Effect of chemical modifications of tryptophan residues on the folding of reduced hen egg-white lysozyme

The effects of chemical modifications of Trp62 and Trp108 on the folding of hen egg-white lysozyme from the reduced form were investigated by means of the sulfhydryl-disulfide interchange reaction at pH 8 and 40 degrees C. The folding of reduced lysozyme was monitored by following the recovery of the original activity. Under the conditions employed, the apparent first-order rate constant for the folding of reduced lysozyme was not changed by the modifications of both Trp62 and Trp108 and the folding was completed within 30 min. However, the extent of the correct folding was changed by the modification of Trp62 but not by that of Trp108. Native and oxindolealanine108 lysozymes recovered 80 and 81% of their original activities after 30-min refolding, respectively, but Trp62-modified lysozymes recovered their activities to a lesser extent than native and oxindolealanine108 lysozymes. The recovered activities of Trp62-modified lysozymes after 30-min refolding were 63% for oxindolealanine62 lysozyme, 65% for delta 1-carboxamidomethylthiotryptophan62 lysozyme, and 52% for delta 1-carboxymethylthiotryptophan62 lysozyme. These results suggest that Trp62 is important for preventing the misfolding of reduced lysozyme, but that neither Trp62 nor Trp108 is involved in the rate-determining step (the slowest step) in the folding pathway. A decrease in the hydrophobic nature of Trp62 seems to increase the misfolding and thus to decrease the extent of the correct folding of reduced lysozyme. A mechanism for the involvement of Trp62 in the folding pathway of reduced lysozyme is proposed.     

Morphological and Biochemical Studies on the Cerebral Cortex from Reeler Mutant Mice: Development of Cortical Layers and Metabolic Mapping by the Deoxyglucose Method

Abstract: Cerebral cortex from reeler mutant mice was examined morphologically and biochemically. The sequential process of postnatal cell migration in the cerebral cortex of reeler (rl/rl) was examined morphologically. The dense cellular cortical plate lies below the molecular layer near the cerebral surface just after birth in normal mice while in reeler most of the cells are concentrated in the center of the cortex. In the cortex of adult reeler, the broad laminar structure of the neurons could be seen to form inverted positions in the cortical layers. The total wet weight, and the concentration of DNA and RNA in the pallium cerebri from reeler did not differ significantly from those in the control. As to the protein profiles of the pallium cerebri detected by SDS- polyacrylamide gel electrophoresis, no significant differences were observed. Activities of CNPase (2',3'-cyclic nucleotide 3'-phosphohydrolase), which is a myelin enzyme of CNS, and choline acetyltransferase were at the same level in both the reeler and the control. Therefore, reeler mutation does not appear to affect the genetically determined cell numbers, number of cholinergic fibers, and myelination. By autoradiographic observation of the cerebral cortex after intraperitoneal injection of [¹⁴C]2-deoxyglucose, it was revealed that 2-deoxyglucose was incorporated intensively into the fourth layer (granular layer) of the cerebrum from the control. In reeler it was also incorporated into the granular layer but in a more widespread distribution. We conclude that terminals to the granular layer make metabolically active synapse, perhaps even in a manner inverted from normal.     

The reaction of the mutagen 1,1′-hexamethylene-bis[(5-p-chlorophenyl)-biguanide] with guanosine and cysteine

The mutagen 1,1′-hexamethylene-bis[(5-p-chlorophenyl)-biguanide] reacts at 37°C with guanosine and guanine to yield xanthosine or xanthine and oxidizes cysteine to cystine. After treatment of a guanosine-labelled DNA sample from Escherichia coli with the mutagen xanthine could be detected as a reaction product. At a slow rate the mutagen is hydrolysed spontaneously yielding urea, 1,6-hexanediol and 4-chloroaniline. The reaction mechanisms both of the hydrolysis and of the reaction with cysteine and guanosine are discussed.     

