p-Chloromercuribenzoate-induced dissociation of cytoskeletal proteins in red blood cells of rats

Effects of p-chloromercuribenzoate (PCMB) on the cytoskeletal organization of rat red blood cells were studied. Upon incubation with 50 microM PCMB in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, 80% of actin and 45% of spectrin were released from the ghosts, resulting in the fragmentation of ghost membranes. Addition of 2 mM Mg2+ or 0.1 M KCl, or lowering incubation temperature to 0 degree C substantially inhibited the solubilization of the cytoskeletal proteins and the fragmentation of ghost membranes, which enable to examine the effects of PCMB on the interaction between transmembrane proteins and the peripheral cytoskeletal network. Decreased recoveries of transmembrane proteins, such as band 3 and glycophorin, in Triton shell fraction were observed in the ghosts incubated with PCMB either in the presence of Mg2+ or at 0 degree C. PCMB also inhibited the in vitro association of purified spectrin with spectrin-depleted inside-out vesicles through interaction with proteins in the vesicle, such as bands 2.1 and 3. In the PCMB-treated ghosts, intramembrane particles were highly aggregated, which further supports the PCMB-induced dissociation of the transmembrane proteins from the cytoskeletal network. The decreased recovery of glycophorin in the Triton shell fraction also observed in intact red blood cells upon incubation with PCMB. These results suggest that the main action of PCMB on red cell membranes under physiological condition, at higher ionic strength and in the presence of Mg2+, is to dissociate transmembrane proteins from the peripheral cytoskeletal network, which may modify functions of these proteins.     

Brefeldin A inhibits oligosaccharide processing of glycoproteins in mouse hypothyroid pituitary tissue at several subcellular sites

We have studied the effects of brefeldin A (BFA) and monensin on the processing of the oligosaccharides of thyrotropin (TSH), free alpha-subunits, and cellular glycoproteins of mouse pituitary tissue to clarify the subcellular sites of action of BFA. BFA was previously shown to inhibit the translocation of glycoproteins from the rough endoplasmic reticulum to the Golgi apparatus but action at other sites was possible. Pituitaries from hypothyroid mice were incubated with [35S]methionine, [3H]mannose, [3H]galactose, [3H]fucose, N-[3H]acetylmannosamine, or [35S]sulfate for 2 hr in the absence or presence of 5 micrograms of BFA/ml or 2 microM monensin. TSH and free alpha-subunits were immunoprecipitated from tissue lysates and analyzed by sodium dodecyl sulfate-gel electrophoresis. The tryptic glycopeptides of TSH were separated using high-performance liquid chromatography. Total glycoproteins in cell lysates were precipitated using trichloroacetic acid. Labeled oligosaccharides were released from the tryptic glycopeptides of TSH and cellular glycoproteins by endoglycosidase H and they were analyzed by paper chromatography. Compared with control incubations, BFA caused the intracellular accumulation of glycoproteins having less than expected amounts of Man9GlcNAc2 units, but with excess Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2 units. There was a lesser accumulation of glucose-containing oligosaccharides, especially Glc1Man9GlcNAc2. Monensin also caused the accumulation of certain high mannose species, but the pattern differed from that seen for BFA, since Man9GlcNAc2 units were preserved and there was less excess of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2 units. BFA did not block the initial attachment of oligosaccharides at any of the three Asn-glycosylation sites of TSH, but caused the accumulation of Man5-8GlcNAc2 units at each site. Both monensin and BFA inhibited fucosylation, sulfation, and sialylation more markedly than mannose incorporation. Thus, in addition to its previously described action of inhibiting rough endoplasmic reticulum to Golgi transport, BFA appears to partially inhibit the glucose-trimming enzymes as well as some Golgi enzymes.     

A theory of gene-culture coevolution of parental preference and sex ratio in humans

A theory on the evolution of human primary sex ratio is proposed. Effects of parental preference for sons, reflected in birth control based on offspring sex ratio and female biased infanticide, on the evolution of primary sex ratio are analyzed. Both are shown to select for female bias in primary sex ratio. The gene-culture coevolution of female infanticide and primary sex ratio is also studied and it is shown that female infanticide develops more in societies in which the father plays a more important role in the transmission of culture than the mother does.     

