Conformational transition between native and reactive center cleaved forms of alpha 1-antitrypsin by Fourier transform infrared spectroscopy and small-angle neutron scattering

alpha 1-Antitrypsin (alpha 1-AT) is the best-characterized member of the serpin superfamily of plasma proteins. Protease inhibitor members of this family undergo a characteristic reactive-center cleavage during expression of their inhibitory activity. The physical basis of this transition in alpha 1-AT from the stressed native conformation to the more stable reactive center cleaved (split) form was studied by Fourier transform infrared (FT-IR) spectroscopy and neutron scattering. The FT-IR spectra show that, while split alpha 1-AT has three intense well-resolved components associated with the presence of antiparallel beta-sheet and alpha-helix conformations, the amide I band of native alpha 1-AT has only one intense component, associated with the presence of beta-sheet structure. 1H-2H exchange within the polypeptide backbone, studied by FT-IR and NMR spectroscopy, shows that the native form undergoes greater exchange than the split form. Under the same conditions, neutron scattering shows no differences in the radius of gyration RG of the native and the split forms. In contrast, in high concentrations of phosphate approaching those used for crystallization, the native form (unlike the split form) undergoes dimerization. These data indicate that the conformational transition largely involves localized secondary and tertiary structure rearrangements. We propose that the energetically stressed native alpha 1-AT structure is the consequence of a significantly reduced number of hydrogen bonds in secondary structure components and that reactive-site cleavage between Met358 and Ser359 is the key for the development of the fully hydrogen bonded more stable serpin structure.     

Synthesis and degradation of fatty acid ethyl esters by cultured hepatoma cells exposed to ethanol

Fatty acid ethyl esters are a family of neutral lipids that are the products of esterification of fatty acids with ethanol. Unlike other pathways of ethanol metabolism, ethyl esters are present in numerous human organs which are the targets of ethanol-induced damage. In the present study, we have shown that fatty acid ethyl esters are synthesized by a hepatoma cell line in tissue culture when exposed to ethanol concentrations easily attained by man during social drinking. Unlike alcohol dehydrogenase, the enzyme(s) responsible for synthesis of ethyl esters are membrane-bound and concentrated in the microsomal fraction of rat hepatocytes. In addition, fatty acid ethyl esters are hydrolyzed to free fatty acids and ethanol by membrane-bound enzyme(s) that are enriched in the microsomal and mitochondrial-lysosomal fractions. Intracellular hydrolysis of fatty acid ethyl esters release free fatty acids which are preferentially incorporated into cellular cholesterol esters. Thus, we have shown that a hepatocellular line exposed to concentrations of ethanol easily achieved in man by social drinking utilize endogenous fatty acids to form long-lived ethanol metabolites, fatty acid ethyl esters. Importantly, this family of neutral lipids may act as biochemical mediators of ethanol-induced cell damage, including the changes in cholesterol metabolism noted in chronic alcoholics.     

Habitat assortment of sexes and water balance in a dioecious grass

Summary Eggs of the carabid beetle Pterostichus oblongopunctatus kept under optimum conditions in petri dishes had a mean mortality of 8% (n=200), while the mortality of eggs incubated in fresh litter was 83% (n=250). Heating the litter up to 70° C and thus eliminating other organisms from the litter fauna reduced mortality to 18% (n=250). When eggs were deposited by the females themselves in a perspex cylinder containing an undisturbed soil sample, the mean egg mortality was 70% (n235), but when predators had been eliminated the mortality was only 7% (n235). The assumption of Grüm (1975) that egg mortality can be neglected does not hold for P. oblongopunctatus. The predators of the eggs must be common species, but it is not yet known to which family they belong.     

Persistence of predator-prey systems in an uncertain environment

Summary The time derivatives of prey and predator populations are assumed to satisfy a set of inequalities, instead of a precise differential equation, reflecting an uncertain environmental and/or lack of knowledge by the modeler. A system of differential equations is found whose solution gives the boundary of a persistent set, which is positive flow invariant for any system satisfying the inequalities. Conditions are given for the persistent set to be bounded away from both axes, which show that resonance effects cannot drive either predator or prey to extinction if that does not happen for an autonomous system satisfying the inequalities. In general predator-prey systems are more persistent when there is strong asymptotic stability, when there is correlation between prey and predator dynamics, when the effect of perturbations is density dependent, and are more persistent under perturbations of the prey than of the predator.     

