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PubMed1994

Detection of point mutations in exon 2 of the G6PD gene in Chinese G6PD variants.

In the past few years, a total of 6 different mutations of the G6PD gene have been reported in China. One of these, the C6 mutation (A95-->G), accounted for about 15.4% of the Chinese G6PD variants. In order to develop a strategy for rapid detection of mutation-containing exons of the G6PD gene, we applied the single-strand conformation polymorphism (SSCP) technique to the detection of mutations in exon 2 of this gene. We observed four patients with abnormal migration patterns of the exon 2 band among 20 cases of G6PD variants. Direct PCR sequencing confirmed a T to C substitution in exon 2 that has previously been reported. This procedure is therefore of particular importance for the rapid detection of mutation-containing exons in the G6PD gene.

Base Sequence↗
PubMed1994

Multiple fluorescence-based PCR-SSCP analysis.

Multiple fluorescence-based polymerase chain reaction single-strand conformation polymorphism (MF-PCR-SSCP) was developed. The target sequence was amplified by PCR using forward and reverse primers labeled with two different fluorescent dyes at their 5' ends. The amplified products were then heat-denatured, mixed with internal standard DNA markers labeled with a third fluorescent dye and applied to a temperature-controlled gel in an automated DNA sequencer, with a gel-temperature-controlling system. Mutations were detected as positional shifts of two-colored peaks in the electrophoretogram. The image data were analyzed by the computer program GENESCAN 672. The peak positions were standardized to internal DNA size markers. MF-PCR-SSCP analysis of 7 human tumor cell lines with 7 different single base mutations of the human K-ras oncogene detected all mutations even under the same electrophoresis conditions. Complete loss of heterozygosity was detected in two cell lines simultaneously. A gel temperature at 20 degrees C and polyacrylamide concentration of 10% gave the best separation. MF-PCR-SSCP is superior to the current PCR-SSCP in several ways: it does not involve radioactivity, migration patterns are standardized to internal standard DNA markers, there is a strict temperature-controlling system and the higher percentage of the gel enables better separation with resultant 100% detection of mutations most likely under one set of electrophoresis conditions.

Base Sequence↗
PubMed1993

[Selected properties of strains of a new species of Proteus penneri from the second American collection].

The second collection of the novel species Proteus penneri consists of 25 strains from which only two have shown rough from properties in the tests differentiating S and R variants of bacteria. The migration pattern of their lipopolysaccharides in gel electrophoresis was leader-like, typical for smooth organisms. 13 out of 25 lipopolysaccharide preparations showed strong-reactivity with anti-0 sera in semi-quantitative precipitation test. Serological similarity between the strains within species Proteus penneri is discussed.

Electrophoresis, Agar Gel↗
PubMed1993

Tubulovesicular transcytotic pathway in rat biliary epithelium: a study in perfused liver and in isolated intrahepatic bile duct.

Morphometric ultrastructural analysis of horseradish peroxidase-containing structures has been performed in vivo, in rat liver and, in vitro, in isolated bile ducts to determine whether a transcytotic vesicle pathway exists in biliary epithelial cells. In vivo, horseradish peroxidase (100 mg/kg body wt) was given by intraportal injection in normal rats (n = 15) or 1 hr after administration of 600 mg/kg valproic acid (n = 15). Ultrastructural morphometric analysis was conducted on livers between 1 and 40 min after horseradish peroxidase injection. In vitro, bile ducts were isolated on collagenase digestion, incubated in horseradish peroxidase for 3 min and prepared for electron microscopy immediately or after incubation for another 5, 10, 15 or 20 min in horseradish peroxidase-free medium at 37 degrees C. In four experiments, colchicine (10(-5) mol/L) or beta-lumicolchicine (10(-5) mol/L) was added to the culture medium 2 hr before horseradish peroxidase. In a separate series of experiments, 50 mumol/L taurocholic acid or 500 mumol/L ursodeoxycholic acid was added to the culture medium 12 min before horseradish peroxidase. The volume density (percent area) of horseradish peroxidase-containing structures was analyzed in the 1-microns-wide area of basolateral or apical cytoplasm. In vivo, horseradish peroxidase-containing structures maximally increased from the basolateral to the periluminal region over a 20-min interval (percent area increased from 0.09 +/- 0.12 to 2.02 +/- 0.33; p < 0.001) and over a 10-min interval in valproic acid-treated animals (from 0.17 +/- 0.11 to 2.05 +/- 0.36; p < 0.001). In vitro, horseradish peroxidase immediately labeled vesicles in the basolateral cytoplasm. Within 15 min, the vesicles were labeled in the periluminal region (percent area increased from 0.36 +/- 0.08 to 1.90 +/- 0.17; p < 0.001). Colchicine but not beta-lumicolchicine decreased the volume density of labeled structures in the apical cytoplasm (percent area at 15 min, 1.94 +/- 0.24 after beta-lumicolchicine and 1.04 +/- 0.29 after colchicine; p < 0.01). Taurocholic or ursodeoxycholic acid did not change the migration pattern of labeled vesicles, but peroxidase tended to appear earlier in the apical cytoplasm, especially after taurocholic acid. In addition, taurocholic acid increased the percentage of labeled tubules in the apical cytoplasm. These studies show that a polarized tubulovesicular transcytotic pathway exists in rat biliary epithelium and is microtubule dependent. These tubulovesicular structures are labeled with horseradish peroxidase, which is rapidly transported from the cell periphery to the luminal area.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Geographic distribution of calcium pyrophosphate (CPPD) deposition disease in pre-Columbian North America: independent validation of CPPD criteria.

