Comparison between closed-loop portal and peripheral venous insulin delivery systems for an artificial endocrine pancreas

To establish the ideal insulin delivery route for an artificial endocrine pancreas, we examined the effectiveness of closed-loop portal insulin delivery. We investigated the effects of the route of insulin delivery on net hepatic glucose balance (NHGB) in dogs under pancreatic clamp conditions with somatostatin plus basal glucagon and insulin infusions. A constant rate of suprabasal insulin was infused via the portal vein or a peripheral vein, and glucose was infused into the portal vein for 180min. The mean net hepatic glucose uptake (NHGU) values in the portal insulin infusion group (PI group) were significantly greater than those in the peripheral venous insulin infusion group (VI group); the changes from the baseline values at 180min were 3.54 ± 0.66 and 2.45 ± 0.82mgkg^–1min^–1 in the PI and VI groups, respectively, P < 0.05. Furthermore, dogs under pancreatic clamp conditions were controlled after a 2-g/kg oral glucose load by applying the closed-loop intraportal (PO) or intravenous (IV) insulin infusion algorithm. There were no significant differences in glycemic control and insulin requirements between these algorithms. However, the maximum peripheral venous and arterial plasma insulin concentrations with the PO algorithm were significantly lower than those with the IV algorithm [305.1 ± 68.9 and 468.1 ± 66.9pmol/l (peripheral vein) and 305.3 ± 62.9 and 469.6 ± 85.1pmol/l (artery) with the PO and IV algorithms, respectively, P < 0.05]. On the other hand, the maximum portal plasma insulin concentration with the PO algorithm was significantly higher than that with the IV algorithm (619.9 ± 101.7 and 414.3 ± 79.9pmol/l with the PO and IV algorithms, respectively, P < 0.05). The mean NHGU values with the PO algorithm were significantly greater than those with the IV algorithm. Our results confirmed that closed-loop portal insulin delivery is feasible with regard to both insulin profiles and hepatic glucose handling in vivo, and indicated that the portal vein is the most suitable insulin delivery route for the artificial endocrine pancreas.     

Analysis of cytosolic and lysosomal pH in apoptotic cells by flow cytometry

Several reports indicate that the cytosol is acidified during apoptosis although the mechanism is not yet fully elucidated. The most acidic organelle found in the cell is the lysosome, raising the possibility that lysosomal proton release may contribute to the cytosolic acidification. We here describe methods for measurement of the cytosolic and lysosomal pH in U937 cells by a dual-emission ratiometric technique suitable for flow cytometry. Cytosolic pH was analysed in cells loaded with the fluorescent probe BCECF, while lysosomal pH was determined after endocytosis of FITC-dextran. Standard curves were obtained by incubating cells in buffers with different pH in the presence of the proton ionophore nigericin. Apoptosis was induced by exposure of cells to 10ng/ml TNF- for 4h, and apoptotic cells were identified using a fluorescent marker for active caspases. By gating of control and apoptotic cells, the cytosolic and lysosomal pH were calculated in each population. The cytosolic pH was found to decrease from 7.2 ± 0.1 to 5.8s±0.1 and the lysosomal increased from 4.3±0.4 to 5.2±0.3. These methods will be useful in future attempts to evaluate the involvement of lysosomes in the acidification of the cytosol during apoptosis.     

Molecular and cytogenetic organization of the 5S ribosomal DNA array in chicken (Gallus gallus)

The 5S ribosomal (r) RNA genes encode a small (120-bp) highly-conserved component of the large ribosomal subunit. The objective of the present research was to study the molecular and cytogenetic organization of the chicken 5S rDNA. A predominant 2.2-kb gene (5S) consisting of a coding and intergenic spacer (IGS) region was identified in ten research and commercial populations. A variant gene repeat of 0.6kb (5S) was observed in some of the populations. Genetic linkage analysis and cytogenetic localization by fluorescence in-situ hybridization assigned the 5S rDNA to chromosome 9. The 5S rDNA array was determined to be 80.2±7.0kb upon electrophoretic sizing following EcoRV digestion. Sequence analysis of 5S IGS regions revealed considerable conservation between chicken subspecies (98.4% identity) as well as homology with vertebrate Pol III promoter and regulatory sequence motifs. Minor intraindividual sequence variation within 1000bp of IGS was observed in four cloned Red Jungle Fowl (Gallus gallus gallus) 5S repeats (95.5% identity in this region). Sequence comparisons between IGS regions of 5S and 5S genes indicated two short continuous (>20bp) and many short non-continuous homologous regions as well as other conserved features such as promoter and termination motifs.     

