[A longitudinal study on the progression and influencing factors of myopia in primary school students from grade one to grade three in Hubei Province].

Objective: To investigate the longitudinal epidemiological characteristics of myopia in primary school students from grade one to grade three in Hubei province, and to analyze the main factors affecting the occurrence and development of myopia, so as to provide the direction and theoretical basis for the prevention and control of myopia in children and adolescents. Methods: Longitudinal study. A total of 13, 244 primary school students from grade one to grade three in 17 prefectures and forest districts of Hubei province were included in the study by means of random stratified cluster sampling. Among them, there were 7, 331 boys and 5, 913 girls with an age of (7.7±1.0) years. The same group of students were followed up for two years from October 2017 to October 2019 to complete three data collections. All included subjects underwent visual acuity examination, which required further automatic computer optometry after using cyclopentolate hydrochloride eye drops if the visual acuity was less than 1.0. In the questionnaire, the general situation and eye behavior of the included subjects were collected. The Chi-square test was used to compare the prevalence of myopia among different genders,different reproductive history and grades. The Kruskal-Wallis H test was used to compare the difference in the mean spherical equivalent refraction, and logistic regression was used to analyze the influencing factors of myopia. Results: The prevalence of myopia was 23.10% at baseline in 2017, 28.67% after one year's follow-up and 33.26% after two years' follow-up among primary school students in grade one to grade three in Hubei province. The overall prevalence of myopia increased with time (χ²=307.47, P<0.05). It also increased with the increase of grades. After two years of follow-up, the myopia rate of students in grades one through three was 25.62%, 35.07% and 41.05%, respectively (χ²=200.98, P<0.05). In 2017, the prevalence of myopia was 21.62% in boys and 24.93% in girls, and it increased to 31.20% and 35.69% after 2 years of follow-up, respectively. Both the prevalence of myopia and the spherical equivalent refraction were higher in girls than in boys. Moreover, myopia was associated with parents' myopia (OR=1.17), less time for outdoor activities (OR=1.06), no rest after half an hour's study (OR=1.18), more time for daily exposure to electronic products (OR=1.07), longer time for extracurricular study (OR=1.09), and higher grades (OR=1.78). Children whose parents were more myopic and who ate sweets and fizzy drinks more often had greater levels of myopia. Conclusions: Myopia occurs early in primary school students of grade one to grade three in Hubei province and increases rapidly. More attention should be paid to good habits for eyes, regular screening, outdoor activities and girls. (Chin J Ophthalmol, 2021, 57: 749-756).

[Clinical analysis of the risk factors for No.12p LN metastasis in advanced gastric cancer].

Objective: To analyse the risk factors for periportal lymphnode (No.12p LN) in advanced gastric cancer. Methods: A retrospective analysis of 183 patients with advanced gastric cancer from January 2005 to December 2010, and all patients were underwent D2 lymphadenectomy in addition to No. 12p LN dissections. Potential clinicopathological factors that could influence No. 12p LN metastasis were statistically analyzed. Results: There were 18 cases (9.8%) with periportal lymphnode metastasis. A logistic regression analysis suggested that the Borrmann type (Ⅲ/Ⅳ versus Ⅰ/Ⅱ, P=0.008), tumor size (≥6 cm vs <6 cm, P=0.001), and depth of invasion (pT4 vs pT2/pT3, P=0.049) were associated with 9.5-, 8.5-, and 3.2-fold increases, respectively, for risk of No. 12p LN metastasis. A logistic regression analysis also showed that No. 5 (P=0.002) and No. 12a (P=0.002) LN metastasis were associated with 7.5- and 7.3-fold increases, respectively, for risk of No. 12p LN metastasis. In addition, significant differences in 5-year survival of patients with and without No. 12p LN metastasis were observed (11.1% vs 32.7%, P=0.042). Conclusions: Borrmann type, tumor size and depth of invasion are significant factors for identifying patients with No. 12p LN metastasis. Patients with No. 5 or No. 12a LN metastasis should be higher possibility of No.12p LN metastasis.

