Electron microscopical studies of membrane injuries in blue fox spermatozoa subjected to the process of freezing and thawing

Disintegration of blue fox sperm membranes is studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In unfrozen spermatozoa studied by SEM, the plasmalemma and the acrosome appeared to be intact, except for a few cases of disruption of the former structure at the anterior part of the head. In semen frozen in 0.5-ml plastic straws by use of N2 vapor after dilution with Tris-fructose-citric acid with 8 vol % glycerol and 20 vol % egg yolk and thawed at 70 degrees C for 8 sec, the spermatozoa displayed different degrees of membrane damage. These alterations could be classified into three main categories of which the first included only minor changes in the plasmalemma, but vesiculation and disintegration of the outer part of the acrosomal membrane. In the second category (also the most frequent one) the outer part of the acrosomal membrane was extensively vesiculated, and the plasmalemma was discharged proximal to the equatorial segment. Extensive loss of plasmalemma and complete absence of the outer part of the acrosomal membrane characterized the last category of membrane damage. The functional implications of the three categories of membrane alterations are discussed.     

Determination of the enantiomeric composition of (R*,S*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate, the male-produced aggregation pheromone of Sitophilus granarius

The enantiomeric composition of sitophilate, the granary weevil [Sitophilus granarius (L.)] male-produced aggregation pheromone [(R^*,S^*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate)], was determined by three methods: (1) bioassaying the synthetic (2S,3R) and (2R,3S) enantiomers of the active (R^*,S^*) diastereomer; (2) ¹H NMR spectroscopy of Mosher ester derivatives of the natural pheromone and synthetic (2S,3R)-and (2R,3S)-sitophilate; and (3) capillary GLC comparisons of the retention times of derivatized natural pheromone and the two synthetic enantiomers. The combined methods confirmed the (2S,3R) enantiomer as the active form of sitophilate. Male granary weevils were shown to produce >96% (2S,3R)-sitophilate. No significant attraction of S. granarius by the (2R,3S) enantiomer was observed. Rice and maize weevils [S. oryzae (L.) and S. zeamais Motschulsky] were not attracted by (2S,3R)-sitophilate. S. granarius L. est un déprédateur important des grains stockés. Le (R^*,S^*)-1-éthylpropyl 2-méthyl-3-hydroxypentanoate a été identifié en 1987 comme le principal composé du sitophilate, la phéromone mâle d'agrégation de S. granarius. La composition énantiométrique du sitophilate a été déterminée par 3 méthodes:

New motile anaerobic bacteria growing by succinate decarboxylation to propionate

Three strains of new anaerobic, gram-negative bacteria which grew with succinate as sole source of carbon and energy were isolated from anoxic marine and freshwater mud samples. Cells of the three strains were small, non-spore-forming, motile rods or spirilla. The guanine-plus-cytosine content of the DNA of strain US2 was 52.6±1.0 mol%, of strain Ft2 63.5±1.4 mol%, and of strain Ft1 62.6±1.0 mol%. Succinate was fermented stoichiometrically to propionate and carbon dioxide. The growth yields were 1.2–2.6 g dry cell mass per mol succinate degraded. Strains US2 and Ft2 required 0.05% w/v yeast extract in addition to succinate for reproducible growth. Optimal growth occurred at 30°–37°C and pH 6.8–8.0. Addition of acetate as cosubstrate did not stimulate growth with any strain. Strain Ft2 grew only under strictly anaerobic conditions, whereas strains US2 and Ft1 tolerated oxygen up to 20% in the headspace. Strains US2 and Ft2 grew only with succinate. Strain Ft1 also converted fumarate, aspartate, and sugars to propionate and acetate. This strain also oxidized propionate with nitrate to acetate. Very low amounts of a c-type cytochrome were detected in propionate plus nitrate- or glucose-grown cells of this strain (0.4 g x g protein^-1). Moderate activities of avidin-sensitive methylmalonyl-CoA decarboxylase were found in cell-free extracts of all strains.     

Metal-dependent binding of a nuclear factor to the rat metallothionein-I promoter.