Consideration of prosthesis–patient mismatch and left ventricular mass regression after implantation of the Carpentier–Edwards pericardial valve in elderly Japanese patients: body surface area may be irrelevant

The assessment of prosthesis patient mismatch (PPM) for small aortic annulus is important for prognosis after aortic valve replacement (AVR). Recent investigations have demonstrated that PPM occurs in AVR patients with an indexed effective orifice area (iEOA) of less than 0.85cm²/m². We investigated hemodynamic performance and left ventricular mass (LVM) regression after AVR. Eighteen patients who underwent AVR using a 19-mm Carpentier-Edwards pericardial (CEP) valve without annular enlargement were studied by echocardiography and Doppler examination 4 months after AVR. Patients were divided into two groups on the basis of their body surface area (BSA); the smaller BSA (group S, 1.14–1.36m², nine patients) and the larger BSA (group L, 1.40–1.83m², nine patients). Of these 18 patients, ten underwent isolated AVR, and five underwent AVR with coronary artery bypass graft; (i.e., double valvular replacement, AVR with maze procedure, and AVR with mitral valvulophasty. There were no statistically significant differences between the two groups, except for age (group S, 78.3 ± 2.5 years; group L, 73.6 ± 2.4 years). There was no significant difference for the iEOA during the late phase at rest (group S, 1.10 ± 0.26 cm²; group L, 1.02 ± 0.28cm²). However, there was a significant difference for the LVM regression between the preoperative and postoperative values (group S, 243 ± 23.6mg/cm² [pre], 190 ± 16.9mg/cm² [post]; group L, 302 ± 13.7mg/cm² [pre], 199 ± 16.7mg/cm² [post]). In elderly Japanese patients with a BSA of less than 18m², we demonstrated LVM regression and avoidance of PPM after implantation of the aortic 19-mm CEP valve.This paper was presented on November 1, 2003, at the 41st Annual Meeting of the Japanese Society of Artificial Organs, Sendai, Japan     

Mother-Baby psychiatric units (MBUs): National data collection in France and in Belgium (1999–2000)

Summary The French version of the Marcé checklist was used to collect data for 176 joint admissions to 11 psychiatric mother-baby units in 1999 and 2000. Mean age of the babies at admission ranged from 4 to 16 weeks. Two units also admitted older children. Mothers admitted were diagnosed with schizophrenia or chronic delusional disorders (n=44), acute transitory psychosis Bouffée délirante (n=20), bipolar disorders (n=20), depressive illness (n=38), personality disorders or intellectual disability (n=39), and other disorders (n=15). The mean duration of hospitalisation was 11 weeks. Units that also offered day-care admission in the same or a near-by unit had shorter mean admissions. More than half the womens partners (or babies fathers) had mental health problems. Women with schizophrenia or chronic delusional disorders and personality disorders or intellectual disability remained hospitalised longer, improved less, and were more often separated from their babies, or discharged with supervision, than women admitted with other diagnoses.     

