Three molecular forms of a rat asialoglycoprotein receptor

It has been reported that a rat asialoglycoprotein receptor is composed of three polypeptide chains with molecular masses of 43, 54, and 64 kilodaltons (43, 54, and 64-Kd forms) and that the first has a different primary structure from the latter two forms. Incorporation of [3H]leucine into these forms showed that no precursor-product relationship is found between the 54-Kd and 64-Kd forms. The half-life of the 43-Kd form (25 h) was shorter than those of the 54-Kd and 64-Kd forms (66 and 70 h, respectively). Glycopeptides of the three forms were prepared from rat livers previously labeled in vivo with [3H]glucosamine. Gel filtration analysis of the glycopeptides before and after endo H treatment revealed that they were all resistant to endo H. Alkali treatment did not change the elution position appreciately. These results indicate that the three molecular forms contained only complex oligosaccharide chains. The receptor was prepared from rat livers previously treated with tunicamycin in vivo and subjected to SDS-PAGE. A distinct band with a molecular mass of 33 Kd was observed. The receptor was also immunoprecipitated from rat hepatocytes in primary culture previously labeled with [35S]methionine and analysed by SDS-PAGE and fluorography. In addition to the major 43-Kd form, a band with a molecular mass of 41 Kd was found and tunicamycin treatment gave rise to a 33-Kd component, which is in good agreement with the receptor purified from tunicamycin treated rats. It is suggested that the 43-Kd form is synthesized as a 33 Kd polypeptide, cotranslationally glycosylated to form the 41 Kd component and then processed to the final 43-Kd form. We also think that the 43-Kd form could bind to asialoorosomucoid-Sepharose 4B without its carbohydrate chains.     

Functional Analysis of Tcl1 Using Tcl1-Deficient Mouse Embryonic Stem Cells

Tcl1 is highly expressed in embryonic stem (ES) cells, but its expression rapidly decreases following differentiation. To assess Tcl1’s roles in ES cells, we generated Tcl1-deficient and -overexpressing mouse ES cell lines. We found that Tcl1 was neither essential nor sufficient for maintaining the undifferentiated state. Tcl1 is reported to activate Akt and to enhance cell proliferation. We found that Tcl1 expression levels correlated positively with the proliferation rate and negatively with the apoptosis of ES cells, but did not affect Akt phosphorylation. On the other hand, the phosphorylation level of β-catenin decreased in response to Tcl1 overexpression. We measured the β-catenin activity using the TOPflash reporter assay, and found that wild-type ES cells had low activity, which Tcl1 overexpression enhanced 1.8-fold. When the canonical Wnt signaling is activated by β-catenin stabilization, it reportedly helps maintain ES cells in the undifferentiated state. We then performed DNA microarray analyses between the Tcl1-deficient and -expressing ES cells. The results revealed that Tcl1 expression downregulated a distinct group of genes, including Ndp52, whose expression is very high in blastocysts but reduced in the primitive ectoderm. Based on these results, we discuss the possible roles of Tcl1 in ES cells.     

Illicit Drug Use Is a Significant Risk Factor for Loss to Follow Up in Patients with HIV-1 Infection at a Large Urban HIV Clinic in Tokyo

Background

Loss to follow up (LTFU) is an important prognostic factor in patients with HIV-1 infection. The impact of illicit drug use on LTFU of patients with HIV-1 infection is unknown in Japan.

Methods

A single center observational study was conducted to elucidate the impact of illicit drug use on LTFU at a large HIV clinic in Tokyo. LTFU was defined as those who discontinued their visits to the clinic for at least 12 months and were not known to be under the care of other facilities or have died within 12 months of their last visit. Patients who first visited the clinic between January 2005 and August 2010 were enrolled. Information on illicit drug use was collected in a structured interview and medical charts. Comparison of the effects of illicit drug use and no use on LTFU was conducted by uni- and multi-variate Cox hazards models as the primary exposure.

