Morphological identification of hepatitis C virus E1 and E2 envelope glycoproteins on the virion surface using immunogold electron microscopy

It is known that hepatitis C virus (HCV) particles are spherical, 55-65 nm particles with fine surface projections of about 6 nm in length and with a 30-35 nm inner core. We have reported that free HCV particles labeled with gold particles specific to the HCV E1 glycoprotein are located in 1.14-1.16 g/ml fractions from plasma samples with high HCV RNA titers after sucrose density gradient centrifugation. However, the morphology of the HCV E2 glycoprotein on the virion has not yet been elucidated. To visualize HCV E2 localization on the virion, we used the same plasma samples where HCV particles were clearly shown. An indirect immunogold electron microscopic study was carried out using monoclonal and polyclonal anti-HCV E2 antibodies. HCV-like particles specifically reacted with the anti-HCV E2 antibodies. Moreover, to evaluate the localization of the HCV E1 and E2 glycoproteins on the virion surface, an immunogold electron microscopic study using double labeling with anti-HCV E1 antibodies and anti-HCV E2 antibodies was also performed. These particles also specifically reacted with both anti-E1 and E2 antibodies. This is the first report showing the presence of both HCV E1 and E2 glycoproteins on HCV virion surface in human plasma samples.     

Axonal regeneration of cat retinal ganglion cells is promoted by nipradilol, an anti-glaucoma drug

Neurons in the CNS can regenerate their axons in an environment of the peripheral nervous system, but this ability is limited. Here we show that an anti-glaucoma drug, nipradilol, at low concentration led to a four-fold increase in the number of cat retinal ganglion cells regenerating their axons into a transplanted peripheral nerve 4 and 6 weeks after axotomy. Nipradilol also increased the number of three main regenerating retinal ganglion cell types (alpha, beta, not alpha/beta), and enhanced the rate of axonal regeneration of these retinal ganglion cells. Nipradilol is a donor of nitric oxide and an antagonist of alpha-1, beta-1 and -2 adrenoreceptors, and we therefore examined whether one of these pharmacological effects might be more important in promoting axon regeneration. A nitric oxide donor increased the number of regenerating retinal ganglion cells, but not the rate of axonal regeneration. Denitro-nipradilol (nitric oxide-deprived nipradilol) or a nitric oxide scavenger injected before nipradilol increased the number of regenerating retinal ganglion cells but did not promote regeneration rate. Blockade of individual alpha- and beta-adrenoreceptors did not increase the number of regenerating retinal ganglion cells or the rate of regeneration. From these results, it is suggested that nitric oxide plays a crucial role in mediating the effects of nipradilol on axon regeneration and neuroprotection, and the metabolite of nipradilol supports the effects.     

Deletion of the CaBIG1 gene reduces beta-1,6-glucan synthesis, filamentation, adhesion, and virulence in Candida albicans

The human fungal pathogen Candida albicans is able to change its shape in response to various environmental signals. We analyzed the C. albicans BIG1 homolog, which might be involved in beta-1,6-glucan biosynthesis in Saccharomyces cerevisiae. C. albicans BIG1 is a functional homolog of an S. cerevisiae BIG1 gene, because the slow growth of an S. cerevisiae big1 mutant was restored by introduction of C. albicans BIG1. CaBig1p was expressed constitutively in both the yeast and hyphal forms. A specific localization of CaBig1p at the endoplasmic reticulum or plasma membrane similar to the subcellular localization of S. cerevisiae Big1p was observed in yeast form. The content of beta-1,6-glucan in the cell wall was decreased in the Cabig1Delta strain in comparison with the wild-type or reconstituted strain. The C. albicans BIG1 disruptant showed reduced filamentation on a solid agar medium and in a liquid medium. The Cabig1Delta mutant showed markedly attenuated virulence in a mouse model of systemic candidiasis. Adherence to human epithelial HeLa cells and fungal burden in kidneys of infected mice were reduced in the Cabig1Delta mutant. Deletion of CaBIG1 abolished hyphal growth and invasiveness in the kidneys of infected mice. Our results indicate that adhesion failure and morphological abnormality contribute to the attenuated virulence of the Cabig1Delta mutant.     

Apoptosis in the developing mouse heart.

