Stereochemical Control in Microbial Reduction. Part 10. Asymmetric Reduction of Alkyl 3-Methyl-2-Oxobutanoate with Immobilized Bakers' Yeast in Hexane

Esters of 3-methyl-2-oxobutanoic acid are reduced with bakers' yeast by three methods: free bakers' yeast in water, immobilized bakers' yeast in water, and immobilized bakers' yeast in hexane. Although (R)-hydroxy esters are obtained in all cases, the enantiomeric excess varies from 3% (reduction of the methyl ester with free bakers' yeast in water) to 93% (reduction of the butyl ester with immobilized bakers' yeast in hexane) depending on the structure of substrate and on the reaction conditions. The mechanism of the present stereochemical control is discussed.     

Enzyme-Histochemical Identification of Lymphatic Vessels by Light and Backscattered Image Scanning Electron Microscopy

The walls of lymphatics are characterized by strong 5'-nucleotidase activity, whereas those of blood capillaries reveal significantly lower or no activity. Alkaline phosphatase activity, on the other hand, is markedly higher in blood capillaries than in lymphatic vessels. On the basis of such characteristics, lymphatics and blood capillaries were distinguished histochemically in rat stomach using 5'-nucleotidase-alkaline phosphatase double staining. The distribution and intensity of lead-demonstrated 5'-nucleotidase activity in lymphatic vessels could be determined by comparing the images of the same histochemically stained cryostat section as seen by light and backscattered image scanning electron microscopy. The specificity of the 5'-nucleotidase reaction was obtained by inhibiting nonspecific alkaline phosphatase by including L-tetramisole in the 5'-nucleotidase incubation medium. The products of the 5'-nucleotidase activity were deposited on the outer surface of the plasma membrane of the lymphatic endothelial cells.     

Chinese hamster ovary cells continuously secrete a cysteine endopeptidase

Summary The protease activity in serum-free conditioned medium of chinese hamster ovary (CHO) cells was measured using peptidyl (or aminoacyl)-4-methylcoumaryl-7-amides (MCAs) as the substrates. Aminopeptidase increased in level as amounts of nonviable cells increased during cultivation in serum-free medium, indicating that the activity seems to be originated from intracellular proteases. The activity toward Boc-Leu-Arg-Arg-MCA, which was strongly inhibited by p-chloromercuribenzonate and N-ethylmaleimide, was the strongest among those toward peptidyl-MCAs in the conditioned medium within 48 h-cultivation in serum-free medium. In contrast to the case of aminopeptidase activity, the endopeptidase activity decreased in level after 48 h-cultivation although amounts of nonviable cells increases. Thus, CHO cells continuously secrete the cysteine proteases. This work was supported by the management of the Research Association for Biotechnology as a part of the R&D of Basic Technology for Future Industries sponsored by NEDO (New Energy and Industrial Technology Development Organization).     

Prosaposin Facilitates Sciatic Nerve Regeneration In Vivo

Abstract: Prosaposin, a multifunctional protein, is the precursor of saposins, which activate sphingolipid hydrolases. In addition to acting as a precursor for saposins, prosaposin has been shown to rescue hippocampal CA1 neurons from lethal ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Here we show that prosaposin, when added to a collagen-filled nerve guide after sciatic nerve transection in guinea pigs, increased dramatically the number of regenerating nerve fibers within the guide. To identify the target neurons of prosaposin during peripheral nerve regeneration, we determined the degree of atrophy and chromatolysis of neurons in the spinal anterior horn and dorsal root ganglia on the prosaposin-treated and untreated side. The effect of prosaposin on large spinal neurons and small neurons of the dorsal root ganglion was more conspicuous. Subsequent immunohistochemistry demonstrated that the atrophy of cholinergic large neurons in the anterior horn is prevented to significant extent by prosaposin treatment. These findings suggest that prosaposin promotes peripheral nerve regeneration by acting on α-motor neurons in the anterior horn and on small sensory neurons in the dorsal root ganglion. The present study raises the possibility of using prosaposin as a tool for the treatment of peripheral nerve injuries.     

Strength of translation initiation signal sequence of mRNA as studied by quantification method: effect of nucleotide substitutions upon translation efficiency in rat preproinsulin mRNA.

Concerning the translation initiation signals in vertebrate mRNAs, both the ATG initiation codon and the sequences flanking the initiation codon are required to direct the position of initiation. A consensus sequence for the signal, (GCC)GCC(A or G)CCATGG, has been proposed, but actual initiation sequences differ from it to a greater or lesser degree. In the present report, the translation initiation signal sequences of rat preproinsulin and its mutant mRNAs were analyzed using a quantification method proposed previously. In this method, each 16 nt sequence in the mRNA was characterized by its sample score, which shows strength of the signal. So far, Kozak has constructed a number of preproinsulin mutant mRNAs in which nucleotides flanking the ATG codon are systematically varied, and measured the translation initiation efficiency in terms of the proinsulin product. Her experimental results were well understood on the basis of the strength of the translation initiation signal sequence.     