Ultrastructural observations on paracnids. I: Coelogynopora axi Sopott (Turbellaria,Proseriata)

The Schlauchdrüsen or paracnids of Coelogynopora axi Sopott, 1972 consist of two components: a muscle cell and a secretory cell.The secretory cell is provided with a tube, which bears a border of microvilli. In the normal position the tube is situated in the interior of the secretory cell, and the microvilli stand at the inner side of the tube. After expulsion of the tube the microvilli are situated at its free surface.The evagination takes place in response to chemical stimuli and is effected by the contraction of the myofibrils of the muscle cell.The paracnids are supposed to be mechanisms of defense.However, conformities with nematocysts and spirocysts of the cnidarians do not exist.The paracnids in other species of the Coelogynoporidae, for example in Invenusta paracnida (Karling, 1966) and Carenscoilia bidentata Sopott, 1972 differ from those of C. axi in many details.Abbreviations bl- basement lamina - ep- epidermis - hd- hemidesmosomes - mc- muscle cell - mt- microtubules - mv- microvilli - nsc- nucleus of the secretory cell - sb- bowl containing secretion granules - sc- secretory cell - sd- septate desmosome-like structures - sg- secretion granules - t- tube - tf- tonofilaments     

Comparative studies on the increase by polyamines of fidelity of protein synthesis in Escherichia coli and wheat germ cell-free systems.

In the presence of polyamines, the fidelity of protein synthesis in a wheat germ cell-free system was increased significantly, while it was increased slightly in an $\text{E. coli}$ cell-free system. The effective concentration of polyamines for the increase in fidelity of protein synthesis was nearly equal to that for the stimulation of protein synthesis in a wheat germ cell-free system.     

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues

Summary A method for the localization of pyruvate kinase isoenzymes type L, M₂ and M₁ in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M₁ was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M₁. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M₂ was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M₂ was detected in high concentrations in the granulation tissue of type M₂ was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M₂ in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.     

Triterpenoid saponins from Fatsia japonica

Five triterpenoid saponins isolated from the flowers, the mature fruits and the leaves of Fatsia japonica were identified as 3-O-[β-d-glucopyranosyl(1→4)-β-d-glucopyranosyl]-hederagenin (1), 3-O-[β-d-glucopyranosyl-(1→4)-α-l-arabinopyranosyl]-oleanolic acid (2), 3-O-[α-l-arabinopyranosyl]-hederagenin (3), 3-O-[β-d-glucopyranosyl]-hederagenin (4) and 3-O-[β-d-glucopyranosyl(1→4)-α-l-arabinopyranosyl]-hederagenin (5). The saponins 1 and 2 are new, naturally occurring, triterpenoid saponins. The distribution of the five saponins in three parts of the plant was investigated. Saponins 2, 3 and 5 were present in the flowers, saponins 1, 3, 4 and 5 were in the mature fruits and saponins 2, 3, 4 and 5 were in the leaves.     

Nucleic acid of flacherie viruses of the silkworm, Bombyx mori

It was reported previously that two spherical flacherie viruses of silkworm, FVS I and FVS II, had been isolated from flacherie silkworm larvae and the nucleic acid of FVS II was RNA as suggested by the experiments of incorporation of [³H]-uracil. In this paper, it has been confirmed by biochemical methods that the nucleic acid of FVS I and FVS II is RNA. FVS I and FVS II were labeled with ³²P in flacherie silkworms, and the viruses were analyzed by sucrose density gradient centrifugation. When the ³²P-labeled compound in the viruses was treated with 0.5 n KOH, the acid-insoluble ³²P-labeled compound changed to acid-soluble compounds. It was determined by paper chromatography and ion-exchange column chromatography that the alkali-decomposed compounds included four ribonucleotides. Therefore, the viral nucleic acid of FVS I and FVS II was determined to be RNA. The correlations between FVS I and FVS II particles were discussed, and it was suggested that FVS I and FVS II might be closely related or were the same viral species.     

Effects of chemotherapeutics on bacterial ecology in the water of ponds and the intestinal tracts of cultured fish, ayu (Plecoglossus altivelis).

Drug-resistant gram-negative bacilli conferred with R factors were isolated with high frequencies from the intestinal tracts of ayu (Plecoglossus altivelis) cultured in ponds, in which chemotherapeutics had often been used, and with relatively low frequencies from ayu which received no administration of chemotherapeutics. Drug-resistant bacteria were also isolated at low frequencies from the intestinal tracts of wild ayu in rivers, as well as from the water of ayu-culturing ponds and some of them carried R factors. The drug-resistant bacteria carrying R factors were Aeromonas liquefaciens, Citrobacter, Enterobacter cloacae, Escherichia coli, Hafnia and unidentified strains. All the R factors were classified as the Fi-(F) type, except the two R factors detected in an E. coli strain and in an unidentified strain.