[The epidemiology of myopia in primary school students of grade 1 to 3 in Hubei province].

Objective: To investigates the visual condition and myopia status in primary school students of grade 1 to 3 in Hubei province and to further analyze the influencing factors of myopia, so as to provide theoretical basis for prevention and control of myopia. Method: Cross-sectional study. A total of 18 532 primary school students from grade 1-3 in 26 primary schools from 17 prefecture-level cities in Hubei province were included in the study from March 2017 to July 2017, by using a random stratified cluster sampling method. In this study, 16 955 people were actually surveyed, with a response rate of 91.49%. All students' Visual acuity was measured, a further retinoscopy refraction test after application of Cyclopentolate Hydrochloride Eye Drops was conducted for those whose visual acuity was less than 5.0. The general situation and eye behavior of all students were investigated by questionnaires. Chi-square test was used to compare the prevalence of myopia between different grades, different regions and different genders, as well as the distribution of myopia correlation between different regions, and Logistic regression model was used to analyze the correlation between myopia and related factors. Results: The prevalence of myopia among primary school students of grade 1 to 3 in Hubei province is 24.15% with 12.67% in the first grade, 24.91% in the second grade, 34.95% in the third grade, and the difference was statistically significant (χ(2)=862.28, P<0.05). According to the study, mild myopia is the most prevailing condition among myopia students, with the prevalence rate of mild myopia, moderate myopia and severe myopia being 19.21%,4.29%, and 0.64% respectively, and the difference is statistically significant (χ(2)=155.62, P<0.05). In addition, the prevalence of myopia is different in each region, which was highest in provincial capitals, followed by non-provincial cities, and the lowest in rural areas, with statistically significant difference (χ(2)=539.57, P<0.05). Myopia is generally related to multiple factors, such as the grade, parents' myopia, outdoor activities, continuous closing reading, exposure to electronic products, and extracurricular reading time are related to myopia (P<0.05). Conclusions: Children's myopia is a public health problem that should not be ignored. The prevalence of myopia among 1-3 grade pupils is 24.15% with an increasing tendency with grade. The occurrence of myopia is related to heredity and eye behavior. (Chin J Ophthalmol, 2018, 54:756-761).

Association study of C1qA polymorphisms with systemic lupus erythematosus in a Han population.

OBJECTIVE: The aim of this study was to evaluate the association of single nucleotide polymorphisms (SNPs) in the C1qA gene region with systemic lupus erythematosus (SLE) in a Chinese Han population. METHODS: Chinese SLE patients (n = 748) and ethnically- and geographically-matched healthy controls (n = 750) were genotyped for the C1qA region SNPs, rs172378 and rs665691, by using the Sequenom MassArray system. RESULTS: The Chinese Han SLE patients and controls had statistically similar frequencies of alleles, genotypes, and haplotypes of C1qA polymorphisms. Moreover, no association signal was detected on different genetic models (additive, dominant, and recessive) or in SLE subgroups stratified by various clinical manifestations. CONCLUSIONS: The C1qA SNPs, rs172378 and rs665691, confer no genetic predisposition to SLE in a Chinese Han population.

Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein.

The rotavirus nonstructural glycoprotein NSP4 is an intracellular receptor that mediates the acquisition of a transient membrane envelope as subviral particles bud into the endoplasmic reticulum. NSP4 also causes an increase in intracellular calcium in insect cells. Purified NSP4 or a peptide corresponding to NSP4 residues 114 to 135 induced diarrhea in young (6 to 10 days old) CD1 mice. This disease response was age-dependent, dose-dependent, and specific. Electrophysiologic data from intestinal mucosa showed that the NSP4 114-135 peptide potentiates chloride secretion by a calcium-dependent signaling pathway. Diarrhea is induced when NSP4, acting as a viral enterotoxin, triggers a signal transduction pathway.

Isolation and partial characterization of a specific alpha-fetoprotein receptor on human monocytes.