Molecular analysis of <Emphasis Type="Italic">Fiji disease virus</Emphasis> genome segments 5, 6, 8 and 10

Summary. The complete sequences of Fiji disease virus (FDV) genome segments 5 (S5), S6, S8 and S10 were obtained and comprised 3150nt, 2831nt, 1959nt and 1819nt, respectively. Each segment contained a single ORF which encoded putative proteins of 115kDa, 97kDa, 69kDa and 63.0kDa, respectively. The putative amino acid sequences encoded by S5 and S6 contained putative leucine zipper motifs while FDV S5 and S8 each contained an ATP-GTP-binding motif. At the amino acid level, FDV S5, S6, S8 and S10 showed most similarity to the corresponding segments of Rice black-streaked dwarf virus. Based on sequence similarities, it is predicted that FDV S8 encodes a minor core protein, while FDV S10 encodes an outer capsid protein. The evolutionary relationships of FDV to other reoviruses are discussed.The nucleotide sequence data for FDV S5, S6, S8 and S10 are available in the DDBJ/EMBL/GenBank databases under the accession numbers AY029521, AF356083, AY297693 and AY297694, respectively.     

Actin filaments around endothelial fenestrae in rat hepatic sinusoidal endothelial cells

The presence of microfilaments in the vicinity of sinusoidal endothelial fenestrae (SEF) suggests that the cytoskeleton of liver sinusoidal endothelial cells (LSEC) plays an important role in the modulation of SEF. In this study, we investigated actin filaments around SEF in LSECs. Monolayers of LSEC culture were established by infusing a rat liver with collagenase for 30min and then culturing in RMPI medium for 24h. Cells were reacted with 0.1% Triton X for 5s and 15% glycerinated PHEM buffer (60mM PIPES, 25mM HEPES, 10mM EGTA, 2mM MgCl, pH 6.9) containing heavy meromyosin for 10min and observed under a transmission electron microscope. By electron microscopy with the modified heavy meromyosin decorated reaction, actin filaments were clearly demonstrated around SEF in LSEC.     

The complete nucleotide sequence of the genome RNA of Lily symptomless virus and its comparison with that of other carlaviruses

Summary. The complete genomic nucleotide sequence and genome structure of Lily symptomless virus (LSV), a lily-infecting carlavirus, have been obtained. The genome of the Korean strain of LSV, LSV-Kr, was 8,394 nucleotides long and contained six open reading frames (ORFs) coding for proteins of Mr 220kDa (1,948aa), 25kDa (228aa), 12kDa (106aa), 7kDa (64aa), 32kDa (291aa) and 16kDa (140aa) from the 5 to 3 end, respectively, which is typical of carlaviruses. Genetic heterogeneity was observed in the ORF1 gene. A total of 221 of 5,847 nucleotides (nt) were heterologous in the ORF1 of replicase; 162nt portions were silent and 59nt resulted in amino acid changes. This heterogeneity indicates that the LSV-infecting lily plants contained a genetically heterogeneous population of LSV (quasispecies). Overall similarities to those of other carlaviruses for the six ORFs of LSV were from 76.1% to 31.6% and from 87.3% to 13.7%, at nucleotide and amino acid levels, respectively. The ORF1 replicase gene of LSV shares 40.9% to 56.8% and 48.9% and 58.6% identities with that of 5 other carlaviruses at the amino acid and nucleotide levels, respectively. LSV was closest to Blueberry scorch virus (BlScV) in this ORF, among the carlaviruses for which sequence information is available. The three triple gene blocks (ORF2-4), ORF5 (coat protein) and 3-proximal 16kDa ORF6 genes were further analyzed, and phylogenetic trees for the coding regions indicate that the LSV was the most closely related to Kalanchoe latent virus and BlScV. This is the first report of the complete nucleotide sequence and genome structure of LSV.Received December 13, 2002; accepted May 14, 2003 Published online July 17, 2003     

A temperature-dependent inhibitory activity of serum on the capacity of Saccharomyces cerevisiae-derived hepatitis B surface antigen to bind to monocytes

Summary. Hepatitis B surface antigen, when produced in yeast (rHBsAg), is capable of binding to cells that express the lipopolysaccharide coreceptor CD14. This interaction is enhanced by a serum protein, the lipopolysaccharide binding protein (LBP). Here we report that most of the rHBsAg particles that attached to monocytes at 0°C, were not endocytosed but were released back into the serum-containing binding buffer at 37°C. Additionally, serum-dependent binding at 37°C was weak when compared to the serum-dependent attachment at 0°C. Pre-incubation at 37°C of cells together with serum did not abolish binding of freshly added rHBsAg at 0°C. However, pre-incubation of rHBsAg with serum at 37°C reduced attachment to cells following incubation at 0°C. Soluble CD14 and LBP, two serum proteins which can act as phospholipid transfer molecules, were shown not to be responsible for the inhibitory effect. Pre-incubation at 37°C of rHBsAg in serum-free hepatoma cell line-conditioned media resulted in a pronounced reduction in subsequent binding to cells at 0°C. These observations suggest that the temperature-dependent inhibitory effect is caused by serum factors that are probably secreted by hepatocytes.     

