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PubMed2001

Heterotrimers formed by tumor necrosis factors of different species or muteins.

Incubation of murine tumor necrosis factor (mTNF) at subnanomolar concentrations results in partial dissociation of the trimers, coinciding with a decrease in bioactivity. Using size-exclusion chromatography, we observed that the conversion of labeled mTNF to monomers is not only prevented by coincubation with an excess of unlabeled mTNF but also with unlabeled human TNF (hTNF). Moreover, after coincubation of mTNF and hTNF four different TNF complexes were revealed by native polyacrylamide gel electrophoresis, viz. homotrimeric mTNF and hTNF, as well as two complexes with an intermediate migration pattern. Analytical gel filtration in combination with native polyacrylamide gel electrophoresis and Western blot immunodetection indicated that these new complexes consisted of heterotrimeric TNF molecules. We conclude that an exchange of monomers takes place during coincubation of two different species of TNF, which results in homotrimeric and heterotrimeric TNF. To assess receptor interaction in vitro, TNF heterotrimeric molecules were used as obtained after incubation of mTNF with labeled hTNF (which only binds to mTNF receptor I) or with labeled mutein mTNF75 (specific for mTNF receptor II). These heterotrimers were retained by both mTNF receptors, which means that the mTNF subunits incorporated in heterotrimeric complexes still can bind to both types of TNF receptor. In addition, the gradual decrease in mTNF bioactivity during preincubation at subnanomolar concentrations was prevented by the presence of mutein mTNF75, which is inactive in an L929 cytotoxicity assay, indicating that heterotrimerization can influence the overall bioactivity.

Animals↗
PubMed2004

Identification of endoglin in rat hepatic stellate cells: new insights into transforming growth factor beta receptor signaling.

Transforming growth factor beta (TGF-beta) signaling is mediated by the cell surface TGF-beta type I (ALK5), type II, and the accessory type III receptors endoglin and betaglycan. Hepatic stellate cells (HSC), the most profibrogenic cell type in the liver, express ALK5, TbetaRII, and betaglycan. To monitor the expression of betaglycan in HSC, we used the commercially available antibody sc-6199 in Western blot analysis. This antibody, raised against a peptide mapping at the carboxyl terminus of the human betaglycan, is claimed to be specific for betaglycan, although it is known that the C-terminal domain is highly conserved in type III receptors. Proteins recognized in HSC by sc-6199 did not match the characteristic migration pattern of betaglycan. Moreover, the determined molecular weight (M(r) 160) and the observed reductant sensitivity after treatment with dithiothreitol resemble those of a closely related type III receptor, endoglin (CD105). Endoglin, a disulfide-linked homodimer, is an accessory component of the TGF-beta receptor complex and mainly expressed on endothelial cells. The presence of endoglin in HSC of rat liver was confirmed by molecular cloning of the endoglin cDNA and immunocytochemistry. The reactivity of sc-6199 with both auxiliary TGF-beta receptors (betaglycan and endoglin) from rats was demonstrated by Western blot and immunocytochemical analysis of cells heterologously expressing these proteins. Furthermore, Northern and Western blotting revealed that both betaglycan and endoglin genes are differentially regulated in HSC and in transdifferentiated myofibroblasts (MFB). By surface labeling and immunoprecipitation experiments, we show that endoglin is found in significant amounts exposed at the plasma membrane of HSC and MFB, which is a pivotal prerequisite for binding of and signaling in response to TGF-beta. In conclusion, we hypothesize that TGF-beta signals in HSC and MFB are tuned by two different interconnected signaling pathways, as it was previously demonstrated for endothelial cells.

Amino Acid Sequence↗
PubMed2000

Torsin A and its torsion dystonia-associated mutant forms are lumenal glycoproteins that exhibit distinct subcellular localizations.