Since a large body of data has suggested a significant role for alpha-fetoprotein (AFP) in the regulation of the immune response at a number of levels, we examined the possibility of a specific receptor for AFP on the immune recognition cell, the monocyte/macrophage. Microscopic autoradiography exhibited an obvious binding of AFP almost exclusively on human peripheral monocytes but not on lymphocytes. In a human monocyte cell line (U937) Scatchard plot analysis indicated the presence of two distinct AFP-specific binding sites with a Kd of 5 x 10(-11) M, 49 binding sites per cell, and 2.5 x 10(-7) M, 7,800 binding sites per cell. 125I-ASD-AFP, AFP-radiolabeled bifunctional photoactivatable thio-cleavable cross-linker, was used to isolate the AFP binding protein from U937 cells. After ultraviolet photoactivation, 125I-sulfosuccinimidyl 2-(p-azido-salicylamido)ethyl-1,3'-dithiopropionate was covalently linked to the putative receptor. Autoradiography of SDS gradient PAGE under reducing conditions showed a major radiolabeled band at between 62 and 65 kD. To confirm the specificity of the finding, recombination of AFP with the isolated receptor was examined in artificially reconstituted membrane vesicles, which also resulted in a single band at approximately 62-65 kD by SDS-PAGE autoradiography. From the data above, we concluded that human monocytes possess a specific AFP binding protein on the membrane, a putative receptor, which may be involved with the physiological regulation of the immune response.

Pathogenesis of rotavirus gastroenteritis.

The outcome of intestinal infection with rotaviruses is more complex than initially appreciated, and it is affected by a complex interplay of host and viral factors. Rotaviruses infect intestinal enterocytes, and the early events in infection are mediated by virus-epithelial cell interactions. Diarrhoea may be caused by several mechanisms including (i) malabsorption that occurs secondary to the destruction of enterocytes, (ii) villus ischaemia and activation of the enteric nervous system that may be evoked by release of a vasoactive agent from infected epithelial cells in the absence of significant pathologic lesions or enterocyte damage, and (iii) intestinal secretion stimulated by the intracellular or extracellular action of the rotavirus non-structural protein, NSP4, a novel enterotoxin and secretory agonist with pleiotropic properties. New studies of rotavirus infection of polarized intestinal epithelial cells show that rotaviruses infect cells differently depending on whether or not they require sialic acid for initial binding, and infection alters epithelial cell functions. NSP4 also affects epithelial cell function and interactions. NSP4 (i) induces an age- and dose-dependent diarrhoeal response in young rodents that is similar to virus-induced disease, (ii) stimulates a Ca(2+)-dependent cell permeability where the secretory response is age-dependent, and (iii) alters epithelial cell integrity. Antibody to NSP4 protects mouse pups from diarrhoea induced by homotypic and heterotypic viruses. These data support a new mechanism of rotavirus-induced diarrhoea whereby a viral enterotoxin triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. This new information about how a gastrointestinal virus causes disease demonstrates common pathogenic mechanisms for viral and bacterial pathogens not previously appreciated. These results also suggest new approaches to prevent or treat rotavirus-induced diarrhoea.

Rotavirus protein expression is important for virus assembly and pathogenesis.

Rotaviruses have a unique morphogenesis in which particles obtain a transient membrane-envelope as newly made subviral particles bud into the endoplasmic reticulum (ER). This process is mediated by a viral nonstructural glycoprotein, NSP4. We have found that NSP4 has pleiotropic properties that became evident following expression of this protein in eukaryotic cells. NSP4 expressed in insect cells bound double-layered rotavirus particles in a manner similar to receptor-ligand interactions and this interaction is thought to trigger the particle budding process. Expression of NSP4 in insect cells also increases intracellular calcium ([Ca2+]i) levels and this effect may explain the toxicity of this protein in eukaryotic cells. Increases in [Ca2+]i levels in insect cells also are observed following exogenous addition to cells of purified NSP4 or of a synthetic peptide of NSP4. Experiments to determine the mechanism by which NSP4 causes an increase in [Ca2+]i showed that Ca2+ is released from a subset of the thapsigargin-sensitive store [endoplasmic reticulum (ER)]. However, exogenously added and endogenously expressed NSP4 use different mechanisms to alter the Ca2+ permeability of the ER membrane. We hypothesize that NSP4-mediated changes in ER membrane permeability trigger viral budding into the lumen of the ER, and eventually induce cell death and release of virus particles from infected cells. We also propose that release of NSP4 following cell lysis and the concomitant stimulation of a Ca2+ signal transduction pathway in neighboring cells contributes to altered ion transport in intestinal epithelium resulting in diarrheal disease.