Apoptosis occurs at high frequency in the myocardium of the developing avian cardiac outflow tract (OFT). Up- or down-regulating apoptosis results in defects resembling human conotruncal heart anomalies. This finding suggested that regulated levels of apoptosis are critical for normal morphogenesis of the four-chambered heart. Recent evidence supports an important role for hypoxia of the OFT myocardium in regulating cell death and vasculogenesis. The purpose of this study was to determine whether apoptosis in the outflow tract myocardium occurs in the mouse heart during developmental stages comparable to the avian heart and to determine whether differential hypoxia is also present at this site in the murine heart. Apoptosis was detected using a fluorescent vital dye, Lysotracker Red (LTR), in the OFT myocardium of the mouse starting at embryonic day (E) 12.5, peaking at E13.5-14.5, and declining thereafter to low or background levels by E18.5. In addition, high levels of apoptosis were detected in other cardiac regions, including the apices of the ventricles and along the interventricular sulcus. Apoptosis in the myocardium was detected by double-labeling with LTR and cardiomyocyte markers. Terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) and immunostaining for cleaved Caspase-3 were used to confirm the LTR results. At the peak of OFT apoptosis in the mouse, the OFT myocardium was relatively hypoxic, as indicated by specific and intense EF5 staining and HIF1alpha nuclear localization, and was surrounded by the developing vasculature as in the chicken embryo. These findings suggest that cardiomyocyte apoptosis is an evolutionarily conserved mechanism for normal morphogenesis of the outflow tract myocardium in avian and mammalian species.     

Comprehensive genetic analysis of relevant four genes in 49 patients with Marfan syndrome or Marfan-related phenotypes

In order to evaluate the contribution of FBN1, FBN2, TGFBR1, and TGFBR2 mutations to the Marfan syndrome (MFS) phenotype, the four genes were analyzed by direct sequencing in 49 patients with MFS or suspected MFS as a cohort study. A total of 27 FBN1 mutations (22 novel) in 27 patients (55%, 27/49), 1 novel TGFBR1 mutation in 1 (2%, 1/49), and 2 recurrent TGFBR2 mutations in 2 (4%, 2/49) were identified. No FBN2 mutation was found. Three patients with either TGFBR1 or TGFBR2 abnormality did not fulfill the Ghent criteria, but expressed some overlapping features of MFS and Loeys-Dietz syndrome (LDS). In the remaining 19 patients, either of the genes did not show any abnormalities. This study indicated that FBN1 mutations were predominant in MFS but TGFBRs defects may account for approximately 5-10% of patients with the syndrome.     

Evaluation of the vestibular evoked myogenic potential during parabolic flight in humans

The purpose of this study was to investigate how gravity affects the vestibular evoked myogenic potential (VEMP). Eight healthy subjects (seven men, one woman; age range 19–45 years) participated in experiments in which three different gravity levels [microgravity (MG), normal gravity (NG), and hypergravity (HG)] were imposed during a parabolic flight procedure. The VEMP was evoked in response to an intense mono-aural click while the subjects kept the sternocleidomastoid (SCM) muscle contracted bilaterally. Background electromyographic activity of the SCM during the test was corrected. The p13–n23 amplitude was significantly greater under MG than under NG or HG. There was no difference in p13 latency between the three gravity levels. Possible mechanisms related to this phenomenon are discussed.     

Normal development of the gut-associated lymphoid tissue except Peyer's patch in MyD88-deficient mice

MyD88 is a key adaptor molecule for signalling via Toll-like receptors (TLRs) and the response to gut commensal microbes. To investigate the role of TLRs/MyD88 pathway in the development of the gut-associated lymphoid tissue (GALT), we examined the development of Peyer's patches (PPs) and cryptopatch (CP), and also one of effector compartment, intraepithelial lymphocyte (IEL) in MyD88-/-, TLR2-/- and TLR4-/- mice. In MyD88-/- mice, the organogenesis of PPs was not disturbed. However, PPs in 2-week-old MyD88-/- mice were significantly smaller than those in MyD88+/- mice. Also, in 2-week-old TLR4-/-, but not TLR2-/- mice, PPs did not develop rapidly. The development of PPs in MyD88-/- and TLR4-/- mice was completely recovered in 10 weeks. PP cells from MyD88-/- mice showed significant decrease in proliferation when stimulated with lipopolysaccharide. The development of CP and IEL was also normal in 10-week-old MyD88-/- mice. These results suggest that the TLRs/MyD88 pathway might be involved in the development of PPs only at early postnatal stage, and TLRs/MyD88-independent signalling is critically involved in the development of GALT in adult mice.     