Calcium pyrophosphate deposition disease (CPPD), a form of crystalline arthritis, has a unique distribution in the Early to Middle Archaic Periods (3000 to 8000 years before the present) of North America, contrasting with the generalized geographic distribution of osteoarthritis. Incursion of CPPD into specific regions suggests possible migration patterns. The disparate geographic distribution of CPPD and osteoarthritis provide independent verification of the validity of diagnostic criteria utilized for the identification of CPPD.

Arthritis↗
PubMed1993

Somatic mutations in c-myc intron I cluster in discrete domains that define protein binding sequences.

The activated c-myc allele in Burkitt's lymphoma tumor cells is associated with a clustering of somatic mutations within intron I near the exon I boundary. We have identified several discrete protein binding sites within this region of c-myc intron I designated as myc intron factor-1 (MIF-1), MIF-2, and MIF-3. In addition to our previous characterization of a 20-nucleotide binding site for MIF-1, we now have identified adjacent 20-nucleotide and 34-nucleotide binding sites for MIF-2 and MIF-3, respectively. All three elements are protected from exonuclease digestion by nuclear protein extracts, and each gives rise to a distinct migration pattern on mobility shift assays. In addition, MIF-1, 2, and 3 share a 5-nucleotide (TTATG) internal sequence, which may account for cross-competition of these binding sites in the exonuclease protection experiment. Deletion mutant analyses showed that selective removal of the MIF-3 binding site alone was sufficient to enhance chloramphenicol acetyltransferase reporter activity similar to that observed with larger deletions of myc intron I. We have demonstrated that somatic mutations in activated c-myc alleles are frequently clustered in discrete domains that define protein recognition sequences.

Base Composition↗
PubMed1993

Isolation and characterization of encephalitic bovine herpesvirus type 1 isolates from cattle in North America.

Nine CNS bovine herpesvirus type 1 (BHV-1) isolates, recovered from bovine brain samples submitted to the Texas Veterinary Medical Diagnostic Laboratories from 1974-1989, were compared by analyzing their DNA restriction endonuclease (RE) fragment migration pattern. Seven had pattern similar to that of the respiratory BHV-1 Cooper strain. The remaining 2 isolates, however, had variant patterns, similar to that of each other, but completely different from patients for the other 7. The RE patterns of these 2 variants were similar to published RE patterns for 2 encephalitic or neuropathogenic BHV-1 strains--the Australian N-569 strain and the Argentine A-663 strain. One of the Texas encephalitic variants (No. 30326) was isolated from the CNS of a calf that died during an epizootic of encephalitis in 1974. The other, designated TX-89, was isolated in 1989 from the CNS of a 7-month-old feedlot steer with acute fatal encephalitis. Microscopic lesions of encephalitis with neuronal degeneration and intranuclear inclusions were observed for 3 of the 9 isolates, the 2 variant isolates (No. 30326 and TX-89), and a respiratory isolate. The remaining 6 CNS isolates, all respiratory subtypes, were recovered from cattle that did not have clinical CNS disease or gross or microscopic CNS lesions; in 5 of these cattle, virus was recovered from at least 1 other organ (lungs) besides the CNS. We conclude that the CNS of calves can be naturally infected with 2 distinct BHV-1 subtypes, the respiratory and the encephalitic, and that the encephalitic subtype (subtype 3 or BHV-1.3) has been present in Texas cattle since at least 1974.