Identification of the minimal replicase and the minimal promoter of (-)-strand synthesis, functional in rotavirus RNA replication in vitro.

An in vitro replication system supporting the initiation and synthesis of complete rotavirus (-)-strands on (+)-strand template RNA (Chen et al., J Virol 68: 7030, 1994) was used to examine several parameters related to rotavirus RNA replication. Coexpression of VP1/2/3 in all possible combinations from baculovirus vectors revealed: [i] Virus-like particles (VLPs) were formed only if VP2 was present, and [ii] VP1/2 and VP1/2/3 VLPs had replicase activity in the in vitro system whereas VP2/3 and VP2 VLPs did not. Thus, the minimal replicase is composed of VP1 and VP2 and replicase activity is associated with VP1. In vitro replication reactions, using T7 transcripts of porcine rotavirus OSU genome segment 9 as reporter template, were performed to map cis-acting elements that regulate replication. Internal deletions and terminal truncations of the reporter RNA localized a replication signal, conferring full template activity, to the 5'-terminal 27 nucleotides (nt 1-27) and the 3'-terminal 26 nucleotides (nt 1037-1062). Further analysis showed that a minimal promoter of (-)-strand synthesis was contained in the 3'-terminal 7 nucleotides (nt 1056-1062); the sequence conserved at the 3'-terminus of all rotavirus genes. Hybrid constructs with this promoter had minimal, but detectable, template activity. This result indicated that upstream sequences between nucleotides 1037-1055 positively regulate the activity of the minimal promoter.

Rotavirus structure: interactions between the structural proteins.

Structural studies on rotavirus using electron cryomicroscopy and computer image analysis have permitted visualization of each shell in the triple-layered rotavirus structure. Biochemical results have aided our interpretation of the structural organization of these layers and protein interactions seen in the three-dimensional structure, and have provided a better understanding of the structure-function relationships of the rotavirus structural proteins.

A novel approach to experimentally estimating the number of reactive epitopes on multivalent antigens.

Various coefficients in radioimmunoassays are expressed in a series of equations. These equations provide an approach to estimating the number of epitopes on a multivalent antigen as well as a macromolecular topogram. In this paper, pig insulin (Ins), horse myoglobin (Mb), human serum albumin (HSA), human alpha-fetoprotein (AFP) and adr-hepatitis B type virus surface antigen (HBsAg) were chosen as objects of study. The number of epitopes was calculated to be Ins = 2, Mb = 5, HSA = 16, AFP = 6, HBsAg = 146. These epitope numbers, calculated by equations, were very similar to those previously identified experimentally. This suggests that our theoretical approach may be useful in predicting the number of epitopes on other macromolecules of biological and clinical interest.

Characterization of a lung-cancer-associated auto-antigen.

An antigen, protein X (Px), was purified from immune complexes isolated from malignant pleural effusions from patients with adenocarcinoma of the lung by EDTA treatment, PEG 8000 precipitation, protein A affinity chromatography, and Sephadex G-200 separation in the presence of 3 M NaCl. The purified antigen had a M(r) 17,000 by SDS-PAGE, and consisted of isoelectric species of pI 6.3 and 6.6. Purified Px recombined with Ig isolated from pleural fluids from patients with lung adenocarcinoma, but not with Ig from patients with breast carcinoma. Using an autologous human and heterologous chicken antibody, Px was found, by immunohistology, in the cytoplasm of some of the well-differentiated lung adenocarcinoma cells, but was not seen in normal lung or a variety of other malignant tissues. A liquid-phase competitive-inhibition RIA was developed. Over 30 ng/ml of Px were found in 9 of 15 pleural fluids from patients with lung carcinoma, none of 20 from patients with breast, ovary, stomach or colon cancer, and in 3 of 15 patients with unknown primary tumor. Our data suggest that Px may be a lung-cancer-associated autoantigen which can elicit a host humoral response in vivo.