Early-onset torsion dystonia is an autosomal dominant hyperkinetic movement disorder that has recently been linked to a 3-base pair deletion in the DYT1 gene. The DYT1 gene encodes a 332-amino acid protein, torsin A, that bears low but significant homology to the Hsp100/Clp family of ATPase chaperones. The deletion in DYT1 associated with torsion dystonia results in the loss of one of a pair of glutamic acid residues residing near the C terminus of torsin A (DeltaE-torsin A). At present, little is known about the expression, subcellular distribution, and/or function of either the torsin A or DeltaE-torsin A protein. When transfected into mammalian cells, both torsin A and DeltaE-torsin A were found to behave as lumenally oriented glycoproteins. Immunofluorescence studies revealed that torsin A localized to a diffuse network of intracellular membranes displaying significant co-immunoreactivity for the endoplasmic reticulum resident protein BiP, whereas DeltaE-torsin A resided in large spheroid intracellular structures exclusive of BiP immunoreactivity. These results initially suggested that DeltaE-torsin A might exist as insoluble aggregates. However, both torsin A and DeltaE-torsin A were readily solubilized by nonionic detergents, were similarly accessible to proteases, and displayed equivalent migration patterns on sucrose gradients. Collectively, these data support that both the wild type and torsion dystonia-associated forms of torsin A are properly folded, lumenal proteins of similar oligomeric states. The potential relationship between the altered subcellular distribution of DeltaE-torsin A and the disease-inducing phenotype of the protein is discussed.

Animals↗
PubMed2003

Insect resistance to Bacillus thuringiensis: alterations in the indianmeal moth larval gut proteome.

Insect resistance to the Cry toxins of Bacillus thuringiensis (Bt) has been examined previously using a number of traditional biochemical and molecular techniques. In this study, we utilized a proteomic approach involving two-dimensional differential gel electrophoresis, mass spectrometry, and function-based activity profiling to examine changes in the gut proteins from the larvae of an Indianmeal moth (IMM, Plodia interpunctella) colony exhibiting resistance to Bt. We found a number of changes in the levels of certain specific midgut proteins that indicate increased glutathione utilization, elevation in oxidative metabolism, and differential maintenance of energy balance within the midgut epithelial cells of the Bt-resistant IMM larva. Additionally, the electrophoretic migration pattern of a low molecular mass acidic protein, which apparently is an ortholog of F(1)F(0)-ATPase, was considerably altered in the Bt-resistant insect indicating that variations in amino acid content or modifications of certain proteins also are important components of the resistance phenomenon in the IMM. Furthermore, there was a dramatic decrease in the level of chymotrypsin-like proteinase in the midgut of the Bt-resistant larva, signifying that reduction of chymotrypsin activity, and subsequently decreased activation of Cry toxin in the insect midgut, is an important factor in the resistant state of the IMM. The proteomic analysis of larval gut proteins utilized in this study provides a useful approach for consolidating protein changes and physiological events associated with insect resistance to Bt. Our results support the hypothesis that physiological adaptation of insects and resistance to Bt is multifaceted, including protein modification and changes in the synthesis of specific larval gut proteins. We believe that increased oxidative metabolism may be an adaptive response of insects that undergo survival challenge and that it could mediate detoxification as well as higher rates of generalized and localized mutations that enhance their resistance and provide survival advantage.

Animals↗
PubMed1998

Loss of heterozygosity on the long arm of chromosome 11 in orbital embryonal rhabdomyosarcoma (OERMS): a microsatellite study of seven cases.

OBJECTIVES. To investigate, by means of microsatellite analysis, regions of chromosome 11 involved in the genesis of embryonal rhabdomyosarcoma (ERMS) localized to the orbit. METHODS. Microsatellite analysis was carried out on seven cases of orbital ERMS by comparing the electrophoretic migration patterns of PCR-amplified microsatellites of chromosome 11 from both constitutional (blood) and tumor genotypes. Five of the tumors analyzed were samples frozen at the time of surgery, and two were paraffin embedded. RESULTS. Overall, microsatellites D11S1396 (11q13.1-q22.3) and D11S976 (11q) showed loss of heterozygosity (LOH) in all tumor samples, thus indicating the presence, on the long arm of chromosome 11, of one or more tumor suppressor genes with a possible role in the genesis of the disease. CONCLUSION. While the role of genes on the short arm of chromosome 11 in the genesis of ERMS is well established, much less is known of the possible involvement of tumor suppressor genes on the long arm of the same chromosome. This is the first report showing the possible involvement of tumor suppressor genes in this portion of the chromosome in ERMS localized to the orbit.