Results

The study subjects were 1,208 patients, mostly Japanese men, of relatively young age, and infected through homosexual contact. A total of 111 patients (9.2%) were LTFU (incidence: 24.9 per 1,000 person-years). Among illicit drug users and non users, 55 (13.3%) and 56 (7.1%) patients, respectively, were LTFU, with incidence of 35.7 and 19.2 per 1,000 person-years, respectively. Uni- and multi-variate analyses showed that illicit drug use was a significant risk for LTFU (HR=1.860; 95% CI, 1.282-2.699; p=0.001) (adjusted HR=1.544; 95% CI, 1.028-2.318; p=0.036). Multivariate analysis also identified young age, high CD4 count, no antiretroviral therapy, and no health insurance as risk factors for LTFU.

Conclusions

The incidence of LTFU among illicit drug users was almost twice higher than that among non users. Effective intervention for illicit drug use in this population is warranted to ensure proper treatment and prevent the spread of HIV.     

Human immune responses elicited by an intranasal inactivated H5 influenza vaccine

Intranasally administered influenza vaccines could be more effective than injected vaccines, because intranasal vaccination can induce virus-specific immunoglobulin A (IgA) antibodies in the upper respiratory tract, which is the initial site of infection. In this study, immune responses elicited by an intranasal inactivated vaccine of influenza A(H5N1) virus were evaluated in healthy individuals naive for influenza A(H5N1) virus. Three doses of intranasal inactivated whole-virion H5 influenza vaccine induced strong neutralizing nasal IgA and serum IgG antibodies. In addition, a mucoadhesive excipient, carboxy vinyl polymer, had a notable impact on the induction of nasal IgA antibody responses but not on serum IgG antibody responses. The nasal hemagglutinin (HA)-specific IgA antibody responses clearly correlated with mucosal neutralizing antibody responses, indicating that measurement of nasal HA-specific IgA titers could be used as a surrogate for the mucosal antibody response. Furthermore, increased numbers of plasma cells and vaccine antigen-specific Th cells in the peripheral blood were observed after vaccination, suggesting that peripheral blood biomarkers may also be used to evaluate the intranasal vaccine-induced immune response. However, peripheral blood immune cell responses correlated with neutralizing antibody titers in serum samples but not in nasal wash samples. Thus, analysis of the peripheral blood immune response could be a surrogate for the systemic immune response to intranasal vaccination but not for the mucosal immune response. The current study suggests the clinical potential of intranasal inactivated vaccines against influenza A(H5N1) viruses and highlights the need to develop novel means to evaluate intranasal vaccine-induced mucosal immune responses.     

Photoreactivities of 5-Bromouracil-containing RNAs

5-Bromouracil (^BrU) was incorporated into three types of synthetic RNA and the products of the photoirradiated ^BrU-containing RNAs were investigated using HPLC and MS analysis. The photoirradiation of r(GCA^BrUGC)₂ and r(CGAA^BrUUGC)/r(GCAAUUCG) in A-form RNA produced the corresponding 2′-keto adenosine (^ketoA) product at the 5′-neighboring nucleotide, such as r(GC^ketoAUGC) and r(CGA^ketoAUUGC), respectively. The photoirradiation of r(CGCG^BrUGCG)/r(C^mGCAC^mGCG) in Z-form RNA produced the 2′-keto guanosine (^ketoG) product r(CGC^ketoGUGCG), whereas almost no products were observed from the photoirradiation of r(CGCG^BrUGCG)/r(C^mGCAC^mGCG) in A-form RNA. The present results indicate clearly that hydrogen (H) abstraction by the photochemically generated uracil-5-yl radical selectively occurs at the C2′ position to provide a 2′-keto RNA product.     