Studies on the plant cytoskeleton using miniprotoplasts of tobacco BY-2 cells

Vacuoles in plant cells can be eliminated by centrifugation of protoplasts through a density gradient. In this review, properties of evacuolated protoplasts, named ‘miniprotoplasts’, and the significant roles in plant cytoskeleton studies are described. Miniprotoplasts, prepared from tobacco BY-2 cells whose cell-cycle had been synchronized at late anaphase, continued to divide to form two daughter cells. In the presence of cytochalasin B cytokinetic cleavage was enhanced, suggesting a role of actin filaments in plant cytokinesis. In the cytoplasmic extract of miniprotoplasts both tubulin and actin could be polymerized to form microtubules (MTs) and actin filaments (AFs), respectively. A purification method for tubulin, actin and related proteins was developed using the extract. To investigate the interaction between cortical microtubules and the plasma membrane, an experimental system in which MTs were reconstructed on membrane ghosts was developed by combination of membrane ghosts and the extract.     

Genetically polymorphic α-l-fucosidase (FUCA1) isozymes detected in blood plasma

The main isozyme patterns of desialylated blood plasma or serum -l-fucosidase (FUCA) were found to be almost identical to those of semen, urine, placental extracts, and leukocyte lysates, when detected by polyacrylamide gel isoelectric focusing, and activity staining using the fluorogenic substrate 4-methylumbelliferyl--l-fucopyranoside. Three phenotypes (1, 2-1, and 2) determined from plasma samples were identical to the phenotypes from urine and leukocyte lysates from the same individuals. A population study of plasma samples collected from 485 Japanese individuals indicated that the frequencies of the FUCA11 ^* and FUCA12 ^* alleles were 0.7505 and 0.2495, respectively. The mean plasma enzyme activities (+SD) of the three phenotypes were 318.8 ± 116.7 nmol/ml per h for type 1, 268.0 ± 108.3 nmol/ml per h for type 2-1, and 233.2 ± 84.4 nmol/ml per h for type 2. The mean activities of types 1 and 2 suggest that, on average, the FUCA11 ^* gene product in plasma has about 1.4 times the activity of FUCA12 ^*.     

Production and composition of extracellular polysaccharide from cell suspension cultures of Mentha

A suspension culture of Mentha was established from callus which formed on the tips of young shoots of a Mentha hybrid (M. arvenis × M. spicata). Changes in growth parameters during a culture cycle were recorded. The general appearance of cells during division and growth, including the changes in cell form, was also represented.Suspension-cultured cells of Mentha hybrid released a large amount of extracellular polysaccharides (ECP) mainly at the logarithmic phase of the growth cycle. The ECP contained galacturonic acid as major components and arabinose, galactose, glucose, xylose, rhamnose and mannose as minor components. The ratio of the uronic acid content to total sugar content in the ECP was below 40% at day 7, but increased up to 90% at day 21. The relative contents of xylose and glucose in the ECP decreased during the culture period, while the arabinose content increased and those of rhamnose, mannose and galactose remained constant.The IR spectrum suggested that the ECP were low-methoxylated pectic polysaccharides. The presence of lignin and related compounds in the ECP was not detected. The protein content of the ECP was about 10% and the main amino acids were alanine, proline, hydroxyproline, valine, asparticacid and serine, in that order.     

A rice (Oryza sativa L.) mutant having a low content of glutelin and a high content of prolamine

Among the mutant lines of rice that have been selected for morphological characters, one line, NM67, was found to have a low content of glutelin and a higher content of prolamine in its seed protein than other Japanese cultivars. This mutant is a semi-dwarf and partially sterile line, and its leaves turn yellow before heading. Genetic analysis after backcross to the original cultivar, Nihonmasari, revealed the following: (1) the character of low glutelin content was always accompanied by the character of high prolamine content; (2) the low glutelin (and high prolamine) character seemed to be manifested by a single dominant gene; and (3) semi-dwarfness, low fertility and early yellowing leaf of the mutant, which might also be pleiotropy, were controlled by a single recessive gene independent of the gene for protein content. The protein character of NM67 was genetically separated from semi-dwarfness and low fertility, and a new line having low glutelin content and high prolamine content with normal morphological characters comparable to those of the original cultivar was obtained from the progenies of the cross. The possible use of this line as a low protein rice cultivar is discussed.     

Endogenous methionine enkephalin may play an anticonvulsant role in the seizure-susceptible El mouse

After the intracisternal injection of three protease inhibitors which prevent the degradation of methionine enkephalin (amastatin, Des-Pro²-bradykinin, and phosphoramidon) and a mixture of these protease inhibitors, we investigated the effect on convulsive seizures in the seizure-susceptible El mouse. We also measured the cerebral methionine enkephalin content by high-performance liquid chromatography coupled with radioimmunoassay. Protease inhibitors significantly decreased both the incidence of seizures and the seizure score in El mice in a dose-dependent manner. This anticonvulsant effect was reversed by naloxone (2 mg/kg, sc). The cerebral methionine enkephalin content increased significantly after the administration of protease inhibitors in comparison with saline injection. These findings suggest that it was not protease inhibitors but instead increase of endogenous methionine enkephalin that reduced the incidence of seizures and the seizure score in El mice. Together with our previous data, the present findings support our hypothesis that a deficit in anticonvulsant endogenous methionine enkephalin is involved in the pathogenesis of seizures in the El mouse.