Scanning electron microscopy of taxonomic diagnostic criteria of the freshwater sponge,Heteromeyenia tubisperma (Potts, 1881) (Porifera : Spongillidae)

Taxonomic diagnostic criteria of the spongillid freshwater sponge, Heteromeyenia tubisperma (Potts, 1881) were examined using scanning electron microscopy. The species is characterized by a gemmule which bears an unusually long, prominent porous tube. The application of SEM to the systematic studies of the freshwater sponges provides diagnostic capabilities not available with the light microscope. It is desirable that a key, coupled with a reference atlas of scanning electron micrographs illustrating taxonomic diagnostic criteria of freshwater sponge species, particularly utilizing type specimens, be developed.     

Serological and chemical interrelationship of antigens from Leptospira interrogans serovar canicola

Antigens of the outer envelope from Leptospira interrogans serovar canicola (Hond Utrecht IV) were extracted by 50% (v/v) ethanol or by sodium dodecyl sulphate and serological analysis suggested that they were identical. The "fraction 4" extracted by alkali was found to contain glycoproteins of high (retentate) and low (filtrate) molecular weight; the latter behaved like a hapten in serology and in animal immunization experiments. Antibodies were raised in rabbits against this hapten by conjugating it to bovine albumin fraction V. The antiserum was found to react with both the low molecular weight and high molecular weight glycoproteins. This anti-hapten serum contained little or no whole-cell-agglutinating antibodies. The fraction 4 retentate behaved like a complete antigen in serological and immunization studies. Fraction 4 retentate and the outer envelope preparations were serologically related but they were not identical. Chemical studies revealed similarities between the carbohydrate component of the outer envelope obtained by ethanol extraction and fraction 4. The outer envelope extracted by ethanol, fraction 4 and its low and high molecular weight glycoproteins contained arabinose, rhamnose, fucose, xylose, mannose, galactose, glucose, glucosamine and glucuronic acid. Three unidentified peaks were observed in gas-liquid chromatographic analysis of the O-trimethylsilyl derivatives of methyl glycosides of all these samples and one of these peaks co-eluted with the O-trimethylsilyl derivative of 3-O-methylmannose.     

Corticosterone binding in AtT-20 pituitary tumor cell cytosol: evidence for one class of binding site for both natural and synthetic glucocorticoids.

The AtT-20 mouse pituitary cell is an established, cloned cell line which produced adrenocorticotrophic hormone in a glucocorticoid-suppressible manner. A receptor for glucocorticoids was identified in cytosol prepared from these cells using the natural mouse glucocorticoid, corticosterone, as the labeled ligand. The question of whether this binding component is identical to the one detectable using labeled triamcinolone acetonide was addressed by comparing their physicochemical characteristics and by detailed studied of binding specificity using both ligands. The corticosterone and triamcinolone acetonide binding components behaved similarly on sucrose density gradient analysis and DEAE-cellulose ion-exchange chromatography. Scatchard analysis with corticosterone detected 30% fewer binding sites than a similar analysis with triamcinolone acetonide, probably because corticosterone binding was of lower affinity (Kd = 8.6 . 10(-9)M vs. 1.4 . 10(-9)M) and hence less stable. The relative glucocorticoid binding affinities of thirteen unlabeled steroids were obtained using either labeled steroid as ligand. Both ligands yielded similar results, suggesting that they both detected a similar binding site. The results suggest that AtT-20 cell cytosol contains a single class of binding site which detects both natural and synthetic glucocorticoids.     

The taxonomic and ecological status of the environmentally restricted spongillid species of North America. IV. Spongilla heterosclerifera Smith 1918

The taxonomic status, present distribution and specific threats to the existence of the environmentally restricted freshwater sponge, Spongilla heterosclerifera Smith 1918 were investigated. This species, collected only from Oneida Lake, New York, has not colonized other habitats and continues to exhibit typical diagnostic characteristics, thus qualifying as a valid environmentally restricted species. Although the sponge presently colonizes two sites in the lake, both near the northwestern shore, the total absence of sponge fauna from other lake regions near more heavily populated areas of this species. Physicochemical data from Oneida Lake greatly extend the known environmental parameters of Spongilla heterosclerifera.     

Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland.

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87--93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.