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Biomedical subjects

J M Gilbert

Publications and source records attributed to J M Gilbert.

At least 19 recordsLinked to original sources
PubMed2000

The dhfr oribeta-binding protein RIP60 contains 15 zinc fingers: DNA binding and looping by the central three fingers and an associated proline-rich region.

Initiation of DNA replication occurs with high frequency within oribeta, a short region 3' to the Chinese hamster dhfr gene. Homodimers of RIP60 (replication initiation-region protein 60 kDA) purified from nuclear extract bind two ATT-rich sites in oribeta and foster the formation of a twisted 720 bp DNA loop in vitro. Using a one hybrid screen in yeast, we have cloned the cDNA for human RIP60. RIP60 contains 15 C(2)H(2)zinc finger (ZF) DNA binding motifs organized in three clusters, termed hand Z1 (ZFs 1-5), hand Z2 (ZFs 6-8) and hand Z3 (ZFs 9-15). A proline-rich region is located between hands Z2 and Z3. Gel mobility shift and DNase I footprinting experiments show hands Z1 and Z2 independently bind the oribeta RIP60 sites specifically, but with different affinities. Hand Z3 binds DNA, but displays no specificity for RIP60 sites. Ligation enhancement, DNase I footprinting, and atomic force microscopy assays show that hand Z2 and a portion of the associated proline-rich region is sufficient for protein multimerization on DNA and DNA looping in vitro. Polyomavirus origin-dependent plasmid replication assays show RIP60 has weak replication enhancer activity, suggesting that RIP60 does not harbor a transcriptional transactivation domain. Because vertebrate origins of replication have no known consensus sequence, we suggest that sequence-specific DNA binding proteins such as RIP60 may act as accessory factors in origin identification prior to the assembly of pre-initiation complexes.

3T3 Cells↗
PubMed2000

Surgical management of polypoid lesions of the gallbladder.

BACKGROUND: With improvements in ultrasonography more polypoid lesions of the gallbladder (PLGs) are being detected. The management of these is controversial. METHODS: The demographic, radiological and pathological data of 38 patients with ultrasonographically detected PLGs were reviewed. A Medline search for such lesions was performed and a review of the literature is presented. RESULTS: Thirty-four patients underwent cholecystectomy and four were advised against or declined operation. Of the 34 who had cholecystectomy, 11 had macroscopic and histopathologically proven PLGs. Of these, seven had cholesterol polyps, two had adenomas, one had a carcinoid tumour and one had an adenocarcinoma of the gallbladder. One patient had a histopathologically normal gallbladder. The remainder had chronic cholecystitis with or without gallstones. All of the patients with neoplastic lesions of the gallbladder had solitary polyps greater than 1.0 cm in diameter. CONCLUSION: A protocol for the management of ultrasonographically detected PLGs is proposed. In this protocol it is suggested that patients with a PLG should undergo surgery if they are symptomatic, or if the PLG is 1.0 cm or more in diameter.

Adult↗
PubMed2000

The central proline of an internal viral fusion peptide serves two important roles.

The fusion peptide of the avian sarcoma/leukosis virus (ASLV) envelope protein (Env) is internal, near the N terminus of its transmembrane (TM) subunit. As for most internal viral fusion peptides, there is a proline near the center of this sequence. Robson-Garnier structure predictions of the ASLV fusion peptide and immediate surrounding sequences indicate a region of order (beta-sheet), a tight reverse turn containing the proline, and a second region of order (alpha-helix). Similar motifs (order, turn or loop, order) are predicted for other internal fusion peptides. In this study, we made and analyzed 12 Env proteins with substitutions for the central proline of the fusion peptide. Env proteins were expressed in 293T cells and in murine leukemia virus pseudotyped virions. We found the following. (i) All mutant Envs form trimers, but when the bulky hydrophobic residues phenylalanine or leucine are substituted for proline, trimerization is weakened. (ii) Surprisingly, the proline is required for maximal processing of the Env precursor into its surface and TM subunits; the amount of processing correlates linearly with the propensity of the substituted residue to be found in a reverse turn. (iii) Nonetheless, proteolytically processed forms of all Envs are preferentially incorporated into pseudotyped virions. (iv) All Envs bind receptor with affinity greater than or equal to wild-type affinity. (v) Residues that support high infectivity cluster with proline at intermediate hydrophobicity. Infectivity is not supported by mutant Envs in which charged residues are substituted for proline, nor is it supported by the trimerization-defective phenylalanine and leucine mutants. Our findings suggest that the central proline in the ASLV fusion peptide is important for the formation of the native (metastable) Env structure as well as for membrane interactions that lead to fusion.

Alpharetrovirus↗
PubMed1999

Production and characterization of a soluble, active form of Tva, the subgroup A avian sarcoma and leukosis virus receptor.