Genomic footprinting studies in vivo and experiments using synthetic metal regulatory elements (MREs) in vitro suggest protein binding to the MREs of the mouse and rat metallothionein I (MT-I) genes. Using gel-retardation assays of promoter fragments, we observe a cadmium-dependent binding factor for the rat MT-I promoter in rat hepatoma cells. This factor is present in extracts from both uninduced and cadmium-induced cells, but requires the presence of cadmium to bind to the promoter. The formation of a cadmium-dependent complex is competed by an oligonucleotide containing two MREs. This competition is lost when when one of the MREs is mutated, indicating a requirement for at least two MREs for binding of this factor. The cadmium-dependent factor dissociates more rapidly from the MT-I promoter than does a factor that binds to a consensus Sp1 site present on the same DNA fragment. UV crosslinking analysis using nuclear extracts from cadmium induced cells, in the presence of an oligonucleotide probe containing both 5-bromodeoxyuridine and 32P-deoxycytidine, identifies a 39 kDalton protein associated with the metal inducible complex.     

Mechanism of activation of 1,2-dehydro-N-acetyldopamine for cuticular sclerotization

The mechanism of oxidation of 1,2-dehydro-N-acetyldopamine (dehydro NADA) was examined to resolve the controversy between our group and Andersen's group regarding the reactive species involved in β-sclerotization. While Andersen has indicated that dehydro NADA quinone is the β-sclerotizing agent [Andersen, 1989], we have proposed quinone methides as the reactive species for this process [Sugumaran, 1987; Sugumaran, 1988]. Since dehydro NADA quinone has not been isolated or identified till to date, we studied the enzymatic oxidation of dehydro NADA in the presence of quinone traps to characterize this intermediate. Accordingly, both N-acetylcysteine and o-phenylenediamine readily trapped the transiently formed dehydro NADA quinone as quinone adducts. Interestingly, when the enzymatic oxidation was performed in the presence of o-aminophenol or different catechols, adduct formation between the dehydro NADA side chain and the additives had occurred. The structure of the adducts is in conformity with the generation and reactions of dehydro NADA quinone methide (or its radical). This, coupled with the fact that 4-hydroxyl or amino-substituted quinones instantly transformed into p-quinonoid structure, indicates that dehydro NADA quinone is only a transient intermediate and that it is the dehydro NADA quinone methide that is the thermodynamically stable product. However, since this compound is chemically more reactive due to the presence of both quinone methide and acylimine structure on it, the two side chain carbon atoms are “activated.” Based on these considerations, it is suggested that the quinone methide derived from dehydro NADA is the reactive species responsible for cross-link formation between dehydro NADA and cuticular components during β-sclerotization.     

Human metallothionein genes: structure of the functional locus at 16q13

The functional human metallothionein (MT) genes are located on chromosome 16q13. We have physically mapped the functional human MT locus by isolation and restriction digest mapping of cloned DNA. The mapped region contains all sequences on chromosome 16 that hybridize to metallothionein gene probes and comprises 14 tightly linked MT genes, 6 of which have not been previously described. This analysis defines the genetic limits of metallothionein functional diversity in the human genome.     

Nerve growth factor and neuronal cell death

The regulation of neuronal cell death by the neuronotrophic factor, nerve growth factor (NGF), has been described during neural development and following injury to the nervous system. Also, reduced NGF activity has been reported for the aged NGF-responsive neurons of the sympathetic nervous system and cholinergic regions of the central nervous system (CNS) in aged rodents and man. Although there is some knowledge of the molecular structure of the NGF and its receptor, less is known as to the mechanism of action of NGF. Here, a possible role for NGF in the regulation of oxidant--antioxidant balance is discussed as part of a molecular explanation for the known effects of NGF on neuronal survival during development, after injury, and in the aged CNS.     

Localized insertion of new S-layer during growth of Bacillus stearothermophilus strains

Bacillus stearothermophilus strains PV 72 and ATCC 12980 carry a crystalline surface layer (S-layer) with hexagonal (p6) and oblique (p2) symmetry, respectively. Sites of insertions of new subunits into the regular lattice during cell growth have been determined by the indirect fluorescent antibody technique and the protein A/colloidal gold technique.During S-layer growth on both bacillus strains the following common features were noted: 1. shedding of intact S-layer or turnover of individual subunits was not seen; 2. new S-layer was deposited in helically-arranged bands over the cylindrical surface of the cell at a pitch angle related to the orientation of the lattice vectors of the crystalline array; 3. little or no S-layer was inserted into pre-existing S-layer at the poles, and 4. septal regions and, subsequently, newly formed cell poles were covered with new S-layer protein.