rec11, the linear element gene rec10, the pairing gene meu13, the double-strand-break formation genes rec6, rec7, rec12, rec14, rec15, and mde2, and the recombination gene dmc1. The double-mutant strains rec8 rec11 and rec8 rec12 were also assayed. Most of the single and both double mutants showed advancement of bulk DNA synthesis, start of nuclear movement (horsetail stage), and meiotic divisions by up to 2 hr. Only mde2 and dmc1 deletion strains showed wild-type timing. Contrasting behavior was observed for rec8 deletions (delayed by 1 hr) compared to a rec8 point mutation (advanced by 1 hr). An hypothesis for the role of cohesin and recombination proteins in the control of the G₁-to-S transition is proposed. Finally, differences between azygotic meiosis and two other types of fission yeast meiosis (zygotic and pat1-114 meiosis) are discussed with respect to possible control steps in meiotic G₁.     

LTA4-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB4 receptor of human polymorphonuclear leukocytes

5-Oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A₄ (LTA₄) in addition to 5,12-dihydroxy-(6E,8E,10E,14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E,11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA₄ was found to be pH-dependent. After incubation of LTA₄ in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA₄ in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC₅₀ of 250 nM, as compared to values of 3.5 nM for leukotriene B₄ (LTB₄) and >500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB₄ totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB₄. The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB₄ receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB₄ receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z,14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB₄.     

Determination of Pseudomonas aeruginosa by Biochemical Test Methods

A rapid and simple test method for the detection of acylamidase activity of Pseudomonas aeruginosa was devised. One loopful of a nutrient agar overnight culture of a test organism was inoculated into 1 ml of a test medium consisting of 0.2% KH₂PO₄, 0.01% MgSO₄·7H₂O, 0.5% NaCl and 0.1% acetamide (final pH 6.8). After aerobic incubation at 37C for 6 hr, one drop of Nessler's reagent was dropped into the test medium. A reddish-brown sediment appeared immediately if results were positive. Of 40 test strains of P. aeruginosa 39 gave strongly positive results. A strain showed a weakly positive result after 6 hr incubation, but the reaction became stronger after 18 hr culture. Other species of Pseudomonas, and various species of bacteria such as genera Vibrio and Aeromonas, and family Enterobacteriaceae were negative in this test. From these experimental results, the acylamidase test was considered to be highly specific for strains of P. aeruginosa, and therefore useful as a reliable method for the identification of this species.     

Biodegradation of alkylpyridines by bacteria isolated from a polluted subsurface

Ten bacterial strains were isolated fromalkylpyridine polluted sediments 7.6 m below thesurface. These strains were able to degrade 11different alkylpyridine isomers. Degradation ratesdepended on number and position of the alkyl group. Isomers with an alkyl group at position 3 were moreresistant to microbial attack. Of the 10 strains, 6isolates were selected for detailed study. Theseisolates mineralized the isomers to CO₂,NH₄ ⁺, and biomass. All strains weregram-negative rods with a strict aerobic metabolism. Characterization of physiological and biochemicalproperties revealed similarity between strains. Eeachstrain however, had a limited substrate range whichenabled it to degrade no more than 2 to 3 compounds ofthe 14 alkylpyridine isomers tested. Examination ofthe genetic variability among cultures with therandomly amplified polymorphic DNA technique revealedhigh levels of genomic DNA polymorphism. The highestsimilarity between 2 strains (0.653) was observedbetween 2-picoline and 3-picoline degrading cultures. The molecular basis of the differences in substratespecificity is under investigation.     

Levels of aflatoxin B1, bacteria and fungi in feed and food in 1971 — 1987

Totally 39% out of 8371 feed and their component samples were contaminated by aflatoxin B₁. Mean contamination was 36μg/kg with maximum yield 10100 μg/kg. Contamination of samples by total count of organisms, mean contamination and maximum yield, respectively was: 1) bacteria 99%, 2.2×10⁶, 2.4×10⁸; 2) proteolytic bacteria 94%, 1.2×10⁵, 3.0×10⁶;3) moulds 98%, 1.3×10⁵, 9.0×10⁶; 4) yeasts 44 %, 3.3×10⁴, 3.6×10⁶. The samples were contaminated in 92 % byAspergillus spp, in 71% byAspergillus flavus, in 83% byPenicillium spp, and in 20% byFusarium spp with mean contamination 8.3×104, 1.1×10³, 4.2×10⁴, 5.0×10³ , and maximum yield 6.8×10⁶, 1.0×10⁵, 5.0×10⁶, 1.5×10⁶, respectively. Totally 8.5% of strains were aflatoxinogenic and 4.4% of the strains were isolated from feed and 21 % of the strains from grain/nut.     

