Influence of Methanogenic Populations in Holocene Lacustrine Sediments Revealed by Clone Libraries and Fatty Acid Biogeochemistry

Methanogenic populations were investigated in subsaline Laguna Potrok Aike sediments, southern Argentina. Microbial density and activity were assessed via cell count and in situ ATP detection for the last ～11K years. Methanogen phylogenetics highlighted species stratification throughout depth, whereas CO₂ reduction was the major pathway leading to methane production. Organic substrates, characterized using pore water analysis, bulk organic fractions and saturated fatty acids, showed a clear link between sediment colonization and initial organic sources. Concentrations and δ¹³C compositions of methane and fatty acids provided final evidence of a microbial imprint on Holocene organic proxies in the most colonized intervals.     

Capacitation of bovine sperm by heparin: inhibitory effect of glucose and role of intracellular pH

Bovine sperm incubated with heparin for 7.5-8.5 h underwent an acrosome reaction in the absence but not the presence of glucose (5 mM). When sperm were incubated under capacitating conditions with heparin for 4 h, glucose inhibited sperm penetration of oocytes (p less than 0.01) and lysophosphatidylcholine (LC) induced acrosome reactions. Addition of glucose for the last 0.25 h of a 4.25-h incubation with heparin had no effect on ability of sperm to acrosome-react in response to LC. Nonmetabolizable sugars 3-O-methyl glucose, 2-deoxyglucose, sucrose, and sorbitol did not inhibit capacitation as judged by sperm sensitivity to LC or fertilization (p greater than 0.05), but capacitation was inhibited by the glycolyzable substrates glucose, mannose, and fructose (p less than 0.05). The glycolytic inhibitor, fluoride, reversed glucose inhibition of capacitation in a dose-dependent manner similar to its effect on glucose uptake by sperm. Extracellular pH declined from 7.4 to 7.2 during a 4-h incubation of sperm with heparin and glucose. The decline of extracellular pH during sperm incubation with glucose did not affect capacitation, since only an extracellular pH below 7.02 inhibited capacitation. The intracellular pH (pHi) of sperm increased 0.40 units over a 5-h incubation under capacitating conditions. The change in pHi was inhibited by glucose. Incubation of sperm with heparin and glucose for 12 h resulted in capacitated sperm as judged by both LC sensitivity and fertilizing ability. These studies demonstrate that glycolyzable substrates delay capacitation of bovine sperm and suggest the effect is in delaying an alkalinization of pHi.     

Isolation of heparin-binding growth factors from bovine, porcine and canine hearts

Fresh bovine, porcine and canine hearts were homogenized and mitogens for mesoderm-derived cells were purified in three different steps. Extraction by two different ammonium sulfate precipitations was followed by cation-exchange chromatography and by heparin-Sepharose affinity chromatography. A heparin-Sepharose fraction from heart (eluted at 1.1 M NaCl) increased mitotic activity in serum-deprived cultures of porcine aortic endothelial and smooth muscle cells, and in human fibroblasts. This mitogenic activity is potentiated by heparin and inhibited by gamma-interferon. The heart mitogenic fraction showed one double peak on HPLC at A215 and one polypeptide band on SDS/PAGE. These peaks and bands were identical to those obtained from bovine brain. The heart acidic fibroblast growth factor (aFGF) showed a positive signal in Western blots using antibodies raised against brain aFGF. Gas-phase amino acid sequencing established that the mitogens were identical to aFGF and the N-terminally truncated aFGF. Extraction in the presence of a protease inhibitor (pepstatin A) produced a higher-molecular mass form of aFGF with a blocked amino terminus. Another mitogen, eluted at 1.6 M NaCl from heparin-Sepharose, reacted with polyclonal antiserum against human recombinant basic fibroblast growth factor (bFGF) and showed a 66% (12 from 18 amino acids determined by gas-phase sequencing) similarity with bFGF. This polypeptide increased the mitotic activity of the same cell lines but was more potent than aFGF.     