Animals↗
PubMed1993

p34cdc2 protein is complexed with the c-mos protein in rat skeletal muscle.

We have used fractionation of subcellular components of the skeletal muscle followed by Western blot analyses to study the localization of the c-mos protein in adult rat muscle. We find that p43c-mos is predominantly located in the KCl supernatant fraction. We show that immunoprecipitates of p43c-mos phosphorylate in vitro two polypeptides of about 34 kDa and 80 kDa respectively. Muscle fractionation and immunodetection studies showed that the p34 protein associated with p43c-mos is the cdc2 protein. p43c-mos is coprecipitated with p34cdc2 when using either anti PSTAIR antibody, antibody directed against the conserved COOH terminal region of the p34cdc2 and by binding to beads that contain cross-linked p13suc1, a protein known to bind p34cdc2. Likewise p34cdc2 coprecipitated with p43c-mos when using anti mos antibody. However p43c-mos is not present in histone H1 kinase active p34cdc2 complex precipitated with anti p34cdc2 COOH-terminal peptide antibody. In adult muscle tissue tubulin is not complexed with p34cdc2 and p43c-mos as previously observed in c-mos and v-mos transformed cells. Gel filtration and crosslinking experiments show that a 170 kDa complex contains c-mos and p34cdc2 proteins. In addition during postnatal development of skeletal muscle we observe modifications in the migration pattern of p34cdc2 correlated with the accumulation of p43c-mos. Our findings raise the possibility of a p43c-mos-p34cdc2 complex could play a role in the differentiation process and maintenance of myotubes in Go.

Animals↗
PubMed1993

Single-strand conformation polymorphism analysis is a rapid and effective method for the identification of mutations and polymorphisms in the gene for glycoprotein IIIa.

Glanzmann thrombasthenia (GT) is the most common inherited disorder of platelets. Most of the molecular defects previously identified in GT have been caused by point (or other small) mutations in the genes for glycoprotein (GP) IIb or GPIIIa. We have used single-strand conformation polymorphism (SSCP) analysis to rapidly identify single-base changes in the GPIIIa gene. Using genomic DNA from normal individuals and patients with GT, each GPIIIa exon and a short stretch of flanking intronic sequence was amplified, heat-denatured, and separated in nondenaturing acrylamide gels. Only those fragments with an abnormal migration pattern were isolated and the nucleotide sequence determined. Using SSCP, we detected the polymorphism in the HPA-1 (P1A) system and all three known silent polymorphisms in the GPIIIa gene. Screening 14 GPIIIa exons from 5 patients with GT, one mutant allele was identified. The nucleotide sequence of the abnormal 240-bp SSCP fragment was determined and a G-->A substitution in the splice donor site of exon iv was identified. Analysis of platelet RNA resulting from this mutation showed two mRNA species: one contained a deletion of exon iv, whereas the other had a 27-bp addition to exon iv due to the use of a cryptic splice site in the downstream intron. Single-base substitutions are the most common mutation in GT and often result in abnormal mRNA splicing. SSCP is a rapid and sensitive technique for identifying mutations or polymorphisms in the GPIIIa gene.

Base Sequence↗
PubMed1993

Effects of 5-fluorouracil substitution on the RNA conformation and in vitro translation of thymidylate synthase messenger RNA.

In vitro transcribed thymidylate synthase (TS) mRNA which is 100% substituted with 5-fluorouracil (FUra) was analyzed for changes in mRNA secondary structure, for alterations in translational efficiency, and for evidence of translational miscoding in vitro. FUra substitution in TS mRNA results in an altered migration pattern in non-denaturing RNA gels and in decreased hyperchromicity in RNA melting temperature studies, consistent with a change in mRNA secondary structure. However, no change in the translational efficiency of FUra-substituted TS mRNA is seen compared to control TS mRNA in either rabbit reticulocyte lysate or wheat germ extract in vitro translation systems. Analysis of the in vitro translation product of FUra-substituted TS mRNA by Western immunoblotting, isoelectric focusing, 5-fluoro-2'-deoxyuridine 5'-monophosphate binding, and TS catalytic activity experiments shows no difference compared to control TS mRNA. We conclude that the in vitro translation products of FUra-substituted and control TS mRNA are identical. Our findings do not support the hypothesis that changes in the mRNA template are responsible for the RNA-directed cytotoxicity of FUra.