Loss of the Protease Dimerization Inhibition Activity of Tipranavir (TPV) and Its Association with the Acquisition of Resistance to TPV by HIV-1

Tipranavir (TPV), a protease inhibitor (PI) inhibiting the enzymatic activity and dimerization of HIV-1 protease, exerts potent activity against multi-PI-resistant HIV-1 isolates. When a mixture of 11 multi-PI-resistant (but TPV-sensitive) clinical isolates (HIV_11MIX), which included HIV_B and HIV_C, was selected against TPV, HIV_11MIX rapidly (by 10 passages [HIV_11MIX^P10]) acquired high-level TPV resistance and replicated at high concentrations of TPV. HIV_11MIX^P10 contained various amino acid substitutions, including I54V and V82T. The intermolecular FRET-based HIV-1 expression assay revealed that TPV''s dimerization inhibition activity against cloned HIV_B (cHIV_B) was substantially compromised. The introduction of I54V/V82T into wild-type cHIV_NL4-3 (cHIV_{NL4-3^I54V/V82T}) did not block TPV''s dimerization inhibition or confer TPV resistance. However, the introduction of I54V/V82T into cHIV_B (cHIV_B^I54V/V82T) compromised TPV''s dimerization inhibition and cHIV_B^I54V/V82T proved to be significantly TPV resistant. L24M was responsible for TPV resistance with the cHIV_C genetic background. The introduction of L24M into cHIV_NL4-3 (cHIV_NL4-3^L24M) interfered with TPV''s dimerization inhibition, while L24M increased HIV-1''s susceptibility to TPV with the HIV_NL4-3 genetic background. When selected with TPV, cHIV_{NL4-3^I54V/V82T} most readily developed TPV resistance and acquired E34D, which compromised TPV''s dimerization inhibition with the HIV_NL4-3 genetic background. The present data demonstrate that certain amino acid substitutions compromise TPV''s dimerization inhibition and confer TPV resistance, although the loss of TPV''s dimerization inhibition is not always associated with significantly increased TPV resistance. The findings that TPV''s dimerization inhibition is compromised with one or two amino acid substitutions may explain at least in part why the genetic barrier of TPV against HIV-1''s development of TPV resistance is relatively low compared to that of darunavir.     

Paracoccus pacificus sp. nov., isolated from the Western Pacific Ocean

A Gram-stain negative, short rod-shaped, non-motile, catalase- and oxidase-positive, aerobic bacterium, designated F14^T, was isolated from the Western Pacific Ocean. Phylogenetic and phenotypic properties of the organism supported that it belongs to the genus Paracoccus. The levels of 16S rRNA gene sequences similarity between strain F14^T and other type strains of recognized members of the genus Paracoccus were 93.6–96.5 %. Growth of strain F14^T was observed at 4–40 °C (optimum, 28–30 °C), pH 6.0–10.0 (optimum, pH 7.0–8.0) and in the presence of 0–7 % (w/v) NaCl (optimum, 1–2 %). The major cellular fatty acid was summed feature 8 (C_18:1 ω6c and/or C_18:1 ω7c). The major respiratory quinone was ubiquinone-10. The polar lipid pattern indicated the presence of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and three unknown lipids. The DNA G+C content was 61.4 mol%. On the basis of polyphasic characterization, strain F14^T represents a novel species, for which the name Paracoccus pacificus sp. nov. is proposed. The type strain is F14^T (=CGMCC 1.12755^T=LMG 28106^T=MCCC 1A09947^T).     

Autoradiographische Untersuchungen über den Modus der Proliferation und Regeneration des Samenepithels der Wistarratte