Journal Article↗
PubMed2003

Functional analysis of the single calmodulin gene in the nematode Caenorhabditis elegans by RNA interference and 4-D microscopy.

Calmodulin (CaM), a small calcium-binding protein, is the key mediator of numerous calcium-induced changes in cellular activity. Its ligands include enzymes, cytoskeletal proteins and ion channels, identified in large part by biochemical and cell biological approaches. Thus far it has been difficult to assess the function of CaM genetically, because of the maternal supply in Drosophila and the presence of at least three nonallelic genes in vertebrates. Here we use the unique possibility offered by the C. elegans model system to inactivate the single CaM gene (cmd-1) through RNA interference (RNAi). We show that the RNAi microinjection approach results in a severe embryonic lethal phenotype. Embryos show disturbed morphogenesis, aberrant cell migration patterns, a striking hyperproliferation of cells and multiple defects in apoptosis. Finally, we show that RNAi delivery by the feeding protocol does not allow the efficient silencing of the CaM gene obtained by microinjection. General differences between the two delivery methods are discussed.

Amino Acid Sequence↗
PubMed2004

DNA based classification of food associated Enterobacteriaceae previously identified by Biolog GN Microplates.

Enterobacteriaceae are frequently isolated from food products and it is essential to have methods for correct identification for both food hygiene and epidemiology reasons. Phenotypic methods are not always sufficient and have to be supplemented by DNA based methods. In the present study, 70 strains of Enterobacteriaceae derived from milk, fish and meat that had previously been identified by Biolog GN Microplates were genomically classified together with 15 representative type strains of species of Enterobacteriaceae. The field strains were dominated by Hafnia alvei, Serratia liquefaciens and Rahnella aquatilis. All strains were subjected to temporal temperature gel electrophoresis (TTGE) analysis using amplicons encompassing the V3, V4 and V9 variable regions of the 16S rRNA gene. Selected strains were analysed by ribotyping and partial 16S rDNA sequencing. The type strains were differentiated into 10 different TTGE groups. Two of the groups contained two type strains. Enterobacter aerogenes and Klebsiella planticola were not distinguished due to their identical sequences and Yersinia ruckeri and Citrobacter freundii showed the same migration pattern. The 70 food strains could be differentiated into 14 TTGE groups where 33 strains (47.1%) could be assigned to TTGE groups including type or reference strains. Rahnella strains were dispersed into three TTGE groups of which one group corresponded to Rahnella genomospecies 1 and one to genomospecies 3. The grouping of Rahnella strains was supported by ribotyping and phylogenetic analysis. TTGE can be a useful additional tool for identification on the species level of food related Enterobacteriaceae.

Animals↗
PubMed1993

Some observations on ethnoveterinary medicine in northern Nigeria.

The Fulani are herdsmen of Northern Nigeria. For generations they have been moving their cattle in an annual migration pattern in search of water. The tracks are chosen carefully, however: tsetse-fly-infected areas are avoided. In his ten years of experience in Nigeria's veterinary sector--part of this time as special officer in charge of eradicating rinderpest--the author saw numerous examples of indigenous knowledge and practices that will be useful for the future development of animal health and production.

Animal Husbandry↗
PubMed1992

The molecular epidemiology of rotavirus-associated gastro-enteritis in the Transkei, southern Africa.

