Elevated immunoreactive-somatostatin levels in the brain of ataxic mutant mice

Immunoreactive-somatostatin (IR-SRIF) levels were investigated in the brain of 4 types of ataxic mice (Rolling Mouse Nagoya, Weaver, PCD, Staggerer) with different cerebellar pathologies. IR-SRIF concentrations (ng/mg) were found to be significantly elevated in both cerebellum and cerebrum of all ataxic mutant mice, IR-SRIF (ng/organ) was found to be increased in the cerebellum and cerebrum in Rolling Mouse Nagoya and PCD compared with control mice. The gel-filtration profile (Sephadex G-50) in the cerebellar extracts of Rolling Mouse Nagoya proved to be identical to that of control mice. Three peaks of IR-SRIF were found to be uniformly elevated in Rolling Mouse Nagoya, with the highest peak coinciding with authentic somatostatin-14. The present results suggest that elevated levels of IR-SRIF in the brain may play a role in the mechanism underlying the manifestation of ataxia in ataxic mutant mice, especially in Rolling Mouse Nagoya and PCD.     

Electrical characteristics of ascidian egg fragments

Fragments of ascidian eggs, but at random in any plane and ranging in size from 10 to 90% of the total egg volume, displayed the electrical characteristics of the intact egg, having a resting potential of -86 mV and giving rise to an action potential upon stimulation by electrical current injection. Following insemination, the fragments generated fertilization potentials, comparable to those of intact eggs, although the repolarization phase was shorter. Our data show that there are sufficient ion channels throughout the egg surface to generate action potentials and fertilization potentials in excised egg fragments, irrespective of their global origin. Furthermore, the fertilizing spermatozoon is capable of activating fertilization channels in areas of the egg plasma membrane not destined for sperm entry.     

Sucrose synthase activity in developing wheat endosperms differing in maximum weight

Past research on kernel growth in wheat (Triticum aestivum) has shown that the kernel itself largely regulates the influx of sucrose for consequent starch synthesis in the endosperm of the grain. The first step in the conversion of sucrose to starch is catalyzed by sucrose synthase (EC 2.4.13). Sucrose synthase activity was assayed in developing endosperms from kernels differing in growth rate and in maximum dry weight accumulation. From 10 to 22 days after anthesis, sucrose synthase activity per wheat endosperm remained constant with respect to time in all grains. However, kernels which had higher rates of kernel growth and which achieved greatest maximum weight had consistently and significantly higher sucrose synthase activities at any point in time than did kernels with slower rates of dry matter accumulation and lower maximum weight. In addition, larger kernels had a significantly greater amount of water in which this activity could be expressed. Although the results do not implicate sucrose synthase as the “rate limiting” enzyme in wheat kernel growth, they do emphasize the importance of sucrose synthase activity in larger or more rapidly growing kernels, as compared to smaller slower growing kernels.     

Cloning and expression of Mycobacterium bovis BCG DNA in "Streptomyces lividans"

The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions. Random DNA fragments from M. bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S. lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S. lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E. coli. M. bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S. lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator. The results suggested that "S. lividans" uses M. bovis BCG translational signals almost as efficiently as its own signals. Moreover, several hybrid proteins with an M. bovis BCG-derived amino terminus seemed to be reasonably stable in "S. lividans." These experiments indicate that "S. lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals. This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents. The vectors may also have wider applications for the analysis of gene expression in Streptomyces.     

Characterization of a diphosphonopentaosylceramide containing 3-O-methylgalactose from the skin of Aplysia kurodai (sea hare)

The complete structure is proposed for a ceramide (Cer), bis(2-aminoethylphosphono)-pentaoside, isolated from the skin of Aplysia kurodai. This new phosphonoglycosphingolipid was purified using two systems of column chromatography on silicic acid. The purity of the glycolipid was confirmed by thin-layer chromatography, analysis of its composition, and proton magnetic resonance spectrometry. The component carbohydrates were glucose, galactose, N-acetylgalactosamine, and 3-O-methylgalactose. Most (90%) of the fatty acid was palmitic acid and the major sphingosine bases were octadeca-4-sphingenine (51%) and anteisononadeca-4-sphingenine (38%). 2-Aminoethylphosphonyl-6-galactose was identified after its partial hydrolysis. From studies by methanolysis, permethylation, mild acid hydrolysis, hydrogen fluoride treatment, chromium trioxide oxidation combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry, the structure of the glycolipid was concluded to be 3-OMeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)-Gal alpha 1----2](2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1Cer.     

The repetitive structure of the profilaggrin gene as demonstrated using epidermal profilaggrin cDNA

Filaggrin is the histidine-rich basic protein that aggregates keratin filaments in fully differentiated cells of the epidermis. Filaggrin is synthesized in the granular cell layer as a high molecular weight precursor protein (profilaggrin) that consists of multiple repeated copies of filaggrin. cDNA clones for rat and mouse epidermal profilaggrin have been constructed from sucrose gradient-enriched RNA in order to study the repetitive structure of profilaggrin. These clones hybridize to high molecular weight epidermal mRNA (23 kilobase pairs, rat and 19 kilobase pairs, mouse) and exhibit limited cross-hybridization between species. Several rat clones direct the synthesis of a portion of rat profilaggrin in bacteria. One of these, rat profilaggrin cDNA clone R4D6, is 2400 base pairs in length. The R4D6 cDNA is shown to contain repetitive sequence by restriction mapping and southern hybridization analysis of restriction digests of this plasmid, using subfragments of the plasmid as hybridization probes. Southern hybridization analysis of rat genomic DNA, digested to completion with several restriction enzymes, reveals a simple hybridization pattern of fragments equal in size to those of the cDNA. Partial digestion of rat genomic DNA results in a ladder of bands based on a 1200-base pair repeat, equal to the size of the repeating unit of the cDNA clone, and consistent with the expected repeating size of profilaggrin. Together, these results show that the profilaggrin mRNA and gene have repetitive structure and that the gene apparently lacks introns in the coding region.     