Zusammenfassung Um den Modus der Proliferation und Regeneration des Samenepithels bei der Winterratte zu finden, wurden die Generationszeiten und Teilphasen der Spermatogonienarten A₁, A₂, A₃, A₄, Im und B mit der Methode der markierten Mitosen gemessen. Nach einmaliger Injektion von H-3-Thymidin wurden 48 Wistarratten zwischen 2 Std und 14 Tagen getötet. Auf Autoradiogrammen wurde dann der Prozentsatz markierter Mitosen der einzelnen Spermatogonienarten bestimmt.Insgesamt wurden für die einzelnen Spermatogonienarten über 30 Maxima markierter Mitosen gefunden (Abb. 2). Aus der Lage der ersten beiden Maxima post inj. ergibt sich für die A₁-Spermatogonien eine Generationszeit von 5,9 Tagen. Die übrigen Spermatogonien (A₂, A₃, A₄, Im, B) haben fast gleiche Generationszeiten von 39–42 Std (Tabelle 1).Unter der Voraussetzung, daß sich die A₁-Spg von den Im-Spg (oder A₅-Spg) ableiten, kann die Cyclusdauer aus der Summe der Generationszeiten der Spermatogonienarten A₂+A₃+A₄+Im (oder A₅)+A₁ = 12,8 Tage erklärt werden. Dieser Wert stimmt ausgezeichnet mit Werten überein, die bei früheren Untersuchungen mit anderen Methoden gefunden wurden.Es wird auf einige Beobachtungen eingegangen, die mit diesem einfachen Regenerationsschema nicht übereinstimmen. Es wird dann im einzelnen diskutiert, wie diese Widersprüche durch eine Erweiterung des Regenerationsschemas evtl. beseitigt werden könnten. In diesem Zusammenhang wird unter anderem versuchsweise die Annahme gemacht, daß die Bildung von A₁-Spermatogonien von allen A-Spermatogonien ausgehen kann.

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On proliferation and regeneration of the seminiferous epithelium in the Wistar rat. (Autoradiographical study)

Summary To find the mode of proliferation and regeneration of the seminiferous epithelium of the Wistar rat, the life spans and subphases of the spermatogonial types A₁, A₂, A₃, A₄, Im, and B were determined by the method of labeled mitoses. After a single injection of H-3-thymidine 48 rats were sacrificed between 2 hours and 14 days. By means of autoradiographs the percentage of labeled mitoses was determined.More than 30 maxima of labeled mitoses were found (Fig. 2). The position of the first two maxima after injection gives the life span of A₁-spermatogonia as 5.9 days. The other spermatogonial types have almost identical life spans of 39–42 hoursUnder the assumption that the A₁-spermatogonia are the descendents of Im-spermatogonia (or A₅-spermatogonia), the duration of the cycle of the seminiferous epithelium can be explained by the addition of the life spans of the spermatogonial types A₂+A₃+A₄+Im (or A₅ alternatively)+A₁ as 12.8 days. This value is in good agreement with the values of the duration of the cycle determined by earlier investigations.Some observations are discussed which do not correspond with this simple scheme of regeneration. An extension of the regeneration scheme has been proposed to resolve these differences. In this context the hypothesis is made that from all types of A-spermatogonia can rise A₁-spermatogonia.

     

Lithoautotrophic growth of sulfate-reducing bacteria,and description of Desulfobacterium autotrophicum gen. nov., sp. nov.

The capacity of mesophilic sulfate-reducing bacteria to grow lithoautotrophically with H₂, sulfate and CO₂ was investigated with enrichment cultures and isolated species. (a) Enrichments in liquid mineral media with H₂, sulfate and CO₂ consistently yielded mixed cultures of nonautotrophic, acetate-requiring Desulfovibrio species and autotrophic, acetate-producing Acetobacterium species (cell ratio approx. 20:1). (b) By direct dilution of mud samples in agar, various non-sporing sulfate reducers were isolated in pure cultures that did grow autotrophically. Two oval cell types (strains HRM2, HRM4) and one curved cell type (strain HRM6) from marine sediment were studied in detail. The strains grew in mineral medium supplemented only with vitamins (biotin, p-aminobenzoate, nicotinate). Carbon autotrophy was evident (i) from comparative growth experiments with non-autotrophic, acetate-requiring species, (ii) from high cell densities ruling out a cell synthesis from organic impurities in the mineral media, and (iii) by demonstrating that 96–99% of the cell carbon was derived from ¹⁴C-labelled CO₂. Autotrophic growth occurred with a doubling time of 16–20 h at 24–28°C. Formate, fatty acids up to palmitate, ethanol, lactate, succinate, fumarate, malate and other organic acids were also used and completely oxidized. The three strains possessed cytochromes of the b-and c-type, but no desulfoviridin. Strain HRM2 is described as a new species of a new genus, Desulfobacterium autotrophicum. (c) The capacity for autotrophic growth was also tested with sulfate-reducing bacteria that originally had been isolated on organic substrates. The incompletely oxidizing, non-sporing types such as Desulfovibrio and Desulfobulbus species and Desulfomonas pigra were confirmed to be obligate heterotrophs that required acetate for growth with H₂ and sulfate. In contrast, several of the completely oxidizing sulfate reducers were facultative autotrophs, such as Desulfosarcina variabilis, Desulfonema limicola, Desulfococcus niacini, and the newly isolated Desulfobacterium vacuolatum and Desulfobacter hydrogenophilus. The only incompletely oxidizing sulfate reducer that could grow autotrophically was the sporing Desulfotomaculum orientis, which obtained 96% of its cell carbon from ¹⁴C-labelled CO₂. Desulfovibrio baarsii and Desulfococcus multivorans may also be regarded as types of facultative autotrophs; they could not oxidize H₂, but grew on sulfate with formate as the only organic substrate.     