Nano-scaled hydroxyapatite/polymer composite IV. Fabrication and cell adhesion properties of a three-dimensional scaffold made of composite material with a silk fibroin substrate to develop a percutaneous device

Nano-scaled sintered hydroxyapatite (HAp) particles with an a-axis length of 87 ± 23nm, a c-axis length of 236 ± 81nm, and a mean aspect ratio (c/a) of 2.72 were covalently linked onto a silk fibroin (SF) substrate chemically modified by graft polymerization with -methacryloxypropyl trimethoxysilane (MPTS). Graft polymerization with poly(MPTS) on SF was conducted by free-radical initiation in a water solvent with pentaethylene glycol dodecyl ether as a nonionic surfactant. The alkoxysilyl groups of the graft polymers avoided hydrolysis and maintained their activity in coupling with the hydroxyl groups on the HAp surface despite the use of water as the reaction solvent. The weight gain of poly(MPTS) on SF increased with increasing the reaction time, eventually reaching a plateau value of about 15wt% after 50min of reaction time. After HAp covalent coating, the particles separated or aggregated into several crystals, as shown by scanning electron microscopic observation. L929 fibroblast cells adhered more plentifully on HAp-coated SF compared to untreated SF and hydrolyzed poly(MPTS)-grafted SF during 24h or 48h of incubation. The cells adhered only on the HAp surface but not at all on the dehydrated grafted surface of SF without HAp. A button-shaped prototype for a percutaneous device was manufactured by transplantation of HAp-coated SF fibers of about 100µm in length onto silicone moldings using an adhesive, and the device showed good cell adhesiveness.     

Detection of enteric viruses and bacterial indicators in German environmental waters

Summary. A German mining lake and the supplying surface waters, which are located downstream of a sewage plant, were examined regarding their microbiological and virological quality. Between October 2002 and September 2003, specific PCR methods were used to determine the occurrence of enteric viruses in 123 water specimens drawn at different sites downstream of the waste water treatment plant and in 9 samples from the sewage plant influent. Detection rates in sewage plant effluents and surface water samples depended on sampling sites and were: 29–76% for enterovirus (EntV), 24–42% (astrovirus, AstV), 15–53% (norovirus, NV), 3–24% (rotavirus, RoV), 5–20% (hepatitis A virus, HAV) and 20% (adenovirus, AdV). AstV genome load of selected samples was between 3.7×10³ to 1.2×10⁸ genome equivalents per liter (gen.equ./l), depending on sampling location; NV average genome load ranged from 1.8×10⁴ to 9.7×10⁵gen.equ./l. Cell culture methods showed that three out of 18 PCR positive samples contained infectious EntV. Even though microbiolical parameters such as Escherichia coli, enterococci and coliphages indicated acceptable microbiological water quality, the virological data of this study suggest the possibility that surface waters may be a source for enteric viral infections.     

The relationship between HPV16 and expression of CD44v6, nm23H1 in esophageal squamous cell carcinoma