Blotting, Western↗
PubMed1995

Prevalence of serotypes G6 and G10 group A rotaviruses in dairy calves in Quebec.

Fecal samples from diarrheic and nondiarrheic dairy calves (1 to 3 weeks old) from 12 regions of Quebec, collected between 1992 and 1994, were screened for group A bovine rotavirus (BRV) using a combination of 2 VP6-specific monoclonal antibodies (MAbs) in an enzyme-linked immunosorbent assay (ELISA). The overall prevalence of BRV infection was 26.4% (107/405). In diarrheic calves, BRV infection reached 74.3% (55/74), but only 15.7% (52/331) in nondiarrheic calves. BRV-positive samples were serotyped by enzyme-linked immunosorbent assay (ELISA) using G6 and G10 specific MAbs. The analysis of 107 field samples revealed that, in diarrheic calves, 34.5% (19/55) were G6, 27.2% (15/55) were G10, 9% (5/55) were G6 and G10 positive, and 29.9% (16/55) were G6 and G10 negative. In nondiarrheic calves, 19.2% (10/52) were G6, 19.2% (10/52) were G10, 7.6% (4/52) were G6 and G10 positive, and 53.6% (28/52) were G6 and G10 negative. Rotavirus dsRNA was extracted from BRV-positive samples and examined by polyacrilamide gel electrophoresis (PAGE). Of 107 samples tested, 74 (69.1%) were positive, and all the samples demonstrated a typical group A rotavirus migration pattern.

Animals↗
PubMed1995

Persistence of a single electropherotype and serotype (G6P5) of bovine rotavirus in calves on a closed dairy farm from 1990 to 1993.

A virologic survey was conducted on calves with diarrhea associated with bovine rotavirus (BRV) on a closed dairy farm. The BRV was detected from 32 of 219 (14.6%) fecal specimens repeatedly collected from 56 calves born during the years 1992-1993, regardless of whether they had diarrhea. Most of the 32 strains were isolated from fecal specimens obtained from 2- to 6-week-old calves. After electrophoresis of double-stranded viral RNA from the 32 strains, genomic RNA migration patterns were similar to those of the predominant BRV strains isolated at the same farm during the years 1990-1991. All representative strains were identified as G serotype 6 (G6) and P type 5 (P5) by results of the virus-neutralization test and polymerase chain reaction procedure. Thus, BRV had no change in genomic RNA electropherotypes and serologic antigenicities in a closed dairy herd over a period of several years.

Animals↗
PubMed1996

Preservation of functional and regulatory domains of expressed bcl-2 genes in non-Hodgkin's lymphoma.

The most common translocation in human lymphoma, t(14;18)(q32;q21), recombines the bcl-2 gene with the immunoglobulin (Ig) heavy-chain locus leading to the production of high levels of chimeric RNAs and the resulting 26 kDa bcl-2 protein. The oncogenic role of the bcl-2 gene has been shown by the suppression of a variety of programmed cell deaths (apoptosis). Bcl-2 is able to interact with other members of the bcl-2 family through at least one of its conserved dimerization domains. Although overproduction of the wild-type protein appears sufficient for conferring a selective growth or a survival advantage to hematopoietic cells, the mode of activation of the proto-oncogene remains to be elucidated. In a first step, we examined and quantitated the expression of the bcl-2 gene in primary biopsies of non-Hodgkin's lymphomas (NHL) as well as in cell lines derived from NHLs. The results show that bcl-2 expression is found in a variety of hematopoietic lineages, but is most strongly associated with the B cell lineage. Within the B cell lineage, the expression levels vary depending on the differentiation as well as on the t(14;18) rearranged status. The quantitative measurements show high steady-state mRNA levels in early and in t(14;18) arranged B cells, whereas bcl-2 expression decreases with further B cell maturation and differentiation. In a second step we analyzed the bcl-2 mRNA for secondary genetic alterations, which may alter regulatory regions rendering it more tumorigenic. For this purpose, we chose a combined RT-PCR/SSCP method in order to screen out mutations of alleles which are not expressed. Different migration patterns of SSCP products were found only in two cell lines and subsequent sequencing revealed that the functional domains are not affected. Our data suggest that the dimerization properties of this protein are preserved in tumor cells and that modifications of the bcl-2 gene by the somatic hypermutation mechanism are not involved and do not influence the pathobiology of NHL.