Single nucleotide polymorphism (SNP) in growth hormone gene of Jakhrana,a prominent milk goat breed in India

PCR-SSCP patterns on non-degenerative PAGE revealed 6 amplicons in caprine GH exon-4 and 3 alleles A₄, B₄ and C₄ were identified. In exon-5, six SSCP variants revealed three alleles A₅, B₅, and C₅. Out of 54 AA sites of GH-4 coding region, six codons were polymorphic. At codon-6, nucleotide substitution of G/A resulted in to genotypes 6_RR, 6_HH and G/C into genotypes 6_PP, 6_RP. At codon-36, A/G nucleotide substitution resulted in to newer genotypes 36_GG from that of 36_DD in reference Genbank sample. At codon-54, C/T nucleotide substitution caused change of amino acid (AA) from arginine (R) to tryptophan (W) resulted into a new genotype of 54_WW in comparison to 54_RR of Genbank reference sample. In exon-5, out of 67 AA sites 8 codons were polymorphic, but the codons 14 and 60 were preponderant. At codon-14, A/G substitution resulted into 3 genotypes 14_KK, 14_EE and 14_KE with frequency of 0.52, 0.38 and 0.10, respectively. At codon-60, G/C and G/A substitutions resulted in to 3 genotypes 60_GG, 60_RR and 60_GR with frequencies of 0.48, 0.42 and 0.10, respectively. Synonymous mutations as compared to Genbank accession D00476.1 were present at codons 25, 31 and 62 in all the animals of Jakhrana goats. The high genetic variability in GH gene exon-4 and exon-5 may be useful in exploring their associations with milk and growth traits in goat for further genetic improvement.     

Genetic association of a GABAA receptor γ2 subunit variant with severity of acute physiological dependence on alcohol

The ultimate goal of quantitative trait locus (QTL) mapping is to identify the genes affecting complex traits. Using animal models, we recently identified QTLs on mouse Chromosomes (Chrs) 1, 4, and 11 affecting genetic predisposition to acute alcohol withdrawal. Among mice derived from the C57BL/6J (B6) and DBA/2J (D2) inbred strains, the locus identified on Chr 11 (∼20 cM) accounted for 12% of the genetic variability in withdrawal liability. Candidate genes in proximity to this QTL encode the γ₂, α₁, and α₆ subunits of GABA_A receptors. The present studies identify a polymorphism between the B6 and D2 strains in the γ₂ subunit gene, Gabrg2, and expand genotypic analysis to their BXD recombinant inbred strains. This polymorphism predicts a difference in amino acid sequence (Ala-11 vs. Thr-11) within the extracellular amino-terminal region of the γ₂ subunit. Analysis using BXD strain means for acute alcohol withdrawal severity suggests that the γ₂ subunit polymorphism is genetically correlated with alcohol withdrawal severity. This is the first report that QTL mapping for an alcohol-related trait has successfully led to the identification of a polymorphic candidate gene product that is genetically associated with the trait. Received: 15 September 1998 / Accepted: 8 October 1998     

Cytological studies on mixoploidy and embryonic development in mosaic silkworms induced by low temperature treatment

Mosaicism in silkworms is easily inducible by applying a cold shock to the hybrid eggs between two strains with different markers at the early developmental stage. When BF₁ eggs from the cross between a tetraploid female (which had been induced from the cross between a red egg and a white egg strain) and a male of the red egg strain were exposed to a cold shock, hexaploid eggs and two types of mosaic eggs were obtained: black-black (BB-) mosaic eggs, which possessed two kinds of black serosa cells with different-sized nuclei, and black-red (BR-) mosaic eggs. Cytological studies on the embryos from these mosaic eggs revealed that the BB-mosaic type was a 3n/6n mixoploid, while the BR-mosaic type was mixoploid with n/2n/3n, n/3n, or 2n/6n constitution. The hatchability of BB- and BR-mosaic eggs was considerably lower than that of either n/2n or 2n/4n mixoploid eggs obtained by a cold treatment of diploid eggs. Since abnormal development was observed at a high frequency in the embryos from BB- and BR-mosaic eggs at the blastokinesis stage, it is speculated that the coexistence of hexaploid or triploid cells may be a factor in the induction of such abnormality in early embryogenesis.