Summary. The esophageal squamous cell carcinoma (ESCC) has high incidence in Shaanxi Province of China. More and more researches indicated that human papillomavirus type 16 (HPV16) might play an important role in carcinogenesis of ESCC but the relationship between HPV16 and CD44v6, nm23H1 has not been elucidated. HPV16 was detected by amplifying HPV16 E6 gene through polymerase chain reaction (PCR) method and the expression of CD44v6, nm23H1 in 40 ESCCs and fifteen normal esophageal mucosa (NEM) from Shaanxi Province was examined by Streptavidin-Peroxidase (SP) method using monoclonal antibody specific to CD44v6 and nm23H1. The positive rates of HPV16 E6 gene, CD44v6 and nm23H1 were 60% (24/40), 65% (26/40) and 45% (18/40) respectively in ESCCs and 26.67% (4/15), 33.33% (5/15) and 86.67% (13/15) respectively in NEMs. There exited statistical difference for HPV16, CD44v6 and nm23H1 between NEMs and ESCCs respectively (p<0.05). The relationship between HPV16 and the expression of CD44v6 in ESCCs was statistical significance (P=0.021), but no significant correlation was found between HPV16 and the expression of nm23H1 (P=0.436) in ESCCs. The infection rate of HPV16 had no statistical difference in all pathological features we observed, but the expression rates of CD44v6 and nm23H1 had statistical correlation with invasion (p=0.001, 0.013) and lymph nodes metastasis (p=0.014, 0.002) respectively. In different histology grade of ESSCs, the relationship between HPV16 and CD44v6 was statistical significance in grade I (p=0.044) but was not in grade II (p=0.165) and grade III (p=0.658), however as to the expression of nm23H1 there exited no statistical significance in all histology grades of ESCC (p>0.05). The expression rates of CD44v6 and nm23H1 were statistically different between grade I and II (p=0.004, 0.016) respectively and between grade I and grade III (p=0.014, 0.020), but not statistically different between grade II and III (p=0.792, 0.943) respectively. Our data firstly suggested that there existed the statistical relationship between the infection of HPV16 and the expression of CD44v6 in ESCCs and that HPV16 may be involved in invasion and metastasis of ESCC.     

Sequence analysis of the fragment of the phosphoprotein gene of Polish distemper virus isolates

Summary. The nucleotide sequence analysis of the 429bp fragment of the P gene of 11 Polish field isolates of Canine distemper virus (CDV), reference strains and other virus isolates available in the GenBank was the aim of the studies. High homology between all dog strains from east-southern region of Poland and reference strains of CDV was demonstrated. It was estimated as 97–100% for CDV-OND; 96.7–99.8% for CDV-Rock; 96.7–99.8% for CDV-LED and 96.3–97.9% for A75-17. The 100% homology of the nucleotide sequence was observed between CDV Pulawy 92, CDV Pulawy 97 and the reference CDV-OND. The homology between CDV-OND and viruses isolated from the mink and ferret was estimated as 97.7% and 98.4%, respectively. Virus strains isolated from blue foxes demonstrated the highest homology to CDV-OND – equal to 97.7% for DV 79 and 99.5% for DV 92. The fox isolate from 1992 had higher level of homology to dog isolates (96.5–99.5%) than the strain isolated from the fox in 1979 (97.2–98.8%). The phylogenetic tree has two main lineages representing two separated genetic groups: I containing PDV and II containing all distemper virus strains isolated from terrestrial carnivores. CDV strains isolated from dogs from Pulawy region between 1992–1998 and from the fox (DV 92) formed the separate lineage containing also reference strains. They differed from the native isolates from the mink and ferret as well as from Japanese strains of CDV.Received October 29, 2002; accepted April 2, 2003 Published online June 11, 2003     

Relapse of depression during pregnancy following antidepressant discontinuation: a preliminary prospective study

Summary. Objective: Pregnancy has frequently been referred to as a time of emotional well-being for patients. However, systematic data about the risk for relapse of depression during pregnancy are sparse.Method: We completed a longitudinal cohort study of thirty-two (N=32) women with histories of depression who were euthymic at conception and who either discontinued or attempted to discontinue antidepressant therapy proximate to conception. Subjects were prospectively followed across pregnancy once per trimester using structured clinical interviews. Rates of relapse and time to relapse were examined. Factors distinguishing the population with respect to risk for relapse including demographic characteristics and illness history were also examined.Results: Seventy-five percent (N=24) of patients relapsed during pregnancy. The majority of relapses (79%, N=19) occurred in the first trimester, and relapse was more prevalent in women with histories of more chronic depression.Conclusions: Pregnancy is not protective with respect to risk for relapse of depression. Careful treatment planning is necessary for those women on antidepressants who plan to conceive or who become pregnant.     

