Enhanced thermodynamic stabilities of yeast iso-1-cytochromes c with amino acid replacements at positions 52 and 102.

We have determined the structures and thermodynamic stabilities of the wild type Asn-52 and unusually thermostable mutant Ile-52 yeast iso-1-cytochromes c (Das, G., Hickey, D. R. McLendon, D., McLendon, G., and Sherman, F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 496-499). Although both structures were similar, Water-166, buried within the wild type protein, is excluded from the Ile-52 mutant, which substantially reorganizes the local hydrogen bonding. Wild type Cys-102 was replaced with alanine or serine to eliminate dimerization in vitro. The Cys-102 (wild type), Ala-102, and Ser-102 proteins were equally stable, whereas the chemically modified Cys-102-SCH3 was less stable. The order of stability observed with replacements at positions 52 and 102 was as follows: Ile-52 Ala-102 greater than Ala-52 Ala-102 greater than Asn-52 Ala-102 ("normal") greater than Gly-52 Ala-102. No significant stabilization was attributed to potential energy interactions expressed as helix-forming propensities of replacements at position 52. A high correlation between differences in free energy changes and transfer free energies suggests hydrophobic interactions are the main factor for enhancing stability in the Ile-52 mutant. Additional possible contributions to the thermostability of the Ile-52 variant are energetic effects due to packing and hydrogen bonding changes surrounding position 52.     

Structural transition of alpha 1-antitrypsin by a peptide sequentially similar to beta-strand s4A

Crystal structure studies have shown that cleaved and intact serpins differ essentially in the topology of beta-sheet A. This is five-stranded in the intact molecules and six-stranded after cleavage by insertion of strand s4A whose C-terminus has become free [L?bermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. (1984) J. Mol. Biol. 177, 531-556; Wright, T. H., Qian, H. X. & Huber, R. (1990) J. Mol. Biol. 213, 513-528]. The structural transition is accompanied by changes in spectral properties and an increase in thermal stability. We show here that an N alpha-acetyl-tetradecapeptide with the amino acid sequence of strand s4A, residues 345-358 of human alpha 1-antitrypsin, associates with intact alpha 1-antitrypsin and forms a stoichiometric complex with properties very similar to cleaved alpha 1-antitrypsin. Complex generation has the characteristics of a folding process.     

Pyrolytic Methylation-Gas Chromatography of Whole Bacterial Cells for Rapid Profiling of Cellular Fatty Acids

A novel, on-line derivatization technique has been developed which enables generation of fatty acid methyl ester (FAME) profiles from microorganisms by gas chromatography-mass spectrometry without the need for laborious and time-consuming sample preparation. Microgram amounts of bacterial cells are directly applied to a thin ferromagnetic filament and covered with a single drop of methanolic solution of tetramethylammonium hydroxide. After air drying, the filament is inserted into a special gas chromatograph inlet equipped with a high-frequency coil, thus enabling rapid inductive heating of the ferromagnetic filament. This so-called Curie-point heating technique is shown to produce patterns of bacterial FAMEs which are qualitatively and quantitatively nearly identical to those obtained from extracts of methylated lipids prepared by conventional sample pretreatment methods. Relatively minor differences involve the loss of hydroxy-substituted fatty acids by the pyrolytic approach as well as strongly enhanced signals of FAMEs derived from mycolic acids. This type of pyrolysis enables on-line derivatization and thermal extraction of volatile derivatives for analysis, whereas the residual components remain on a disposable probe (ferromagnetic wire) of a pyrolytic device. The reduced sample size (micrograms instead of milligrams) and the lack of sample preparation requirements open up the possibility of rapid microbiological identification of single colonies (thus overcoming the need for time-consuming subculturing) as well as analysis of FAME profiles directly from complex environmental samples.     

Role of glycerol 3-phosphate and glycerophosphate acyltransferase in the nutritional control of hepatic triacylglycerol synthesis

