HPLC-studies on nonmercapt-mercapt conversion of human serum albumin

Human mercaptalbumin (HMA) and nonmercaptalbumin (HNA) could be separated by high-performance liquid chromatography (HPLC) at neutral pH. Using HPLC, the present authors found the nonmercapt-mercapt conversion (HNA----HMA) during hemodialysis and the mercapt-nonmercapt conversion (HMA----HNA) after hemodialysis in chronic renal failure, indicating HMA as the covalent carrier protein for sulfur-containing amino acids.     

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.     

Effect of pH on preferential antibacterial-activity of ethylenediaminetetraacetic acid (EDTA)]

Ethylenediaminetetraacetic acid (EDTA), a chelating agent, was examined for the antibacterial activity against 15 species of bacteria by treating with a 10mM solution at pH adjusted to 5.0, 7.0 or 9.0. All bacterial species tested were classified into three groups; tentatively named the pH5 EDTA-sensitive group comprising Vibrio cholerae and Staphylococcus aureus, the pH9 EDTA-sensitive group comprising Escherichia coli and Pseudomonas aeruginosa and the EDTA-nonsensitive group comprising Proteus mirabilis. The EDTA-sensitivity grouping may be used as a tool for preferential decontamination of certain bacteria in live edible fishes, although further experiments are needed to characterize more strains and also species of bacteria.     

Expression of the pS2 gene in human gastric cancer cells derived from poorly differentiated adenocarcinoma

NH2-terminal amino acid sequence of the pS2 protein produced and secreted by human gastric cancer cells, MKN-45, was determined to be identical to that of MCF-7 cells. A clone encoding pS2 protein was isolated from the cDNA library constructed from MKN-45 cells. The nucleotide sequence was identical to that of pS2 cDNA previously isolated from human breast cancer cells, MCF-7, except for one nucleotide in the 3' untranslated region. Thus, in this cell line, the pS2 gene product is translated and secreted as in MCF-7 cells. RNA blot hybridization analysis revealed that pS2 gene was expressed well in two (MKN-45 and KATO-III; derived from poorly differentiated adenocarcinoma) but not in three cell lines (MKN-1, MKN-28 and MKN-74; from well differentiated adenocarcinoma), suggesting that expression of the pS2 gene depends on the state of cell differentiation. These results suggest that pS2 is expressed in human gastric cancer cells in an estrogen-independent manner and is possibly associated with the malignant state of cells.     

Studies on the transport of vitamin D and of 25-hydroxyvitamin D in human plasma.

A study was conducted to investigate whether human plasma contains one or more than one protein for the transport of vitamin D and of 25-hydroxyvitamin D (25-OH-D).Serum was labeled in vivo with a mixture of radioactive vitamin D3 (derived from orally administered tracer vitamin D3) and of endogenously synthesized labeled 25-OH-D3. Samples of such serum were subjected to several different protein fractionation procedures. Only a single peak of protein-bound radioactivity was observed after each of these procedures. The fraction comprising the ascending and the descending limbs of the single peak of protein-bound radioactivity (after each procedure) were separately pooled. In each instance the ratio of radioactive 25-OH-D3 to radioactive vitamin D3 was found to be almost identical in both the ascending and the descending limbs. Taken together, these findings provide strong evidence that human serum contains only a single binding protein responsible for the normal transport of both vitamin D and 25-OH-D. Plasma labeled in vitro with added 3H-labeled 25-OH-D3 was subjected to gel filtration on Sephadex G-200 and to chromatography on columns of DEAE-cellulose and of SP-Sephadex. After each of these procedures a single peak of protein-bound radioactivity was observed, with elution profiles of protein and of radioactivity that were identical with those observed with in vivo labeled serum. These data indicate that tracer 25-OH-D3 added to plasma in vitro binds to the same plasma protein normally responsible for the transport of vitamin D and of 25-OH-D.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

Protein A-Mouse Acidic Mammalian Chitinase-V5-His Expressed in Periplasmic Space of Escherichia coli Possesses Chitinase Functions Comparable to CHO-Expressed Protein

Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)₆ tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N′-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.     

Amelioration of pneumonia with Streptococcus pneumoniae infection by inoculation with a vaccine against highly pathogenic avian influenza virus in a non-human primate mixed infection model

Background  Highly pathogenic avian influenza virus (HPAIV) infection has a high mortality rate in humans. Secondary bacterial pneumonia with HPAIV infection has not been reported in human patients, whereas seasonal influenza viruses sometimes enhance bacterial pneumonia, resulting in substantial morbidity and mortality. Therefore, if HPAIV infection were accompanied by bacterial infection, an increase in mortality would be expected. We examined whether a vaccine against HPAIV prevents severe morbidity caused by mixed infection with HPAIV and bacteria.
Methods  H7N7 subtype of HPAIV and Streptococcus pneumoniae were inoculated into cynomolgus macaques with or without vaccination of inactivated whole virus particles .
Results  Vaccination against H7N7 HPAIV decreased morbidity caused by HPAIV and pneumonia caused by S. pneumoniae . Bacterial replication in lungs was decreased by vaccination against HPAIV, although the reduction in bacterial colonies was not significant.
Conclusions  Vaccination against HPAIV reduces pneumonia caused by bacterial superinfection and may improve prognosis of HPAIV-infected patients.     

Synergistic effects of TAL1 over-expression and PHO13 deletion on the weak acid inhibition of xylose fermentation by industrial Saccharomyces cerevisiae strain

In the industrial production of bioethanol from lignocellulosic biomass, a strain of Saccharomyces cerevisiae that can ferment xylose in the presence of inhibitors is of utmost importance. The recombinant, industrial-flocculating S. cerevisiae strain NAPX37, which can ferment xylose, was used as the parent to delete the gene encoding p-nitrophenylphosphatase (PHO13) and overexpress the gene encoding transaldolase (TAL1) to evaluate the synergistic effects of these two genes on xylose fermentation in the presence of weak acid inhibitors, including formic, acetic, or levulinic acids. TAL1 over-expression or PHO13 deletion improved xylose fermentation as well as the tolerance of NAPX37 to all three weak acids. The simultaneous deletion of PHO13 and the over-expression of TAL1 had synergistic effects and improved ethanol production and reduction of xylitol accumulation in the absence and presence of weak acid inhibitors.