Pattern of <Emphasis Type="Italic">N</Emphasis> gene-mediated systemic hypersensitive response and turnover of viral replicase protein in tobacco

Summary. We examined the pattern of the N gene-mediated systemic hypersensitive response (HR), which was induced by tobacco mosaic virus upon temperature shift, and analyzed the distribution of the coat protein and the virus-encoded 126kDa replicase protein (126K protein) by immunoblot analysis. In the middle- and lower-positioned leaves, HR occurred in the advancing edge of the infected area, where we detected both the coat protein and the 126K protein. In the areas between the main vein and the advancing edge of these leaves, we observed no HR and did not detect 126K protein, though virus was present in these areas. In the upper-positioned mosaic leaves, patterns of the HR were different depending on the leaf age. In these mosaic leaves, the mechanism preventing the virus from invading dark green tissue seemed to be broken down in 8–14 days old leaves, and HR was observed only in the tissue just invaded by the virus, where we detected the 126K protein. Those results suggested that the viral 126K protein was present when the viral replication was taking place, and easily degraded when the amount of the virus was saturated in the cells.     

Development of an artificial synapse using an electrochemical micropump

Improving the resolution of artificial sensory organs requires an interface that receives external information from electronic circuits and stimulates appropriate neurons individually in response to that information. The method of electric stimulation in available artificial sensory organs is fairly nonselective; therefore, we developed a method of chemical stimulation of neurons using a neurotransmitter containing an electrochemical micropump powered by the bubbling that occurs during water electrolysis. The micropump contains a glass nozzle with a tip 10µm in diameter. Two blackened platinum electrodes for the electrolysis were inserted into the body of the pump, which was filled with neurotransmitter solution. The distance between a neuron of the gastropod Aplysia and the tip of the nozzle was adjusted to about 100µm. A potential difference of 3.0V was applied to the electrodes to propel the solution toward the neuron while its membrane potential was monitored. Administration of 1-mM acetylcholine to a resting neuron caused neural firing only when the voltage was applied for 0.5s and without a time lag. During administration of 50-mM -aminobutyric acid to spontaneously firing neurons, the firing disappeared with a time lag of 1s after application of 3.0V. We concluded that an electrochemical micropump can be applied for rapid neurotransmitter administration to control the excitation and inhibition of neurons. This simple pump can be miniaturized to create synapses in artificial sensory organs.     

Myocardial microvascular perfusion after transfusion of liposome-encapsulated hemoglobin evaluated in cross-circulated rat hearts using tracer digital radiography

The effect of hemodilution with Neo Red Cell (NRC, liposome-encapsulated hemoglobin) on myocardial perfusion was evaluated in cross-circulated rat hearts under 300-bpm pacing and 100-mmHg perfusion pressure. In NRC-transfused hearts (n = 5), NRC volume fraction and hematocrit were 9% ± 3% and 22% ± 4%, respectively; the latter decreased from 43% ± 3% before NRC transfusion. Coronary perfusion rate and left ventricular isovolemic developed pressure increased after NRC transfusion to 4.6 ± 1.0ml/min/g and 127 ± 32mmHg from basal values of 2.5 ± 0.3ml/min/g and 115 ± 28mmHg, respectively. In contrast, the flow increase during reperfusion following 30-s flow cessation decreased from 74% ± 24% to 64% ± 24%. The arteriovenous difference in O₂ saturation was slightly higher after NRC transfusion. Within-layer regional flow distributions from subepicardium to subendocardium assessed by tracer digital radiography (100-µm resolution) showed that coefficients of variation of flows in 400 × 400-µm regions were 0.41 ± 0.10 in NRC-transfused hearts and 0.54 ± 0.11 in nontransfused hearts (n = 5); i.e., the myocardial flow distribution was more uniform in NRC-transfused hearts. These results suggest that NRC is superior to erythrocytes in terms of the homogenization of O₂ delivery, indicating its potential therapeutic value in myocardial microcirculatory failure.     