The receptor for the subgroup A avian sarcoma and leukosis viruses [ASLV(A)] is the cellular glycoprotein Tva. A soluble form of Tva, sTva, was produced and purified with a baculovirus expression system. Using this system, 7 to 10 mg of purified sTva per liter of cultured Sf9 cells was obtained. Characterization of the carbohydrate modification of sTva revealed that the three N glycosylation sites in sTva were differentially utilized; however, the O glycosylation common to Tva produced in mammalian and avian cells was not observed. Purified sTva demonstrates significant biological activity, specifically blocking infection of avian cells by ASLV(A) with a 90% inhibitory concentration of approximately 25 pM. A quantitative enzyme-linked immunosorbent assay, developed to assess the binding of sTva to ASLV envelope glycoprotein, demonstrates that sTva has a high affinity for EnvA, with an apparent dissociation constant of approximately 0.3 nM. Once they are bound, a very stable complex is formed between EnvA and sTva, with an estimated complex half-life of 6 h. The soluble receptor protein described here represents a valuable tool for analysis of the receptor-envelope glycoprotein interaction and for structural analysis of Tva.

Alpharetrovirus↗
PubMed1999

Controlled intraoperative water testing of left-sided colorectal anastomoses: are ileostomies avoidable?

Anastomotic leakage is a major problem in colorectal surgery, and previous studies have suggested that intraoperative identification of leaks allows repair at the time of surgery. This study examined whether testing allowed a defunctioning ileostomy to be safely omitted. A series of 102 consecutive patients underwent left-sided colorectal resection, 52 males and 50 females, mean age 65.7 years (range 16-89 years). After completion of the anastomosis, its integrity was tested by running saline into the rectum, using a manometer, to a maximum distending pressure of 30 cmH2O. Any leaks were repaired and the anastomosis retested. A defunctioning ileostomy was only performed if the anastomosis could not be shown to be leak-proof on testing. Patients underwent a contrast enema on the 8th postoperative day. Twenty-one (20.6%) patients failed the initial leakage test and 3 (3%) patients failed a second test. Two of these 21 patients went on to have a clinical leak, both of which were treated conservatively. Two defunctioning ileostomies were performed at the time of surgery. Sixteen (16.2%) had a leak on radiological testing, and there was clinical evidence of a leak in 5 (4.9%) patients. There were 3 (2.9%) deaths, but none of these had a leak on radiological testing. Incomplete anastomoses were successfully corrected intraoperatively. A defunctioning ileostomy was avoided in 98% of cases. Intraoperative testing to a pressure of 30 cmH2O is helpful in anterior resection, but does not guarantee that an intact anastomosis will remain intact postoperatively.

Adolescent↗
PubMed1998

Biological effects of gamma-irradiation on laboratory and field isolates of Eimeria tenella (Protozoa; Coccidia).

Sporulated oocysts of a field strain (FS-111) and a laboratory strain (WIS) of Eimeria tenella were exposed to 0, 50, 100, 150, or 200 Gy of gamma-radiation from a 60Co source. Irradiated oocysts of WIS and FS-111 were not significantly more fragile after irradiation as shown by the release of sporocysts after 5-105 s of vortex agitation with glass beads. Excystation was normal in both strains after treatment of the sporocysts with trypsin and sodium taurodeoxycholate, even in groups exposed to 200 Gy of radiation. Sporozoite release from irradiated sporocysts was more rapid than that from unirradiated sporocysts, primarily because of a shorter lag phase during the first 30 min. Irradiated sporozoites were slower to parasitize cultured chick kidney cells than were control sporozoites (4 h postinoculation), but after 24 h there was no significant difference (P < 0.05) between irradiated and control groups except for the WIS treated with 200 Gy. After 48 h, developing schizonts were reduced by 77-94% on exposure to 50-200 Gy. Strain FS-111 did not develop as well as WIS in vitro, but the effect of irradiation was similar. When irradiated oocysts of WIS or FS-111 were inoculated into chickens the prepatent period was unaffected, but fewer oocysts were produced, lesion scores were lower, and the weight gain was less strongly affected in proportion to the doses of radiation. These results suggest that the effects of radiation damage were largely confined to the mechanism of nuclear and cellular reproduction rather than other physiological processes.

Animals↗
PubMed1998

Cleavage of rhesus rotavirus VP4 after arginine 247 is essential for rotavirus-like particle-induced fusion from without.

