Enhancement of 1,4-dihydroxy-2-naphthoic acid production by Propionibacterium freudenreichii ET-3 fed-batch culture

The production of 1,4-dihydroxy-2-naphthoic acid (DHNA) was investigated using a fed-batch culture of Propionibacterium freudenreichii ET-3. DHNA is a precursor of menaquinone (MK) and is transformed to MK by combination with an isoprenoid unit. We found that ET-3 stopped MK production and increased DHNA production in an anaerobic fed-batch culture by maintaining the lactose concentration at approximately zero. The maximum DHNA concentration observed in the anaerobic fed-batch culture was markedly higher than the maximum DHNA concentration observed in an anaerobic batch culture. Moreover, MK or DHNA production was affected by the lactose feeding rate; this suggests that lactose metabolism participates in the syntheses of these products. On the other hand, accumulation of propionate was found to inhibit DHNA production in the fed-batch culture. Based on the fact that ET-3 increases DHNA production in an aerobic culture by consuming propionate, we carried out a cultivation experiment in which an anaerobic fed-batch culture was switched to an anaerobic batch culture and found that the DHNA production was increased to a greater extent than the DHNA production in an anaerobic fed-batch culture. These results suggest that DHNA production by ET-3 is markedly influenced by carbon source limitation and the oxygen supply.     

Release of transforming plasmid DNA from actively growing genetically engineered Escherichia coli

We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria.     

Treatment with leucine stimulates the production of hepatocyte growth factor in vivo

Hepatocyte growth factor (HGF) has pleiotropic effects. Up-regulation of HGF activity in vivo may be beneficial. Branched-chain amino acids (BCAAs) are known to modulate various cellular functions. When starved rats received intraperitoneal injections of valine, leucine or isoleucine, only leucine treatment increased both hepatic and circulating levels of HGF in a dose-dependent manner, up to 1.5 and 2.3 times higher, respectively, than in controls. When young growing rats with free access to food were injected with leucine once a day for a week, HGF levels and liver weights were significantly higher than those of control rats. Furthermore, 1 week of leucine treatment of adult rats resulted in elevated serum albumin levels with an increase in HGF levels. Taken together with our previous report showing that leucine stimulates HGF production by hepatic stellate cells in culture, leucine, among BCAAs, may induce an increase in HGF production by the liver in vivo.     

Time Course of Optical Quality and Intraocular Scattering after Refractive Lenticule Extraction

Purpose

To assess the time course of optical quality and intraocular scattering in relation to visual acuity after femtosecond lenticule extraction (FLEx) for the correction of myopia.

Methods

This study evaluated 36 eyes of 36 patients with spherical equivalents of −4.38±1.53 D [mean ± standard deviation] who underwent FLEx. Before surgery, and 1 week and 1, 3 and 6 months after surgery, we assessed the modulation transfer function (MTF) cutoff frequency, Strehl ratio, objective scattering index (OSI), and OQAS values (OVs), using a double-pass instrument. We also investigated the relationship of the OSI with corrected distance visual acuity (CDVA) preoperatively and postoperatively.

Results

The mean changes in MTF cutoff frequency, Strehl ratio, OSI, OV100%, OV20%, and OV9% preoperatively and 6 months postoperatively were −5.51±15.01, −0.03±0.07, 0.35±0.83, −0.17±0.48, −0.14±0.38, and −0.09±0.22, respectively. We found no significant preoperative correlation between the OSI and logMAR CDVA (Spearman rank correlation coefficient r = 0.068, p = 0.69), and modest, but significant correlations 1 week and 1, 3, and 6 months postoperatively (r = 0.572, r = 0.562, r = 0.542, r = 0.540, p<0.001, respectively).

Conclusions

FLEx induced a transient decrease in optical quality in association with an increase in intraocular scattering in the early postoperative period, possibly due to mild interface haze formation, but gradually recovered with time. It is suggested that this transient degradation in optical quality related to an increase in the intraocular scattering may result in a slight delay of CDVA recovery in the early postoperative period.     

A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.     

Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.     

Rapid Identification of Mutans Streptococcal Species

Mutans streptococci are considered the predominant pathogens in dental caries. Three methods, i.e. dot blot hybridization analysis, PCR analysis and SDS-blue dextran-PAGE, were examined for identifying mutans streptococcal species. In dot blot hybridization, DNA probe derived from the dextranase gene (dexA) of Streptococcus mutans hybridized with different intensities under the condition of low stringency against each species of mutans streptococci although the dexA probe was specific for S. mutans under the condition of high stringency. Oligonucleotide primers for polymerase chain reaction (PCR) were designed on the basis of the dexA DNA sequence. The primers amplified species-specific PCR products in the reference species (15 strains of 5 species) of mutans streptococci. An electrophoretic profile of dextranases from the mutans streptococci on SDS-blue dextran-PAGE also showed species-specific behavior. These results suggest that the three identification methods examined here are useful for distinguishing the species of mutans streptococci and also indicate that PCR analysis is suitable for simple, rapid and reliable identification of mutans streptococcal species.     

Mast cells contain spleen-type prostaglandin D synthetase

Prostaglandin D synthetase activity in the cytosol (100,000 x g, 1-h supernatant) fraction of peritoneal mast cells of adult rats (105.0 nmol/min/mg protein) was the highest among such activities in various rat tissues and cells. As judged by the absolute requirement for glutathione for the reaction (Km = 300 microM), the Km value for prostaglandin H2 (200 microM), and insensitivity of the activity to 1 mM 1-chloro-2,4-dinitrobenzene, the enzyme in mast cells was similar to rat spleen prostaglandin D synthetase and differed from rat brain prostaglandin D synthetase or glutathione S-transferase, all of which catalyze the isomerase reaction from prostaglandin H2 to prostaglandin D2. In immunotitration analyses, the activity in mast cells showed a titration curve exactly identical with that of the purified spleen-type enzyme and almost completely absorbed by an excess amount of antibody against this enzyme, but it remained unchanged after incubation with antibodies against the brain-type enzyme and glutathione S-transferase isozymes thus far purified. In Western blot after two-dimensional electrophoresis of crude extracts of mast cells, a single immunoreactive spot was observed with antibody against the spleen-type enzyme at the same position as that of the purified enzyme (Mr = 26,000, pI = 5.2). Furthermore, the immunoreactive protein obtained from mast cells showed the same peptide fingerprints as those of the purified spleen-type enzyme, after partial digestion with Staphylococcus aureus V8 protease or trypsin. In immunoperoxidase staining, the immunoreactivity of the spleen-type enzyme was found in the cytosol of tissue mast cells in various organs such as thymus, intestine, stomach, and skin of adult rats. These findings indicate that prostaglandin D2 is produced by the spleen-type synthetase in mast cells of various tissues.