Development of a dialyzer with enhanced internal filtration to increase the clearance of low molecular weight proteins

Accumulated low molecular weight proteins in hemodialysis patients require a high-flux dialyzer. There have been several methods proposed for enhancing internal filtration, including narrowing the inside diameter of the hollow fibers, lengthening the fibers, and increasing the fiber density ratio. We tried to enhance the internal filtration by increasing the pressure drop in the dialysate compartment through increasing the fiber density ratio. If the fiber density ratio is too high, however, an irregular dialysate path may result, thus decreasing dialysis performance. Therefore, we took note of the shape of the inner housing and added a short taper structure, which improved the dialysate path dramatically. Consequently, we developed an internal filtration-enhanced dialyzer (APS-Prototype) to improve dialysis performance. The internal filtration rate in water (measured by Doppler ultrasound) was 13.2l/session for the APS-Prototype and 5.3l/session for the APS-15E. The amount of 1-microglobulin (1-MG) in bovine plasma was 0.34g for the APS-Prototype and 0.11g for the APS-15E. In addition, the amount of 1-MG in vivo was 29.0% ± 5.8% for the APS-Prototype, significantly higher than that for the APS-15E (13.6% ± 1.9%). The desirable loss of albumin is 2–4g in hemodiafiltration, and it was 3.92 ± 1.03g for the APS-Prototype. The prototype showed excellent solute removal performance with no clinical or engineering problems.     

Identification and Characterization of the UL24 Gene Product of Herpes Simplex Virus Type 2

The UL24 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 281 amino acid protein with a molecular mass of 30.5kDa. In this study, the HSV-2 UL24 gene product has been identified by using a rabbit polyclonal antiserum produced against a recombinant protein containing the full-length UL24 gene product of HSV-2 fused to glutathione-S-transferase. The antiserum reacted specifically with a 32kDa protein in HSV-2 186-infected Vero cells and with 31 and 32kDa proteins in UL24-expressing Cos-7 cells. Accumulation of UL24 protein to detectable levels required viral DNA synthesis, indicating that the protein was regulated as a late gene. UL24 protein was found to be associated with purified HSV-2 virions and C capsids. Indirect immunofluorescence analysis demonstrated that the UL24-specific fluorescence was detected in perinuclear regions of the cytoplasm and/or in the nucleus as small discrete granules from 9h post infection (hpi). Furthermore, the UL24 protein expressed singly was detected predominantly in the nucleus and slightly in the cytoplasm at 24h after transfection, with branch-like cytoplasmic protruding structures. Strong nucleolus staining was visible in partial cells.     

Synthesis of Seoul virus RNA and structural proteins in cultured cells

Summary. Seoul virus is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). The virion has a tripartite (S, M, and L) negative-stranded RNA genome, which is characteristic of the family Bunyaviridae. However, the molecular basis of virus replication is not well known. We established a Northern blot hybridization (NB) procedure using digoxygenin-labeled RNA probes, to quantitate the hantaviral plus- and minus-strand RNAs separately. Virus RNA replication was analyzed in infected Vero E6 cells. When the Vero E6 cells were infected with Seoul virus strain KI-83-262 (KI) at m.o.i.=0.25, the plus-strand RNA was detected within 1h post-infection (hpi), and the minus-strand RNA was detected subsequently. Using laser confocal microscopy, the nucleocapsid protein (NP) was detected within 2hpi, and accumulated as scattered granules in the cytoplasm until 24hpi. In contrast, the G2 protein first appeared at 8hpi, was immediately transported to the Golgi, and accumulated in the Golgi until 24hpi. Infectious virus particles were released into the medium at 24hhpi. These findings indicate that hantavirus RNA replication starts with the appearance of NP at 2hpi, glycoproteins then accumulate gradually in the Golgi, and virion formation is initiated once the viral RNAs and proteins have accumulated.Received October 10, 2002; accepted April 25, 2003 Published online July 17, 2003