A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.

Previous studies have shown that the nonstructural glycoprotein NSP4 plays a role in rotavirus pathogenesis by functioning as an enterotoxin. One prediction of the mechanism of action of this enterotoxin was that it is secreted from virus-infected cells. In this study, the media of cultured (i) insect cells infected with a recombinant baculovirus expressing NSP4, (ii) monkey kidney (MA104) cells infected with the simian (SA11) or porcine attenuated (OSU-a) rotavirus, and (iii) human intestinal (HT29) cells infected with SA11 were examined to determine if NSP4 was detectable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-Western blotting, immunoprecipitation and N-terminal amino acid sequencing identified, in the early media from virus-infected cells, a secreted, cleavage product of NSP4 with an apparent molecular weight of 7,000 that represented amino acids 112 to 175 (NSP4 aa112-175). The secretion of NSP4 aa112-175 was not affected by treatment of cells with brefeldin A but was abolished by treatment with nocodazole and cytochalasin D, indicating that secretion of this protein occurs via a nonclassical, Golgi apparatus-independent mechanism that utilizes the microtubule and actin microfilament network. A partial gene fragment coding for NSP4 aa112-175 was cloned and expressed using the baculovirus-insect cell system. Purified NSP4 aa112-175 increased intracellular calcium mobilization in intestinal cells when added exogenously, and in insect cells when expressed endogenously, similarly to full-length NSP4. NSP4 aa112-175 caused diarrhea in neonatal mice, as did full-length NSP4. These results indicate that NSP4 aa112-175 is a functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants.

Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VPI/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VPl, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.

The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production.

Rotavirus infection is the leading cause of severe diarrhea in infants and young children worldwide. The rotavirus nonstructural protein NSP4 acts as a viral enterotoxin to induce diarrhea and causes Ca2+-dependent transepithelial Cl- secretion in young mice. The cellular basis of this phenomenon was investigated in an in vitro cell line model for the human intestine. Intracellular calcium concentration ([Ca2+]i) was monitored in fura-2-loaded HT-29 cells using microscope-based fluorescence imaging. NSP4 (1 nM to 5 microM) induced both Ca2+ release from intracellular stores and plasmalemma Ca2+ influx. During NSP4-induced [Ca2+]i mobilization, [Na+]i homeostasis was not disrupted, demonstrating that NSP4 selectively regulated extracellular Ca2+ entry into these cells. The ED50 of the NSP4 effect on peak [Ca2+]i mobilization was 4.6 +/- 0.8 nM. Pretreatment of cells with either 2.3 x 10(-3) units/ml trypsin or 4.4 x 10(-2) units/ml chymotrypsin for 1-10 min abolished the NSP4-induced [Ca2+]i mobilization. Superfusing cells with U-73122, an inhibitor of phospholipase C, ablated the NSP4 response. NSP4 induced a rapid onset and transient stimulation of inositol 1,4,5-trisphosphate (IP3) production in an IP3-specific radioreceptor assay. Taken together, these results suggest that NSP4 mobilizes [Ca2+]i in human intestinal cells through receptor-mediated phospholipase C activation and IP3 production.

The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activity.