1. Glycerol 3-phosphate content of isolated hepatocytes from starved rats and of glycogen-depleted hepatocytes from fed rats was low and severely limited triacylglycerol synthesis. 2. Raising the glycerol 3-phosphate content by addition of precursors to the cells resulted in a hyperbolic-like relationship between triacylglycerol synthesis and cellular glycerol 3-phosphate content. Statistical analysis of the curves showed no significant differences between the nutritional states either at saturating or at subsaturating glycerol 3-phosphate content. 3. V_max. of glycerophosphate acyltransferase measured in homogenized hepatocytes was decreased by 30–40% in starvation. There was no change in apparent K_m for glycerol 3-phosphate. Since at saturating glycerol 3-phosphate content esterification rates in hepatocytes of both nutritional states were identical, the enzyme is not limiting esterification under this condition. 4. At subsaturating glycerol 3-phosphate content the flux through glycerophosphate acyltransferase necessarily limits esterification. Therefore one would expect a decrease in esterification in starvation under this condition. This was the case when triacylglycerol synthesis was plotted against intracellular glycerol 3-phosphate concentration, calculated from the cellular glycerol 3-phosphate content and the intracellular water space, which was smaller in hepatocytes from starved rats. 5. The data obtained in hepatocytes were extrapolated to the intact liver by using the number of parenchymal cells per g of liver as determined from marker-enzyme analysis and the liver weight per 100g body weight. The extrapolation suggested that glycerol 3-phosphate is limiting esterification in vivo for contents below 0.3–0.4 and 0.5–0.65μmol/g for livers from fed and starved animals respectively. Also for a given fatty acid load and a glycerol 3-phosphate content below 0.3μmol/g the liver may esterify less in the starved state. However, at the glycerol 3-phosphate contents measured in freeze-clamped livers (0.30 and 0.44μmol/g for the fed and starved state respectively), livers in both nutritional states seemed capable of esterifying similar amounts of fatty acids.     

Origines du peuplement mammalien en Afrique du Nord durant le miocène terminal

The late Miocene North African mammalianassemblage is considered here from three viewpoints: survivals, extinctions, and immigrations. The Eurasiatic affinities of the large mammals slightly prevail over the Ethiopian affinities. Amongst the North African large mammals, 4 to 8 taxa are Eurasiatic immigrants, while 4 to 6 are of Subsaharian origin. Contrarily, the micromammalian fauna is highly endemic, with only one species, a murid (Paraethomys miocaenicus), considered here as being related to an Asiatic form (Karnimata darwini). Our study of Eurasian and African Miocene faunas reveals that during the late Astaracian-early Turolian interval, the Saharo-Arabic belt permitted very little latitudinal faunal exchanges. However, during the middle and late Turolian such faunal exchanges became frequent. The micromammal record unequivocally indicates that a brief period of faunal exchange occurred between Africa and western Europe at the end of the Miocene, corresponding with the Messinian Salinity Crisis. The increased intercontinental faunal exchange between Africa and Eurasia during the late Miocene coincides with, and counterbalances the extinction of more than 10 taxa at the Mio/Pliocene boundary.     

Voluntary control of autonomic responses: A case for a dialogue between individual and group experimental methodologies

This paper discusses the general methodological controversy between individual and group research approaches by comparing the main characteristics of these two methods as applied to the specific context of basic research on voluntary heart rate control. A review of the literature published over the past 19 years in this area of study shows an imbalance in the frequency of utilization of these two methods that strongly favors short-term group designs. Implications of this research tendency are discussed. The relevance and the advantages of applying the individual approach to voluntary autonomic control research are outlined. This area is particularly amenable to the individual approach because the phenomena under study seem to be characterized by, among other things, a smaller intrasubject than intersubject variability. It is suggested that the present imbalanced tendency in the choice of a research method be corrected and that researchers adopt a more flexible attitude in the choice of the best method for studying each specific problem.     

Comparitive study of the milk fat globule membrane and the mouse mammary tumour virus prepared from the milk of an infected strain of Swiss albino mice

Milk fat globule membranes and mammary tumour virus particles (d = 1.17 g/cm³) have been obtained from the milk of a Swiss albino mice strain. Comparitive biochemistry shows that these two structures differ significantly in the phospholipid, polypeptide and glycopolypeptide patterns and enzymatic activities. However, the lipid profile and the morphology of both structures suggest a filiation with the plasma membrane. Density fractions obtained from the crude virus preparation have been thoroughly investigated. The results suggest that most of these fractions represent degraded virus and/or atypical virus assembly.     

Superior immunoreactivity of 125I (Des-Tyr-betaAla)-secretin with rabbit anti-secretin sera compared to 125I-secretin and 125 I-6-Tyrosyl secretin.

A secretin analogue in which the normal amino acid sequence had been elongated by a (Des-Tyr-betaAla)-residue was studied as tracer for secretin radioimmunoassay. 125I-(DATA)-secretin exhibited superior immunoreactivity with several rabbit anti-secretin sera compared to 125I-6-Tyr-secretin and also to secretin iodinated at its N-terminal histidyl residue. This may be due, at least in part, to higher conformational integrity of the secretin moiety in the 125I-(DATA)-secretin molecule. Thus, at present, 125I-(DATA)-secretin appears to be most suitable as tracer for sensitive secretin radioimmunoassay.