Rotaviruses were detected in 32.8% (71/216) of stool specimens collected from young children with gastro-enteritis attending the Rehydration Unit at Umtata General Hospital between April 1988 and March 1989. A seasonal distribution was observed with an increase in numbers shedding the virus during the colder months of the year. Based on variations in the migration pattern of the RNA genome segments when passed through polyacrylamide gels, 14 different electrophoretypes were identified: 69% were long patterns and 27% were short patterns. A single dominant long electrophoretype persisted throughout the 12-month study period, whereas 13 other minor types co-circulated at varying intervals. Mixed infections were observed in three cases, including one infant who was infected with both a long (Wa-like) virus and a short (DS-1-like) virus.

Child, Preschool↗
PubMed1977

Marriage and mobility in Britain--secular trends in a nationwide sample.

A nationwide sample survey of 2338 married couples provides information on the birthplaces and residences at meeting of couples first married between 1920 and 1960. Four measures of marital mobility are analysed according to year of marriage and year of first meeting. In their original form all measures show the expected increase in mobility from the 1920s to the 1930s with an exceptionally steep rise in the 1940s and a decline after the Second World War. The role of the war in promoting migration both of civilians and of soldiers is briefly reviewed. Women show rather different migration patterns from those of men. Age at marriage is positively correlated with movement in this sample, but a stronger association is provided by age at first meeting. Mobility averages for each cohort are adjusted for the changes in age at marriage, both real and peculiar to this sample, which have occurred since 1920, and also for variation in the population sizes of birthplaces in the sample over time, and changes in the relative frequencies of different socio-economic classes. After adjustment, little ghange is seen in material mobility over the four decades covered by the survey and many of the differences which do remain after adjustment lack statistical significance. Some socio-economic explanations are offered for these conclusions.

Age Factors↗
PubMed1998

Identification of new and known polymorphisms in glycoprotein IIb and IIIa genes by denaturing gradient gel electrophoresis.

Glycoprotein IIb and IIIa contain antigenic determinants involved in the potential production of allo- or autoantibodies directed against platelets, that may result in severe thrombocytopenia. Most of these epitopes appear to be supported by single nucleotide substitutions. We have used denaturing gradient gel electrophoresis (DGGE) to identify sequence variations within the promoter and the coding regions of the glycoprotein IIb and glycoprotein IIIa genes. Using genomic DNA from 60 unrelated normal individuals, we have amplified short domains that encompass the coding sequences and the exon-intron boundaries of both genes that were further separated according to their melting behaviour during the denaturant electrophoretic migration. Only the fragments with an abnormal migration pattern were sequenced. We confirmed the sensitivity of this method by recognizing both previously described Human Platelet Antigen polymorphisms and mutations affecting either the glycoprotein IIb or the glycoprotein IIIa genes in thrombasthenic patients. We also identified four other polymorphisms. Two were located in the glycoprotein IIb gene, involving intron 21 (C<-->G at nucleotide 10480) and first codon of exon 30 (codon GTC<-->GTT coding for residue Val 990), and two in the glycoprotein IIIa gene (exon 6 CCC<-->CCT coding for residue Pro 268; intron 14 C<--> T at position 37126). The screening of the GPIIIa promoter also revealed three different polymorphisms located at position-468 (A/T polymorphism), -425 (A/C polymorphism) and-400 (A/C polymorphism), which could influence the expression of the complex at the cell surface. Denaturing gradient gel electrophoresis appears to be a sensitive and specific technique for identifying polymorphisms and mutations in the GPIIb and GPIIIa genes.

Journal Article↗
PubMed2005

Families living with HIV.

Given the historical emergence of the AIDS epidemic first among gay men in the developed world, HIV interventions have primarily focused on individuals rather than families. Typically not part of traditional family structures, HIV-positive gay men in Europe and the US lived primarily in societies providing essential infrastructure for survival needs that highly value individual justice and freedom. Interventions were thus designed to focus on at-risk individuals with programmes that were age and gender segregated. As the epidemic has unfolded, the early focus on individuals has become inadequate: families live with HIV, not just individuals. Families' structure, economy, migration patterns, and developmental life cycles are affected by HIV, and these changes radiate throughout the community creating parallel stresses. Family-based, intergenerational models of detection, prevention and treatment services offer enhanced opportunities for effective interventions and suggest very different intervention settings and strategies. However, these models also require addressing the family's basic needs for survival and security in order to be successfully implemented and sustained over time. As HIV was an opportunity for marginalized persons in the developed world to 'turn their life around', the strengths of families in the developing world may be mobilized to contribute to the community's long-term health, survival and security needs.

