Acquisition of ability to utilize Xylitol: disadvantages of a constitutive catabolic pathway in Escherichia coli.

Ribitol+ strains of Escherichia coli acquire the ability to utilize xylitol by mutating to constitutive production of the coordinately controlled ribitol catabolic enzymes ribitol dehydrogenase (RDH) and D-ribulokinase (DRK). Such strains concomitantly acquire toxicity to galacitol and L-arabitol, and to D-arabitol if they are unable to utilize it for growth. Strains selected for resistance to these polyols have DRK structural gene mutations or other mutations that eliminate the constitutive production of DRK, consistent with the view that DRK phosphorylates those polyols to toxic substances. Ribitol+ strains selected for growth on 8 mM xylitol fail to grow on 30 mM xylitol. A product of ribitol and xylitol catabolism represses synthesis of RDH, an enzyme required for growth on xylitol. At 30 mM xylitol, greater than 99% of RDH synthesis is repressed. Strains that grow on 8 mM xylitol can mutate to grow on 30 mM xylitol. Such mutants, relieved of this repression, overproduce RDH, resulting in good growth on the poor substrate, xylitol, but poor growth on the normal substrate, ribitol.     

Receptor-mediated endocytosis and nuclear transport of a transfecting DNA construct

A soluble construct consisting of a plasmid carrying the gene of the SV40 large T-antigen and an insulin-poly-L-lysine conjugate is able to selectively transfect PLC/PRF/5 human hepatoma cells which possess insulin receptors. Transfection can be efficiently competed by excess free insulin. To examine intracellular transport of the construct, it was fluorescently labeled and its accumulation on and in cells visualized by video-enhanced microscopy and quantitative confocal laser scanning microscopy. After 2 h at 37 degrees C, the labeled construct was found predominantly in intracellular acidic compartments, with a substantial portion of fluorescence localized both near and in the cell nucleus. Binding, endocytosis, and nuclear localization of the labeled conjugate could all be competed by excess free insulin, thus indicating that entry of the conjugate into cells was specifically mediated by the insulin receptor.     

Determinants of substrate recognition by poliovirus 2A proteinase.

Poliovirus proteinase 2A (2Apro) is autocatalytically released from the viral polyprotein by cleavage in cis of a Tyr-Gly dipeptide at its own amino terminus, resulting in separation of the P1 structural and P2-P3 nonstructural protein precursors. A second Ty-Gly dipeptide within 3D polymerase is cleaved by 2Apro in trans, but this is not essential for viral proliferation. The mechanism which limits cleavage to only 2 of the 10 Tyr-Gly dipeptides within the poliovirus polyprotein has not been characterized. We have therefore undertaken a systematic mutational analysis of the VP1-2A site to elucidate determinants of substrate recognition by 2Apro. The P2 and P1' positions are important determinants for cis cleavage of this site, whereas a variety of substituents could be tolerated at the P2', P1, and P3 positions. The requirements for trans cleavage of this site were more stringent. We found that the 2Apro of coxsackievirus type A21 and rhinoviruses 2 and 14 have stringent requirements similar to those of poliovirus 2Apro for cleavage in trans.     

Purification and characterization of poliovirus polypeptide 3CD, a proteinase and a precursor for RNA polymerase.

A cDNA clone encoding the 3CD proteinase (3CDpro) of poliovirus type 2 (Sabin), the precursor to proteinase 3Cpro and RNA polymerase 3Dpol, was expressed in bacteria by using a T7 expression system. Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3Cpro and 3Dpol. Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CDpro T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing. This mutant was purified to 86% homogeneity and used for subsequent proteolytic studies. Purified 3CDproM (M designates the cleavage site mutant 3CDpro T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide. Purified 3CDproM demonstrated the same detergent sensitivity in processing experiments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates. Purified 3CDproM did not have any detectable RNA polymerase activity, whereas 3Dpol, separated from 3CDproM by gel filtration in the last step of purification, did. We conclude that 3CDproM can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate. Moreover, cleavage of 3CD to 3Dpol is needed to activate the 3D RNA polymerase.     