Experience with the JMS fully automated dialysis machine

A fully automated dialysis machine has been developed and evaluated clinically. It uses highly pure dialysate (produced by a new dialysate cleaning system) instead of the conventional physiologic saline for the processes of priming, guiding blood to the dialysis machine, replenishing fluid, and returning the blood to the body. The piping for the dialysate is in the shape of a loop, and the dialyzer coupler has no mechanical parts that might become contaminated. As a result of these and certain other improvements in machine design, it is now possible to obtain reasonably clean dialysate. For the priming process, the machine uses a volume of up to 4 L of the dialysate after reverse filtration from the dialyzer. Most foreign matter or eluates can be removed from the dialyzer and the blood channels. Before blood is guided out of the body into the dialysis system, the needles inserted in the artery and vein are simultaneously connected to the blood channel, and the dialysate remaining in the channel is removed from the dialyzer. If the patient's blood pressure falls during dialysis, the dialysate can be replenished at any desired flow rate for reverse filtration. Blood return can be started automatically when the planned dialysis time has elapsed and the target water volume has been removed. The cleaned dialysate is infused from the dialyzer into the blood channel by reverse filtration to allow the blood to be returned to the body via both the artery and the vein at the same time. A total of 216 units of this fully automated dialysis machine have been placed in service at two of our facilities. During the 6 month period beginning in July 2001, they were used for 40,000 hemodialysis sessions in 516 patients. During the dialysate preparation process, the endotoxin levels in the reverse osmosis (RO) water, prefilter dialysate, and reverse filtered dialysate were all less than 1 EU/L. The time required to guide blood into the dialyzer (n = 39) decreased from the 4.6 +/- 1.4 minutes with the conventional machines to 3.2 +/- 0.6 minutes with the new machine (p < 0.01). The time required to return blood to the body also decreased from 8.6 +/- 2.2 minutes with the conventional machines to 6.8 +/- 0.7 minutes with the new machine (n = 34). No mechanical trouble was encountered with the fully automated dialysis machine units during the 40,000 hemodialysis sessions, and the workload of the dialysis unit staff in terms of the time needed to guide out and return blood to the body was significantly reduced. Because the machine simplifies the maneuvers required during hemodialysis, it is expected to contribute greatly to preventing medical accidents and in hospital infections associated with hemodialysis.     

Frequency of CCR5-Delta32 deletion in human immunodeficiency virus type 1 (HIV-1) in healthy blood donors, HIV-1-exposed seronegative and HIV-1-seropositive individuals of southern Brazilian population

The frequency of CCR5-Delta32 allele in human immunodeficiency virus type 1 (HIV-1) infection in the southern Brazilian population was determined in a cross-sectional study carried out from October 2001 to June 2004. Genomic DNA was extracted from peripheral blood cells of 134 healthy blood donors, 145 HIV-1-exposed seronegative individuals, 152 HIV-1-seropositive asymptomatic individuals, and 478 HIV-1-seropositive individuals with AIDS. A fragment with 225 base-pairs of the CCR5 gene was amplified by polymerase chain reaction. The CCR5-Delta32 homozygous deletion was observed in 2 (1.5%) blood donors and in 1 (0.7%) individual HIV-1-exposed seronegative, and was absent among all the HIV-1-seropositive individuals (Fisher's exact test, p=0.0242). The frequency of the homozygous CCR5-Delta32 deletion in the HIV-1-exposed did not differ when compared with that observed in the HIV-1 seronegative blood donors (Fisher's exact test, p=0.6093; OR: 2.18, 95% CI: 0.11-129.6). The wild-type genotype CCR5/CCR5 frequency was higher among the HIV-1-seropositive with AIDS compared to HIV-1 seropositive asymptomatic individuals (Chi-square test, p=0.0263; OR: 2.02, 95% CI: 1.03-3.97). The absence of the homozygous deletion of CCR5-Delta32 among HIV-1-seropositive individuals underscored that this genotype is an important genetic factor associated with the decreased susceptibility to HIV-1 infection. The higher frequency of heterozygosity for the CCR5-Delta32 and the CCR5-Delta32 allele in HIV-1 seropositive asymptomatic compared to HIV-seropositive with AIDS individuals also underscored that this deletion could be associated with the delay of the HIV-1 disease progression in this population. However, the low frequency of CCR5-Delta32 homozygosity observed among HIV-1-exposed seronegative individuals shows that the allele could not explain, by itself, the natural resistance to HIV-1 infection and different mechanisms of protection against HIV-1 infection that must be involved in this population.