Community Health Services↗
PubMed1987

Bleomycin, in contrast to gamma irradiation, induces extreme variation of DNA strand breakage from cell to cell.

Chinese hamster ovary cells grown in vitro were treated with bleomycin or irradiated with high doses of 60Co gamma rays (200 and 400 Gy). DNA strand breaks in single cells were analysed by using our newly introduced microelectrophoretic technique. Bleomycin seems to act in a selective manner so that in some cells the DNA is heavily degraded while in others there is only moderate or no measurable damage. In contrast, a uniform response was found after gamma irradiation. To achieve the same magnitude of DNA fragmentation as in the most severely bleomycin-damaged cells, irradiation with more than 200 Gy is required. Some 8000 double-strand breaks per cell are produced by 200 Gy which will convert the molecular weight of the DNA to the range of 10(8)-10(9) dalton, and free migration of DNA fragments occurs during electrophoresis. We include also a detailed study of the DNA migration pattern following doses of 0-100 Gy gamma rays.

Animals↗
PubMed1994

Effect of gamma-irradiation on the function and viability of alveolar macrophages in mouse and rat.

The tumoricidal activity of C3H mouse alveolar macrophages (AM) was completely abolished by 100 Gy gamma-irradiation, whereas the activities of AM of C57Black/6 (B6) and Balb/c mice were not significantly affected. The viability of AM, determined by both vital staining with crystal violet and by trypan blue exclusion, was decreased markedly by gamma-irradiation only in C3H mouse AM. AM of B6, Balb/c mice and four strains of rat were much more radioresistant than C3H mouse AM with respect to cell viability. On the other hand the effect of irradiation on the colony-forming capacity of AM was essentially the same in three strains of mouse. Morphological examination of irradiated C3H mouse AM and the DNA migration pattern in agarose gel electrophoresis indicated that gamma-irradiation induced high levels of apoptosis in C3H mouse AM. These results suggest that the apoptotic cell death induced by gamma-irradiation at doses that do not affect AM of the other strains of mouse and rat explains the radiosensitivity of C3H mouse AM with respect to cell viability and tumoricidal activity.

Animals↗
PubMed1997

Effect of 6(5H)-phenanthridinone, a poly (ADP-ribose)polymerase inhibitor, and ionizing radiation on the growth of cultured lymphoma cells.

The ability of 6(5H)-phenanthridinone (Phen), a new potent poly(ADP-ribose)polymerase (PARP) inhibitor, to potentiate the effect of ionizing radiation on tumour cells was evaluated. RDM4 murine lymphoma cells were irradiated using a 60Co panoramic source and then examined for their growth, cell cycle distribution and apoptosis. Phen (100 microM) was found to inhibit more than 90% of the PARP activity in control and irradiated cells. Cell proliferation was assessed using Alamar Blue, a new fluorometric assay. Phen was found to sharply increase the radiation-induced inhibition of cell proliferation. Indeed, at 2.5 Gy the relative cell number of Phen-treated cells was 60% below control levels. At the same radiation dose, the G2M arrest was also significantly reinforced by the addition of Phen. Furthermore, this PARP inhibitor was shown to significantly increase the amount of DNA fragmentation as revealed by the DNA migration pattern in agarose gel electrophoresis. Comparable results were obtained with 3-aminobenzamide, another PARP inhibitor, but at concentrations 200-fold higher. Taken together, these results indicate the potential interest of Phen as a valuable pharmacological probe for investigating the role of PARP in cellular responses to radiation. They also suggest a possible use of Phen as an adjuvant in radiotherapy.

