Molecular cloning and sequencing of infC, the gene encoding translation initiation factor IF3, from four enterobacterial species

Abstract Translation initiation factor IF3 plays a crucial role in initiation of protein synthesis in bacteria. In order to elucidate the IF3 structural elements required for these functions, the evolutionary conservation of IF3 and its gene, infC , was investigated. Homologous infC sequences from Salmonella typhimurium, Klebsiella pneumoniae, Serratia marcescens and Proteus vulgaris were amplified by the polymerase chain reaction and sequenced. Analysis of these sequences, as well as that from Bacillus stearothermophilus , revealed several regions (e.g. residues 62–73 and 173–177) of absolute sequence conservation, suggesting an important role for these regions in IF3 function.     

Effects of phosphorylation of P-glycoprotein on multidrug resistance

Cells expressing elevated levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug resistance phenotype. Studies involving protein kinase activators and inhibitors have implied that covalent modification of P-glycoprotein by phosphorylation may modulate its biological activity as a multidrug transporter. Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells independent of, or in addition to their effects on the state of phosphorylation of P-glycoprotein. The protein kinase(s) responsible for P-glycoprotein phosphorylation has(ve) not been unambiguously identified, although several possible candidates have been suggested. Recent biochemical analyses demonstrate that the major sites of phosphorylation are clustered within the linker region that connects the two homologous halves of P-glycoprotein. Mutational analyses have been initiated to confirm this finding. Preliminary data obtained from phosphorylation- and dephosphorylation-defective mutants suggest that phosphorylation of P-glycoprotein is not essential to confer multidrug resistance.     

Biopolitics: the newest synthesis?

On first impression, the disciplines of genetics and political science would appear to be unrelated. And yet, commencing more than 30 years ago, the interdisciplinary field known as Biopolitics has now taken hold. This essay traces the central thrust of the biopolitical research agenda. It describes, analyzes, and assesses how political scientists have sought to show connections between our species' genetic constitution and our species' political behavior. Important bridges between the two are the neurophysiology of the human brain and the role of evolutionary theory in charting man's adaptational political profile. The parameters of the emerging biopolitical literature raise profound policy questions, some of which are also characterized. This revised version was published online in August 2006 with corrections to the Cover Date.     

Susceptibility of heterochromatin to aphidicolin-induced chromosomal breakage

The distribution of aphidicolin-induced chromosomal lesions was analyzed to determine the relative breakage susceptibility of euchromatin and heterochromatin in the cactus mouse, Peromyscus eremicus. The observed breakage was tested against expected distributions corresponding to the karyotypic proportions of autosomal euchromatin, autosomal heterochromatin, X-chromosomal euchromatin, and X-chromosomal heterochromatin. The distribution of induced breakage was independent of sex but dependent on the individual. In all individuals, there was a highly significant (P0.0001) deficiency in the number of breaks observed as compared to expected in autosomal heterochromatin. Sparse observations in the X chromosome and the absence of breaks in the Y chromosome precluded valid statistical tests of the sex-chromosomal distribution of induced breakage. These data indicate that the autosomal heterochromatin of Peromyscus is resistant to aphidicolin-induced chromosomal breakage and argue against a simple relationship between late replication and a general mechanism for chromosomal fragility.     

Initial steps in receptor-mediated endocytosis : The influence of temperature on the shape and distribution of plasma membrane clathrin-coated pits in cultured mammalian cells

We have examined the shape and distribution of clathrin-coated pits in Swiss 3T3 cells at 4 or 37 degrees C using electron microscopy with serial sections and immunofluorescence light microscopy. Both groups were fixed in glutaraldehyde and preserved further using a membrane contrast enhancement technique consisting of sequential osmium-ferrocyanide, thiocarbohydrazide and osmium-ferrocyanide treatment in situ. Concanavalin A-horseradish peroxidase (conA-HRP) was used to identify these structures participating in endocytosis. Two hundred twenty-two clathrin-coated structures were analysed; 126 from cells fixed at 4 degrees C, and 96 from cells fixed after a 3 min warm-up to 37 degrees C. All coated structures labeled with conA-HRP had demonstrable connections to the plasma membrane. These coated structures were morphologically classified into three categories: (a) flat pits; (b) curved pits; and (c) pits with narrow-neck connections to the plasma membrane. At 37 degrees C, 27% of coated pits had narrow neck connections to the plasma membrane whereas at 4 degrees C only 1% had such connections. Receptosomes (endosomes) labeled with conA-HRP were found only after incubation at 37 degrees C, indicating that active endocytosis was occurring in cells at 37 degrees C, but not at 4 degrees C. Immunofluorescence with anti-clathrin antibody was used to quantitate the number of clathrin-coated pits in Swiss 3T3 cells incubated at 4 and 37 degrees C prior to fixation. No difference was detected. There were 426 +/- 122 pits per cell at 37 degrees C and 441 +/- 106 at 4 degrees C. These results support the hypothesis that formation of a narrow neck connected a coated pit to the cell surface is an early step in the mechanism of receptor-mediated endocytosis.     