Structural and dielectric properties of synthetic glycolipids in mixtures with water

Three synthetically produced glycolipids, N-(β-D-glucopyranosyl)-N-octadecyl-stearoylamide (OSGA), N-(β-D-glucopyranosyl-N-octadecyl-oleoylamide (OOGA), N-(β-D-galactopyranosyl)-N-octadecyl-lauroylamide (OLGA) have been studied in different mixtures with water by x-ray diffraction and dielectric measurements with microwaves at 9.4 GHz. The measurements were performed in the temperature range -50-70°C. X-Ray diffraction revealed a direct L_β' → H transition at 20°C, 60°C, and 45°C depending on the glycolipid species but nearly not on the water content. The hexagonal phases are saturated at a water content of ≈20 wt%. The lamellar phase absorbs even less water (< 10 wt%). The dielectric data show that in the H phase the binding of water is stronger than in the L_β' phase. In the temperature range below 0°C, OSGA and OOGA show a “subzero transition” due to the freeze-out of water in a separate ice phase. This transition can be seen in an abrupt decrease of the dielectric function because the dielectric response of ice is much smaller at microwave frequencies. OLGA does not show the subzero transition but an additional transition, hexagonal → distorted hexagonal at 60°C.     

Substance P-like immunoreactivity in the parietal eye visual system of the lizard Uta stansburiana

Summary We examined the parietal eye visual system of the iguanid lizard Uta stansburiana for the presence of substance P-like immunoreactivity by use of both immunofluorescence and peroxidase-antiperoxidase techniques. In the parietal eye no substance P-containing somata were found; however, its plexiform layer contained small (ca. 1 m diam) immunoreactive fibers. These fibers apparently originate outside the parietal eye. Immunoreactive fibers also were found in the parietal nerve, the dorsal sac, and the leptomeninx of the pineal gland. No labeled somata were observed in any of these regions in either normal or colchicine treated animals. Previously we demonstrated that a system of centrifugal fibers to the parietal eye originates from neurons in the dorsal sac (Engbretson et al. 1981). The apparent absence of substance P-containing neurons in the dorsal sac suggests that the substance P-containing fibers in the parietal eye are not the previously observed centrifugal fibers. The source of the substance P-containing fibers in the parietal eye is unknown. The pars dorsolateralis of the left medial habenular nucleus receives a dense substance P-positive projection. No such projection was seen in the right habenula. Simultaneous visualization of the terminals of ganglion cells of the parietal eye (labeled with orthograde intraaxonally transported horseradish peroxidase) and substance P-like immunofluorescence showed that the locus of habenular immunoreactivity is distinct from the projection field of the parietal eye. Thus the substance P-positive terminals in the habenula do not originate in the parietal eye. Transection of the parietal nerve confirmed this conclusion.     

Stimulation of the anaerobic growth ofSalmonella typhimurium by reduction ofl-carnitine,carnitine derivatives and structure-related trimethylammonium compounds

In view of the development of al-carnitine deficiency, the metabolism ofl-carnitine and structure-related trimethylammonium compounds was studied inSalmonella typhimurium LT₂ by means of thin-layer chromatography (TLC).l-Carnitine, crotonobetaine and acetyl-l-carnitine stimulated the anaerobic growth in a complex medium significantly. The stimulation depended on the formation of -butyrobetaine. The reduction ofl-carnitine proceeded in two steps: (1) Dehydration of thel-carnitine to crotonobetaine, (2) hydrogenation of crotonobetaine to -butyrobetaine. The reduction of crotonobetaine was responsible for the growth stimulation. Terminal electron acceptors of the anaerobic respiration such as nitrate and trimethylamine N-oxide, but not fumarate, suppressed the catabolism ofl-carnitine completely. Glucose fermentation, too, inhibited the reduction ofl-carnitine but optimal growth with a high carnitine catabolism was achieved byd-ribose. The esters of carnitine with medium- and long-chain fatty acids inhibited the growth considerably because of their detergent properties.Abbreviations TLC thin-layer chromatography     

A preliminary report of vibriosis in cultured American lobsters,Homarus americanus

A vibrio-like bacterium, designated Vibrio sp. (BML 79-078), was isolated from moribund juvenile Amercan lobsters, Homarus americanus. This is the first report of a vibrio-like bacterium being associated with a disease of lobsters. Koch's postulates have been satisfied for this bacterium. In this study, Vibrio sp. (BML 79-078) and Vibrio anguillarum (ATCC 19264) were found to be pathogenic when injected into juvenile lobsters held at 20°C, while Enterobacter aerogenes was not. An endotoxin was found to be associated with the pathogenesis of the disease syndrome caused by Vibrio sp. (BML 79-087).