During a unique morphogenetic process, rotaviruses obtain a transient membrane envelope when newly synthesized subviral particles bud into the endoplasmic reticulum (ER). As rotavirus particles mature, they lose their transient membrane and a layer of the glycoprotein VP7 forms the virion outer capsid shell. The nonstructural glycoprotein NSP4 functions as an intracellular receptor in the ER membrane (K. S. Au, W. K. Chan, J. W. Burns, and M. K. Estes, J. Virol. 63:4553-4562, 1989), and it has been hypothesized that NSP4 is involved in the removal of the envelope during viral morphogenesis (M. K. Estes and J. Cohen, Microbiol. Rev. 53:410-449, 1989; B. L. Petrie, M. K. Estes, and D. Y. Graham, J. Virol. 46:270-274, 1983). The purpose of the present study was to determine if NSP4 has a direct membrane destabilization activity (MDA) by using liposome leakage assays and electron microscopic visualization of liposome, microsome, and viral envelope disruption. The fluorescent marker (calcein) incorporated into liposomes was released when the liposomes were incubated with purified NSP4. A region corresponding to amino acid residues 114 to 135 of NSP4 also released calcein from liposomes. NSP4(114-135) peptide-specific antibody completely blocked the MDA of the purified NSP4 protein. These results suggest that this region contains at least part of the functional domain of NSP4. Liposomes composed of phosphatidylcholine and microsomes (to simulate ER membranes) were broken when observed by electron microscopy after incubation with NSP4 or the NSP4(114-135) peptide. In contrast, the envelope of Sendai virus, which is derived from cytoplasmic membranes, and erythrocytes were not disrupted by NSP4 and the NSP4(114-135) peptide. These results provide direct evidence that NSP4 possesses MDA and suggest that it can cause ER membrane damage. Therefore, NSP4 might play an important role in the removal of the transient envelope from budding particles during viral morphogenesis. A model for the MDA of NSP4 in viral morphogenesis is proposed.

NSP4 enterotoxin of rotavirus induces paracellular leakage in polarized epithelial cells.

The nonstructural NSP4 protein of rotavirus has been described as the first viral enterotoxin. In this study we have examined the effect of NSP4 on polarized epithelial cells (MDCK-1) grown on permeable filters. Apical but not basolateral administration of NSP4 was found to cause a reduction in the transepithelial electrical resistance, redistribution of filamentous actin, and an increase in paracellular passage of fluorescein isothiocyanate-dextran. Significant effects on transepithelial electrical resistance were noted after a 20- to 30-h incubation with 1 nmol of NSP4. Most surprisingly, the epithelium recovered its original integrity and electrical resistance upon removal of NSP4. Preincubation of nonconfluent MDCK-1 cells with NSP4 prevented not only development of a permeability barrier but also lateral targeting of the tight-junction-associated Zonula Occludens-1 (ZO-1) protein. Taken together, these data indicate new and specific effects of NSP4 on tight-junction biogenesis and show a novel effect of NSP4 on polarized epithelia.

The N terminus of rotavirus VP2 is necessary for encapsidation of VP1 and VP3.

The innermost core of rotavirus is composed of VP2, which forms a protein layer that surrounds the two minor proteins VP1 and VP3, and the genome of 11 segments of double-stranded RNA. This inner core layer surrounded by VP6, the major capsid protein, constitutes double-layered particles that are transcriptionally active. Each gene encoding a structural protein of double-layered particles has been cloned into baculovirus recombinants and expressed in insect cells. Previously, we showed that coexpression of different combinations of the structural proteins of rotavirus double-layered particles results in the formation of virus-like particles (VLPs), and each VLP containing VP1, the presumed RNA-dependent RNA polymerase, possesses replicase activity as assayed in an in vitro template-dependent assay system (C. Q.-Y. Zeng, M. J. Wentz, J. Cohen, M. E. Estes, and R. F. Ramig, J. Virol. 70:2736-2742, 1996). This work reports construction and characterization of VLPs containing a truncated VP2 (VPdelta2, containing amino acids [aa] Met-93 to 880). Expression of VPdelta2 alone resulted in the formation of single-layered delta2-VLPs. Coexpression of VPdelta2 with VP6 produced double-layered delta2/6-VLPs. VLPs formed by coexpression of VPdelta2 and VP1 or VP3, or both VP1 and VP3, resulted in the formation of VLPs lacking both VP1 and VP3. The presence of VP6 with VPdelta2 did not result in encapsidation of VP1 and VP3. To determine the domain of VP2 required for binding VP1, far-Western blot analyses using a series of truncated VP2 constructs were performed to test their ability to bind VP1. These analyses showed that (i) full-length VP2 (aa 1 to 880) binds to VP1, (ii) any N-terminal truncation lacking aa 1 to 25 fails to bind VP1, and (iii) a C-terminal 296-aa truncated VP2 construct (aa 1 to 583) maintains the ability to bind VP1. These analyses indicate that the N terminus of rotavirus VP2 is necessary for the encapsidation of VP1 and VP3.