We recently described our finding that recombinant baculovirus-produced virus-like particles (VLPs) can induce cell-cell fusion similar to that induced by intact rotavirus in our assay for viral entry into tissue culture cells (J. M. Gilbert and H. B. Greenberg, J. Virol. 71:4555-4563, 1997). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection. This VLP-mediated fusion activity was dependent on the presence of the outer-layer proteins, viral protein 4 (VP4) and VP7, and on the trypsinization of VP4. Fusion activity occurred only with cells that are permissive for rotavirus infection. Here we begin to dissect the role of VP4 in rotavirus entry by examining the importance of the precise trypsin cleavage of VP4 and the activation of VP4 function related to viral entry. We present evidence that the elimination of the three trypsin-susceptible arginine residues of VP4 by specific site-directed mutagenesis prevents syncytium formation. Two of the three arginine residues in VP4 are dispensable for syncytium formation, and only the arginine residue at site 247 appears to be required for activation of VP4 functions and cell-cell fusion. Using the recombinant VLPs in our syncytium assay will aid in understanding the conformational changes that occur in VP4 involved in rotavirus penetration into host cells.

Amino Acid Substitution↗
PubMed1997

Comparison of the rotavirus gene 6 from different species by sequence analysis and localization of subgroup-specific epitopes using site-directed mutagenesis.

The nucleotide sequence of gene 6 encoding the rotavirus major capsid protein VP6 of EDIM strain (EW) was determined and compared to that of 20 previously reported strains with known subgroup specificities. Multiple alignments of amino acid sequences exhibited a high level of sequence conservation (87 to 99.2%). Site-specific mutagenesis experiments were undertaken to localize regions involved in subgroup specificity. Amino acid positions 305, 315, and a region 296-299 (or 301 for equine strain H-2) were identified as contributing to subgroup epitopes. A single amino acid mutation at position 305 or 315 was sufficient to change the subgroup specificity of EW VP6 protein from non I/II to subgroup I- or subgroup II-like, respectively. Mutation at these sites may be another important mechanism for subgroup variation, along with gene reassortment.

Amino Acid Sequence↗
PubMed1997

Invasive carcinoma in juvenile polyposis--report of a case in an African patient.

Since the 1980s it has been recognized that the polyps of juvenile polyposis have malignant potential. Since the introduction of colonoscopy this syndrome has been redefined and the hamartoma-adenoma-carcinoma sequence recognized. We believe this case is the first report of an invasive carcinoma associated with juvenile polyposis to be described in an African patient.

Adenocarcinoma, Mucinous↗
PubMed1997

Immune responses to individual rotavirus proteins following heterologous and homologous rotavirus infection in mice.

Serum and intestinal humoral immune responses to rotavirus proteins VP2, VP4, VP6, VP7, NSP2, and NSP4 were quantitatively compared in mice infected with a homologous murine rotavirus (EHPw) or a heterologous simian rotavirus (RRV). Viral protein-specific antibody responses were measured by an immunohistochemistry assay that uses recombinant baculovirus-expressed rotavirus proteins as antigens. In serum, IgG responses to VP6 were dominant and comparable in both RRV- and EHPw-infected groups, but responses to VP2, VP4, VP7, and NSP2 were higher in RRV infection. In feces, IgA responses to VP2, VP4, and VP6 were higher in EHPw-infected mice, but responses to VP7 and NSP2 were detected only in the RRV-infected group. These findings indicate that immune responses to homologous and heterologous rotavirus infection vary both quantitatively and qualitatively. Differences in humoral responses may play a role in the differences in protection induced following homologous or heterologous rotavirus infection.

Animals↗
PubMed1997

Virus-like particle-induced fusion from without in tissue culture cells: role of outer-layer proteins VP4 and VP7.

We recently described an assay that measures fusion from without induced in tissue culture cells by rotavirus, a nonenveloped, triple-protein-layered member of the Reoviridae family (M. M. Falconer, J. M. Gilbert, A. M. Roper, H. B. Greenberg, and J. S. Gavora, J. Virol. 69:5582-5591, 1995). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection of host cells, as the presence of the outer-layer proteins VP4 and VP7 and the cleavage of VP4 are required. Here we present evidence that virus-like particles (VLPs) produced in Spodoptera frugiperda Sf-9 cells from recombinant baculoviruses expressing the four structural proteins of rotavirus can induce cell-cell fusion to the same extent as native rotavirus. This VLP-mediated fusion activity was dependent on trypsinization of VP4, and the strain-specific phenotype of individual VP4 molecules was retained in the syncytium assay similar to what has been seen with reassortant rotaviruses. We show that intact rotavirus and VLPs induce syncytia with cells that are permissive to rotavirus infection whereas nonpermissive cells are refractory to syncytium formation. This finding further supports our hypothesis that the syncytium assay accurately reflects very early events involved in viral infection and specifically the events related to viral entry into the cell. Our results also demonstrate that neither viral replication nor rotavirus proteins other than VP2, VP6, VP4, and VP7 are required for fusion and that both VP4 and VP7 are essential. The combination of a cell-cell fusion assay and the availability of recombinant VLPs will permit us to dissect the mechanisms of rotavirus penetration into host cells.