Purification and characterization of two serine isoacceptor tRNAs from bovine mitochondria by using a hybridization assay method.

For large scale preparation of mitochondrial tRNAs, a new hybridization assay method using synthetic oligodeoxyribonucleotide probes (16-17mer) complementary to individual tRNA sequences was developed and applied for the purification of two serine isoacceptor tRNAs (tRNASerAGY and tRNASerUCN) from bovine mitochondria. It is about 100 times more sensitive than the conventional aminoacylation assay method. 2-4 A260 units each of both tRNASer isoacceptors were purified from 17.5 kg of bovine liver, and they were characterized by means of nuclease digestion, melting profiles and aminoacylation activity. It is suggested that tRNASerUCN possesses the D loop/T loop interaction like usual L-shaped tRNAs, and that tRNASerAGY lacking almost an entire D arm is aminoacylated with an efficiency not very much lower than that of tRNASerUCN.     

400 MHz two-dimensional nuclear Overhauser spectroscopy on anesthetic interaction with lipid bilayer

Interaction between a volatile anesthetic, methoxyflurane, and dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was analyzed by nuclear Overhauser effect (NOE) difference spectroscopy and two-dimensional nuclear Overhauser spectroscopy (NOESY). The NOE difference spectra were obtained by selectively irradiating methoxy protons (hydrophobic end) of the anesthetic: a negative nuclear Overhauser effect of -2.94% was observed with the choline methyl protons of DPPC. The NOESY spectra revealed a cross-peak between the anesthetic methoxy protons and the choline methyl protons. A dipole-dipole interaction exists between the hydrophobic end of the anesthetic and the hydrophilic head group of DPPC. No other cross-peaks were observed. The anesthetic orients itself at the membrane/water interface by interacting with the hydrophilic surface of the DPPC membrane, leaving the hydrophilic end of the anesthetic molecule in the aqueous phase. The preferred residence site of dipolar volatile anesthetics is the membrane/water interface.     

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.     

Expression of goat alpha-lactalbumin in Escherichia coli and its refolding to biologically active protein

A cDNA encoding the mature region of goat alpha-lactalbumin and the 3'-non-coding region was fused to cDNA of the N-terminal half of porcine adenylate kinase which had been placed under the control of the tac promoter in an expression vector in Escherichia coli. In addition, a methionine codon was inserted between the two cDNAs. When the plasmid carried the full-length 3'-non-coding region, little accumulation of the fused protein was observed. However, the deletion of two-thirds of the 3'-non-coding region produced significant expression of the fused protein in E. coli strain JM105. Since goat alpha-lactalbumin contains no methionine residue, the mature goat alpha-lactalbumin was isolated by CNBr digestion of the fused insoluble protein and refolded using thioredoxin. The homogeneous and biologically active goat alpha-lactalbumin was purified by Ca2+ ion-dependent hydrophobic chromatography.     

Movements of the large intestine in the anuran larvae, Xenopus laevis

1. The contractile behavior of the large intestine of Xenopus laevis tadpoles was studied. 2. The large intestine is divided into a colon and rectum, and shows three types of movements: rhythmic ascending (antiperistaltic) waves of contraction originating at the anal end of the large bowel, rhythmic longitudinal contractions in the rectum and colon, and irregular contractions. The first two patterns occur in the large bowel in situ and thus appear mature. The last one occurred only in older preparations, and thus appeared pathological. 3. Antiperistaltic waves of contractions and longitudinal contractions are generated independent of each other, suggesting that circular muscles and longitudinal muscles contract separately. 4. Acetylcholine, adrenaline and noradrenaline augment motility. 5. The premetamorphic motility of the large bowel is similar to that seen in adult frogs. Comparable motility was not observed elsewhere in the larval alimentary tract. The large intestine appears to be the first portion of the anuran alimentary tract to acquire the adult physiological and morphological profile.