Vibrational raman spectra of lipid systems containing amphotericin B

Resonance-enhanced and normal vibrational Raman spectra were observed for both multilamellar and single-wall vesicle assemblies of dimyristoyl phosphatidylcholine containing amphotericin B, a channel-forming polyene antibiotic, and cholesterol. The decrease in the frequency of the polyene antibiotic C  C stretching mode at 1556 cm^?1 and the increase in intensity of the CCH in-plane deformation mode at 1002 cm^?1 indicate that amphotericin B is ordered in a lipid-cholesterol medium similarly to the solid, but is surrounded by a slightly more polar environment. The intensity of the C  C stretching mode I₁₅₅₆ decreases 4-fold during the broadened gel to liquid crystalline phase transition (16–32°C) of dimyristoyl lecithin-cholesterol (4 : 1) multilayers. Other resonance-enhanced vibrations of amphotericin B exhibit similar behavior. For amphotericin B in pure dimyristoyl lecithin multilayer or vesicle systems, however, the vibrational intensity associated with the C  C stretching mode remains constant during the melting of lipid hydrocarbon chains. In addition, a third effect occurs in liquid crystalline egg lecithin-cholesterol (4 : 1, mol ratio) multilayers in which I₁₅₅₆ first increases by 25% between 3 and 25°C, in parallel with the loss of active channels, and then remains constant as the temperature increases from 25 to 42°C. This latter intensity pattern is masked in the dimyristoyl lecithin-cholesterol system by the overwhelming effect upon the C  C mode from changes in the lipid chain packing characteristics which occur during the phase transition.The broadened phase transition in 4 : 1 dimyristoyl lecithin-cholesterol multilayers (16–32°C), as followed by the ratio of intensities at 2880 and 2850 cm^?1 (asymmetric and symmetric methylene C-H stretching modes, respectively) is slightly narrowed by the addition of amphotericin B, and effect from which a binding stoichiometry at 24° of 1 : 1 amphotericin B : cholesterol is estimated. This stoichiometry was confirmed by differential calorimetric scans, which also show the presence of a peak proportional to cholesterol content.Raman I_2880/2850 peak height ratios in pure dimyristoyl lecithin bilayers were increased over the 14–38°C range by amphotericin B, a spectral effect which suggests an ordering of the lipid matrix perhaps as a consequence of the polyene binding to the bilayer surface. For bilayers containing cholesterol, the ratios of intensities of the 2935 cm^?1 feature, composed mainly of acyl chain terminal methyl and underlying methylene C-H stretching modes, to the 2850 cm^?1 feature are significantly increased by amphotericin B. This effect indicates that the antibiotic penetrates the bilayer in the lipid-sterol system.     

Origin of the left anterior descending coronary artery from the pulmonary artery: An unusual cause of angina in a middle-aged woman

A 48-year-old woman was admitted to our institution with angina pectoris and a systolic murmur. At cardiac catheterization, she was found to have an anomalous origin of the left anterior descending coronary artery from the pulmonary trunk. There was also an associated atrial septal defect and a bicuspid aortic valve.     

Interaction of melittin with dimyristoyl phosphatidylcholine liposomes. Evidence for boundary lipid by raman spectroscopy

The interaction of melittin, a polypeptide consisting of 26 amino acid residues, with dimyristoyl phosphatidylcholine bilayers was investigated by vibrational Raman spectroscopy. Spectral peak height intensity ratios, involving vibrational transitions in both the 3000 cm^?1 acyl chain methylene carbon-hydrogen stretching mode region and the 1100 cm^?1 acyl chain carbon-carbon skeletal stretching mode interval, served as temperature profile indices for monitoring the bilayer order-disorder processes. For a lipid : melittin molar ratio of 14 : 1 two order-disorder transitions were observed. In comparison to a gel to liquid crystalline phase transition of 22.5°C for the pure lipid, the lower transition, exhibiting a 2°C width, is centered at 17°C and is associated with a depression of the main lipid phase transition of dimyristoyl phosphatidylcholine. The second thermal transition, displaying a 7°C interval, occurs at approx. 29°C and is associated with the melting behavior of approximately seven immobilized boundary lipids which surround the inserted hydrophobic segment of the polypeptide. For a lipid : melittin molar ratio of 10 : 1 two thermal transitions are also observed at 11 and 30°C. As before, they represent, respectively, the main gel to liquid crystalline phase transition and the melting behavior of approximately four boundary lipids attached to melittin. From these data alternative schemes are suggested for disposing the immobilized lipids around the hydrophobic portion of the polypeptide within the bilayer.     

The preparation of a sarcolemmal fraction from evacuated muscle slices

A novel procedure is described for preparing a plasma membrane fraction from skeletal muscle (i.e., sarcolemma). The procedure entails evacuating the myoplasm from muscle slices as a preliminary step to homogenization and fractionation. The evacuated muscle slices are composed of a stroma-containing sarcolemma, which is then homogenized and fractionated, utilizing a sequence of differential and discontinuous sucrose density gradient centrifugations. On the basis of electron microscopy, selective enzyme markers and α-bungarotoxin binding in innervated and denervated muscles, the fraction most enriched with sarcolemma is recovered from the 0.5/0.7 M interface of a discontinuous sucrose gradient.     

Effects of denervation on the composition and synthesis of sarcolemmal proteins

In vitro synthesis of proteins and changes in polypeptide composition of sarcolemma were studied in innervated and denervated extensor digitorum longus muscle of the rat. A technique of evacuating myoplasm from muscle slices was used as a preliminary step in the preparation of three membrane fractions, M, H and S, containing sarcolemma. On the basis of findings from the previous study and the present investigation, it was concluded that the M fraction was most enriched with extrajunctional sarcolemma.In vitro incorporation of [³H]leucine into membrane proteins of the M fraction showed an apparent linear increase in the rate of protein synthesis from 1–10 days after denervation. The relative increase at 10 days was 137% greater than that of innervated controls. Fractions H and S showed a smaller relative increase.Polypeptide composition of M, H and S fractions based on SDS gel electrophoresis of innervated and denervated muscle, showed qualitative and quantitative changes. The most striking difference was a nominal 29 000 component in M that constituted a disproportionately large peak. Following 10 days of denervation the M fraction underwent significant compositional changes in its electrophoretic profile, the most dramatic of which was a large reduction in the proportion of the 29 000 component. The denervation-induced compositional change is discussed in light of known alterations in the chloride conductance of the muscle plasmalemma.