An immobilized cell reactor with simultaneous product separation. II. Experimental reactor performance

The simultaneous separation of volatile fermentation products from product-inhibited fermentations can greatly increase the productivity of a bioreactor by reducing the product concentration in the bioreactor, as well as concentrating the product in an output stream free of cells, substrate, or other feed impurities. The Immobilized Cell Reactor-Separator (ICRS) consists of two column reactors: a cocurrent gas-liquid "enricher" followed by a countercurrent "stripper" The columns are four-phase tubular reactors consisting of (1) an inert gas phase, (2) the liquid fermentation broth, (3) the solid column internal packing, and (4) the immobilized biological catalyst or cells. The application of the ICRS to the ethanol-from-whey-lactose fermentation system has been investigated. Operation in the liquid continuous or bubble flow regime allows a high liquid holdup in the reactor and consequent long and controllable liquid residence time but results in a high gas phase pressure drop over the length of the reactor and low gas flow rates. Operation in the gas continuous regime gives high gas flow rates and low pressure drop but also results in short liquid residence time and incomplete column wetting at low liquid loading rates using conventional gas-liquid column packings. Using cells absorbed to conventional ceramic column packing (0.25-in. Intalox saddles), it was found that a good reaction could be obtained in the liquid continuous mode, but little separation, while in the gas continuous mode there was little reaction but good separation. Using cells sorbed to an absorbant matrix allowed operation in the gas continuous regime with a liquid holdup of up to 30% of the total reactor volume. Good reaction rates and product separation were obtained using this matrix. High reaction rates were obtained due to high density cell loading in the reactor. A dry cell density of up to 92 g/L reactor was obtained in the enricher. The enricher ethanol productivity ranged from 50 to 160 g/L h while the stripper productivity varied from 0 to 32 g/L h at different feed rates and concentrations. A separation efficiency of as high as 98% was obtained from the system.     

Multiple copies of phosphorylated filaggrin in epidermal profilaggrin demonstrated by analysis of tryptic peptides

The precursor of mouse (c57/B16) epidermal filaggrin (profilaggrin) is a very large (ca. 500 000 daltons), highly phosphorylated protein containing multiple copies of filaggrin (26 000 daltons). The conversion of profilaggrin to filaggrin late in epidermal cell differentiation involves dephosphorylation and proteolysis to yield the unphosphorylated filaggrin, which polymerizes with keratin filaments into macrofibrils. In order to gain insight in the nature of these processes, we compared tryptic digests of profilaggrin with those of filaggrin by reverse-phase liquid chromatography. Approximately 80% of the profilaggrin mass consists of multiple copies of filaggrin. Twenty peptides purified in good yield from both profilaggrin and filaggrin accounted for most of the filaggrin sequence. A detailed analysis of the yield of several peptides provided an estimate of the size and frequency of the repeat unit within profilaggrin. These data indicate that the repeating substructure of profilaggrin contains about 265 amino acids and that about 50 residues are removed per filaggrin domain as the precursor is processed to filaggrin. Assuming a molecular weight of 500 000 (as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis), this indicates there are 16 repeats. Analysis of phosphopeptides isolated from profilaggrin showed that 66% of the phosphate was located on peptides that are unphosphorylated in filaggrin. Analysis of peptide recoveries confirmed the repeat size and showed that every copy of filaggrin was phosphorylated in profilaggrin.     

Sequence of the OXA2 beta-lactamase: comparison with other penicillin-reactive enzymes

The nucleotide sequence of the unusual plasmid-mediated OXA2 beta-lactamase is presented, and compared with other beta-lactamases. The OXA2 enzyme has similar features at the presumed active site, but no other significant regions of homology with other penicillin-reactive enzymes. The active site homology may therefore represent convergent evolution of otherwise dissimilar genes.     

Two positional isomers of sialylheptasaccharides isolated from the urine of a patient with sialidosis

We describe the structures of two positional isomers of sialylheptasaccharide isolated from the urine of a patient with sialidosis with partial deficiency of beta-galactosidase. Based on structural studies including compositional sugar analysis, exoglycosidase digestion, chemical ionization mass spectrometry, proton nuclear magnetic resonance spectrometry, and methylation analysis, their structures were deduced to be as follows: AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----3(Man alpha 1----6)Man beta 1----4GlcNac; AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNac. Sialyloligosaccharide 1 has previously been found in the urine and liver of patients with mucolipidosis I and II and sialidosis, but sialyloligosaccharide 2 has not been found yet in human urine. These two sialyloligosaccharides could not be completely separated by any chromatographic procedures tested. The analytical techniques, including methylation study and NMR spectroscopy, could not clearly detect the differences between them. However, alpha-mannosidase treatment gave important information for the structural analyses of these sialyloligosaccharides.