Animals↗
PubMed2007

A cross-national comparison of youth risk behaviors in Latino secondary school students living in El Salvador and the USA.

OBJECTIVES: As Latin Americans' exposure to the USA increases through migration patterns and US political and economic ties to their countries of origin, they become susceptible to adopting not only the cultural expressions of the USA such as fashion, but also the health-related behaviors of the US population. In assessing potential health risks for Salvadoran youth that may result from the connection between Latin Americans and the USA, this study compared the prevalence of health risk behaviors from four behavior domains (aggression and victimization, depression and suicidal ideation, substance use, and sexual behavior) between Salvadoran and US Latino secondary school students aged 14-17 years. DESIGN: A secondary analysis was performed on two 1999 cross-sectional survey data. In the USA, results were based on 1,063 Latino high school students who answered the nationally representative Youth Risk Behavior Survey (YRBS) conducted by the Centers for Disease Control and Prevention. In El Salvador, results were based on 793 public secondary school students who answered a local YRBS survey conducted in coordination with the Ministry of Education of El Salvador. RESULTS: The prevalence rates for aggression/victimization and for depression and suicidal ideation behaviors were similar between Salvadoran and US Latino adolescents. Substance use prevalence, however, was 10-40% higher for US Latino adolescents. While the prevalence of sexual intercourse was higher among US Latino youth (between 13 and 27% higher, depending on age), the prevalence of condom use was lower among sexually active Salvadoran youth (between 11 and 42% lower, depending on age). CONCLUSIONS: In the context of the transnationalization of the Salvadoran population, with potential for increased influence of the USA in Salvadoran culture, these differences in risk behavior are important for targeting effective interventions for Latino adolescents in El Salvador and in the USA.

Adolescent↗
PubMed2005

Radiostereometric analysis of the double-coated STAR total ankle prosthesis: a 3-5 year follow-up of 5 cases with rheumatoid arthritis and 5 cases with osteoarthrosis.

BACKGROUND: The designs of total ankle prostheses have changed in recent years in order to give better performance. Only a few studies of these ankle prostheses have been published, however, and none on micromotion. PATIENTS AND METHODS: We evaluated 5 patients with rheumatoid arthritis and 5 with osteoarthrosis, 4 (3-5) years after arthroplasty with the double-coated STAR prosthesis. Clinical examination included AOFAS hindfoot score. Standardized a-p and lateral radiographs were taken and RSA analyses were done at regular intervals. RESULTS: There was no difference in results between ankles operated on due to rheumatoid arthritis and due to osteoarthrosis. A rapid initial migration was observed for the tibial components at 6 weeks, but thereafter all but 1 implant seemed stable. The migration pattern for the talar component was similar. Rotation around the 3 axes was observed for the tibial components at 6 weeks, but not thereafter. The talar components became stable for rotation around the longitudinal and sagittal axes, but not around the transverse axis. 8 out of 10 ankles were painless. The median total AOFAS score was 83 and the median range of motion was 32 degrees. None of the 20 components had changed position and there were no signs of bone resorption. INTERPRETATION: Provided the indication is adequate and the prosthesis has been implanted correctly, the double-coated STAR ankle prosthesis will have a satisfactory fixation to underlying bone.

Ankle Joint↗
PubMed1977

Immunochemical evidence for an additional H-2 region closely linked to H-2D.

Anti-H-2 reagents have been tested on solubilized spleen cell preparations in combinations expected to be specific for D region products. Two different types of molecules were detected. One showed the expected reactivity with both antisera to private and antisera to public specificities. However, an additional molecule was detected which reacted only with antisera to public specificities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration patterns indicated that both products have a similar molecular size of approximately 45,000 daltons. The data therefore present chemical evidence for the existence of a third H-2-associated gene product of 45,000 mol wt in addition to the classical H-2K and H-2D antigens.

Animals↗