Animals↗
PubMed1996

Quantification of systemic and local immune responses to individual rotavirus proteins during rotavirus infection in mice.

The purpose of the present study was to develop a quantitative assay that could be used to measure the local and systemic immune responses to specific rotavirus proteins following rotavirus infection of adult mice. To measure these responses, we used an immunocytochemical staining assay of Spodoptera frugiperda (Sf-9) cells which were infected with recombinant baculovirus expressing selected rotavirus proteins. The specificity of the assay was documented by using a series of monoclonal antibodies to individual rotavirus proteins. We observed that the assay had high levels of sensitivity and specificity for a series of VP7- and VP4-specific neutralizing monoclonal antibodies which recognized conformation-dependent epitopes on their target proteins. We also studied immunoglobulin G (IgG) immune responses in serum and IgA immune responses in the stools of mice infected with wild-type murine rotavirus strain EHPw. In both sera and stools, the most immunogenic proteins were VP6 and VP4. VP2 was less immunogenic than VP6 or VP4, and the immune responses to VP7, NSP2, and NSP4 were very low in serum and undetectable in stools.

Animals↗
PubMed1996

Splenectomy in a general hospital.

Splenectomy is often performed in patients with malignant disease or trauma who are at a high risk of complications. In the long term, it increases the risk of infection by encapsulated bacteria. An audit was performed to determine the reasons for splenectomy in a district general hospital, to review the results and complications of surgery, and to see how often the patients were prescribed antibacterial prophylaxis. Twenty-eight patients underwent splenectomy in 3 years. The indication was haematological disease in 13 and trauma in four. In the remaining nine the spleen was removed either as part of a radical gastrectomy or during some other abdominal procedure. Six of the 28 patients had died, one within 30 days from disseminated intravascular coagulopathy following an emergency gastrectomy and splenectomy for haematemesis, two from progressive haematological malignant disease, two from non-haematological malignancy, and one from bronchopneumonia. Of the nine patients (32%) with complications, three required a further laparotomy. Most patients had been prescribed pneumococcal vaccine (85%) and prophylactic antibiotics (93%).

Adolescent↗
PubMed1995

Receptor-induced conformational changes in the subgroup A avian leukosis and sarcoma virus envelope glycoprotein.

We recently reported that Tva, the host cell receptor for subgroup A avian leukosis and sarcoma viruses, binds specifically to the subgroup A envelope glycoprotein (Env-A) (J.M. Gilbert, P. Bates, H. E. Varmus, and J. M. White, J. Virol. 68:5623-5628, 1994). Here we have tested the hypothesis that binding of Tva causes conformational changes in Env-A that correlate with its conversion from a fusion-inactive to a fusion-active state. Conformational changes were examined by both a proteolysis and an immunoprecipitation assay. A temperature-dependent conformational change, demonstrated by the generation of a specific thermolysin digestion product of the surface (SU) subunit, occurred when a soluble form of Tva (sTva) was incubated with Env-A. sTva did not induce this conformational change in Env-C or in a noninfectious precursor form of Env-A, Env-A CL. However sTva did induce the conformational change in Env-A CL that had been pretreated in vitro to produce the SU and transmembrane (TM) subunits. Moreover, interaction of Tva with Env-A at 25 degrees C, but not at 4 degrees C, appeared to reveal a previously buried segment of the putative fusion peptide of Env-A. Our results suggest that binding of Tva to Env-A results in specific conformational changes in the Env-A glycoprotein that are relevant to the activation of its fusion function.

3T3 Cells↗
PubMed1995

Rotavirus-induced fusion from without in tissue culture cells.

We present the first evidence of fusion from without induced in tissue culture cells by a nonenveloped virus. Electron micrographs of two strains of rotavirus, bovine rotavirus C486 and rhesus rotavirus, show that virally mediated cell-cell fusion occurs within 1 h postinfection. Trypsin activation is necessary for rotavirus to mediate cell-cell fusion. The extent of fusion is relative to the amount of virus used, and maximum fusion occurs between pHs 6.5 and 7.5. Fusion does not require virus-induced protein synthesis, as virus from both an empty capsid preparation and from an EDTA-treated preparation, which is noninfectious, can induce fusion. Incubation of rotavirus with neutralizing and nonneutralizing monoclonal antibodies before addition to cells indicates that viral protein 4 (VP4; in the form of VP5* and VP8*) and VP7 are involved in fusion. Light and electron micrographs document this fusion, including the formation of pores or channels between adjacent fused cells. These data support direct membrane penetration as a possible route of infection. Moreover, the assay should be useful in determining the mechanisms of cell entry by rotavirus.

Animals↗