Isolation of Protein Inhibitors of Papain,Trypsin, and α-Amylase in the Grain of Foxtail Millet

The amino acid sequence of Indian peafowl egg-white lysozyme has been identified. The reduced and carboxymethylated lysozyme was digested with trypsin followed by purification of the resulting peptides by reverse-phase HPLC. The tryptic peptides obtained were sequenced using the DABITC/PITC double coupling manual sequencing method. The alignment of the tryptic peptides were deduced by comparison with corresponding peptides of hen egg-white lysozyme. This protein proved to consist of 129 amino acid residues, and a relative molecular mass of 14423 Da was calculated. Amino acid sequence comparison of peafowl lysozyme and other phasianoid bird lysozymes revealed a maximum homology ratio of 98% with turkey lysozyme.     

Immunocytochemical identification of axons containing coexistent serotonin and substance P in the cat lumbar spinal cord

Axons containing both serotonin-like (5-HT)-LI and substance P-like (SP)-LI immunoreactivity were identified in all laminae of the cat spinal cord at the level of the lumbar enlargement. Using an immunologically-specific, double immunofluorescence method, coexistent 5-HT-LI and SP-LI immunoreactivity could be visualized in the same tissue section with appropriate FITC and rhodamine fluorescent filter sets. The fewest number of coexistent axons were observed in the superficial laminae of the dorsal horn, while their number increased in the more ventral dorsal horn laminae. Numerous coexistent axons were observed in the area adjacent to the central canal. The greatest number of coexistent axons was found in the ventral horn, especially in the motoneuronal cell groups. This study demonstrates that axons containing coexistent 5-HT-LI and SP-LI immunoreactivity are found in all laminae of the cat lumbar spinal cord and are thus involved in both sensory and motor functions. Their more frequent occurrence in the ventral horn suggests a greater role for coexistent 5-HT and SP in motor function. Since axons containing coexistent 5-HT and SP, and those containing only 5-HT, likely originate from different populations of neurons, our observations provide evidence for a diverse origin of descending 5-HT afferents to the different spinal laminae.     

Correction of ornithine transcarbamylase (OTC) deficiency in spf-ash mice by introduction of rat OTC gene

We introduced rat ornithine transcarbamylase (OTC) gene into OTC-deficient spf-ash mice by mating spf-ash heterozygotes with transgenic mice which carried recombinant DNA composed of 1.3 kb of the 5' flanking region of the gene fused onto rat OTC cDNA. The liver OTC activity of hemizygous spf-ash mice which carried the transgene was about twice that of nontransgenic spf-ash mice, and the small intestinal OTC activity was 6 times higher; the values being 12% and 27% of the control levels, respectively. The transgenic spf-ash mice showed normal hair growth without sparse fur, nearly normalized urinary orotic acid excretion and normalized serum citrulline concentration.     

Identification of asymptomatic Entamoeba histolytica infection by a serological screening test: A cross-sectional study of an HIV-negative men who have sex with men cohort in Japan

BackgroundAmebiasis, caused by Entamoeba histolytica, is spreading in developing countries and in many developed countries as a sexually transmitted infection. Here, we evaluated the efficacy of serological screening to identify asymptomatic E. histolytica infection as a potential epidemiological control measure to limit its spread.Methodology/Principal findingsThis cross-sectional study was carried out between January and March 2021 in an HIV-negative men who have sex with men (MSM) cohort at the National Center for Global Health and Medicine. Serological screening was performed using a commercially available ELISA kit. For seropositive individuals, we performed stool polymerase chain reaction (PCR) to determine current E. histolytica infection. We performed E. histolytica serological screening of 312 participants. None had a history of E. histolytica infection prior to the study. The overall E. histolytica seropositivity was 6.7% (21/312), which was similar to that found by the rapid plasma reagin test (17/312). We identified current infection in 8 of 20 seropositive participants (40.0%) by stool PCR.Conclusions/SignificanceOur serological screening approach constitutes a potentially practical epidemiological strategy. Active epidemiological surveys, in combination with an effective screening strategy for asymptomatically infected individuals, should be applied to help reduce sexually transmitted E. histolytica infections.