Microtubule and microfilament distribution and tubulin content in the cell cycle of Indian muntjac cells.

Using DAPI, rabbit antitubulin antibody, FITC-labeled goat anti-rabbit IgG, and TRITC-phalloidin to stain individual cells, the microspectrophotometric analysis showed that three markers that represent the nucleus, microtubules (MT), and microfilaments (MF), respectively, could be recognized in individual cells without interference. The phase of the cell cycle was determined by DNA content. We found that in Indian muntjac (IM) cells, the amount of tubulin in G2 and M phases was about twice as much as that in G1 phase. In G2 cells, the cytoplasmic microtubule complex (CMTC) became denser than in G1 cells. The cytoplasmic MT extent in basically the same orientation as MF bundles in interphase. The regions where the MT is denser also have a denser MF distribution.

The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum.

We previously reported that expression of rotavirus nonstructural glycoprotein NSP4 is responsible for an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in Spodoptera frugiperda (Sf9) insect cells (P. Tian, Y. Hu, W. P. Schilling, D. A. Lindsay, J. Eiden, and M. K. Estes, J. Virol. 68:251-257, 1994). The purpose of the present study was to determine the mechanism by which NSP4 causes an increase in [Ca2+]i by measuring the permeability of the cytoplasmic and endoplasmic reticulum (ER) membranes in recombinant-baculovirus-infected Sf9 cells. No obvious change in plasmalemma permeability to divalent cations was observed in cells expressing NSP4 compared with that in cells expressing another rotaviral glycoprotein (VP7) when the influx of Ba2+, a Ca2+ surrogate, was monitored. The basal Ca2+ permeability of the internal Ca2+ store was evaluated by measuring the release of Ca2+ induced by ionomycin, a Ca2+ ionophore, or thapsigargin, an inhibitor of the ER Ca(2+)-ATPase pump, following suspension of the cells in Ca(2+)-free extracellular buffer. Releasable Ca2+ decreased with time to a greater extent in cells expressing NSP4 compared with that in cells expressing VP7, suggesting that NSP4 increases the basal Ca2+ permeability of the ER membrane. To determine the possible mechanism by which NSP4 increases ER permeability, purified NSP4 protein or a 22-amino-acid synthetic peptide consisting of residues 114 to 135 (NSP4(114-135) was added exogenously to noninfected Sf9 cells during measurement of [Ca2+]i. Both NSP4 and the NSP4(114-135 peptide produced a time-dependent increase in [Ca2+]i that was attenuated by prior inhibition of phospholipase C with U-73122. Pretreatment of the cells with thapsigargin completely blocked the increase in [Ca2+]i produced by NSP4(114-135, but the peptide only partially reduced the change in [Ca2+]i produced by thapsigargin. No changes in [Ca2+]i were seen in cells treated with control peptides. These results suggest that (i) exogenous NSP4 increases [Ca2+]i through the activation of phospholipase C, (ii) Ca2+ release by exogenous NSP4 is from a store that is a subset of the thapsigargin-sensitive compartment, and (iii) amino acid residues 114 to 135 of NSP4 are sufficient for this activity. In contrast to exogenous NSP4, the mechanism by which endogenously expressed NSP4 increases [Ca2+]1 appears to be unrelated to phospholipase C, since no effect of U-73122 was seen on the elevated [Ca2+]1 in cells expressing NSP4 and exogenously applied NSP4(114-135) caused a further increase in [Ca2+]1 in cells expressing NSP4 protein.(ABSTRACT TRUNCATED AT 400 WORDS)