Fluorescence energy transfer between cysteine 199 and cysteine 343: evidence for MgATP-dependent conformational change in the catalytic subunit of cAMP-dependent protein kinase

The catalytic subunit of cAMP-dependent protein kinase has two cysteine residues, Cys 199 and Cys 343, which are protected against alkylation by MgATP [Nelson, N. C., & Taylor, S. S. (1981) J. Biol. Chem. 256, 3743]. While Cys 199 is in close proximity to the active site of the catalytic subunit and is probably directly protected against alkylation by MgATP, the mechanism by which MgATP prevents alkylation of Cys 343 is unclear. To determine whether MgATP directly protects Cys 343 from alkylation by being in close proximity to both Cys 199 and the MgATP binding site, fluorescence resonance energy transfer techniques were used to measure the distance between Cys 199 and Cys 343. Two different donor-acceptor pairs containing 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole at Cys 199 as the acceptor and either 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2, 8- dione or N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine at Cys 343 as the donor were prepared following the method described in the preceding paper [First, E. A., & Taylor, S. S. (1989) Biochemistry (preceding paper in this issue)]. From the efficiencies of fluorescence resonance energy transfer for each donor-acceptor pair, the distance between Cys 199 and Cys 343 was estimated to be between 31 and 52 A. Since Cys 199 is close to the MgATP binding site and since MgATP cannot extend beyond a distance of 16 A, it is unlikely that Cys 343 at a distance of at least 31 A from Cys 199 is in direct contact with the bound nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective modification of the catalytic subunit of cAMP-dependent protein kinase with sulfhydryl-specific fluorescent probes

The catalytic subunit of cAMP-dependent protein kinase contains only two cysteine residues, and the side chains of both Cys 199 and Cys 343 are accessible. Modification of the catalytic subunit by a variety of sulfhydryl-specific reagents leads to the loss of enzymatic activity. The differential reactivity of the two sulfhydryl groups at pH 6.5 has been utilized to selectively modify each cysteine with the following fluorescent probes: 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene- 2,8-dione, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, and 4-[N-[(iodoacetoxy)ethyl]-N-methyl-amino]-7-nitrobenz-2-oxa-1,3-diazole. The most reactive cysteine is Cys 199, and exclusive modification of this residue was achieved with each reagent at pH 6.5. Modification of Cys 343 required reversible blocking of Cys 199 with 5,5'-dithiobis(2-nitrobenzoic acid) followed by reaction of Cys 343 with the fluorescent probe at pH 8.3. Treatment of this modified catalytic subunit with reducing reagent restored catalytic activity by unblocking Cys 199. In contrast, catalytic subunit that was selectively labeled at Cys 199 by the fluorescent probes was catalytically inactive. Even though Cys 199 is presumably close to the interaction site between the regulatory subunit and the catalytic subunit, all of the modified C-subunits retained the capacity to aggregate with the type II regulatory subunit in the absence of cAMP, and the resulting holoenzymes were dissociated in the presence of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chlorierte Kohlenwasserstoffe in Brutv?geln aus dem Binnenland Niedersachsens

Zusammenfassung Rückstände chlorierter Kohlenwasserstoffe in Eiern und Lebern von im Binnenland Niedersachsens brütenden Vogelarten — Feldsperling, Mehlschwalbe, Weißstorch, Graureiher, Saatkrähe, Stockente und andere Arten — werden angegeben und deren Abhängigkeit von Brutort, Nahrung und Zugverhalten diskutiert.

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Chlorinated hydrocarbons of some bird species breeding in the inland of Lower Saxony (FRG)

Summary Residues of chlorinated hydrocarbons in eggs and livers of some bird species — Tree Sparrow, House Martin, White Stork, Heron, Rook, Mallard, and further species — are presented. The dependence on place of breeding, food web, and migration is discussed.

     

Acrosome reaction of bovine spermatozoa in vivo: sites and effects of stages of the estrous cycle

Thirty-two cows were inseminated near the uterotubal junction at various stages of the estrous cycle and slaughtered 16 h later to determine the effects of stage of the estrous cycle and tubal site of sperm recovery on the frequency of acrosome-reacted bull spermatozoa. Slaughter times were 46, 70, 144, or 168 h after each cow was injected with prostaglandin (PG) F 2 alpha or during the luteal phase of the estrous cycle. Sperm were recovered from the upper uterus and the isthmus and ampulla of the oviducts and stained for both viability and acrosome reaction. The highest frequency of acrosome-reacted sperm was found in the ampulla ipsilateral to a dominant follicle (largest follicle present) or recent ovulation and primarily at 70 h after PGF2 alpha (P less than 0.05). Also, fewer sperm were acrosome reacted prior to (46 h post-PGF2 alpha) and well after (168 h post-PGF2 alpha) estrus than during or immediately postestrus (70, 90, and 144 h post-PGF2 alpha; P less than 0.05). Except for two cows, one at 46 h and one at 70 h, all cows with more than 50% acrosome-reacted sperm in the ampulla had ovulated before slaughter. These data suggest that capacitated sperm become localized in the ampulla of the oviduct of the ovulatory side around the time of ovulation.