Base Sequence↗
PubMed1995

Electropherotypes of rotavirus strains causing gastroenteritis in infants and young children in Tirana, Albania, from 1988 to 1991.

During 1988-1991, an epidemiological survey was conducted in Tirana (Albania) on group A rotavirus strains which cause gastroenteritis in infants and young children. Rotaviruses were detected in 312 of 1,241 (25.1%) examined specimens from children with acute diarrhoea. Viruses were detected throughout the study period. Among the 72 rotavirus strains tested for double-stranded RNA (dsRNA) electrophoretic migration pattern, 9 different electropherotypes were recognized, 1 of those being more frequent than the others. At the beginning and at the end of the examined period (1988 and 1990-1991) two different long electropherotypes were predominant, whereas in 1989 (middle period) short electropherotypes were common indicating an involvement of virus strains with short electropherotypes in hospitalization-requiring diarrhoeas occurring in the area surveyed in that year.

Age Factors↗
PubMedDate not supplied

Immunomodulation by thalidomide: systematic review of the literature and of unpublished observations.

Three decades of immunological investigations using thalidomide are reviewed. Both in vitro and in vivo investigations are in accordance with the clinical finding that thalidomide does not impede T-cell competence in the control of infection by mycobacteriae. The term immunosuppressant does not apply. The immunomodulatory effects of thalidomide are evident in a myriad of phenomenological changes, and a molecularly defined common denominator of these activities is not known at present. Critical assessment with the objective to account for the clinical activity of thalidomide in specific human diseases leads to a focus on effects of thalidomide on phagocytic leukocytes and endothelia. The former are responsive to thalidomide by modulation of cytokine synthesis in vitro and in vivo; this activity can be shown using monocyte-specific stimuli in peripheral blood mononuclear cells but also in other phagocytic cells like microglia. For technical reasons, endothelial cells have until now been tested primarily in vitro. However, there is solid evidence now from intravital microscopy that the induction of adhesivity in postcapillary venules by LPS is modulated by thalidomide. Altered surface antigen expression has been described on leukocytes obtained from humans and experimental animals treated with thalidomide, but convincing evidence is lacking for in vitro modulation of surface antigen expression on leukocytes (as opposed to the modulation of adhesion antigens on endothelial cells stimulated by LPS or exogenous TNF alpha in the presence of thalidomide). Therefore, in vivo redistribution is likely to account for some, if not all, changes in circulating leukocyte phenotypes. The immunopathological conditions most clearly responsive to thalidomide are vasculitic alterations of post-capillary venules either in the context of mycobacterial infection (in the case of erythema nodosum leprosum) or mucocutaneous aphths. In both instances (as in the majority of focal inflammatory lesions), leukocyte infiltration and cytokine responses, in particular TNF alpha, are present. Thalidomide acts clinically not only by palliation of existing lesions but also by prevention of recurrence. The mechanism operates in skin, mucosa and parts of the nervous system and is most readily explained by synergism of TNF alpha modulation and a separate point of action on leukocyte migration patterns.

Adjuvants, Immunologic↗
PubMedDate not supplied

Hispanic Americans in the United States: Young, dynamic and diverse.

Hispanic Americans are a rapidly growing population group, with numbers that increased 53 percent between 1980 and 1990. In 1994 Hispanics numbered 26.6 million, accounting for 10.3 percent of the civilian noninstitutional U.S. population. The Census Bureau projects continued rapid growth but at a decreasing rate than in the past. The population totals may exceed 31 million by the year 2000 and 96 million by 2050. According to the Census Bureau, Hispanic Americans may surpass African Americans as the largest U.S. minority group by the year 2005. While Hispanics present a varied social and economic portrait, they are likely to be young and economically active. As a group, they tend to trail the rest of the U.S. population in terms of educational attainment, occupational status and income. Hispanics are much more likely to be living below the poverty level and less likely to have health insurance than other U.S. populations. The origins and migration patterns vary both between and among the various groups. Each group has tended to settle in a few states with close to 90 percent of all Hispanics living in just nine states. Low educational attainment, combined at times with a lack of English language and other job skills, tends to confine Hispanics to the lower-paying, less stable and more hazardous occupations. Low income and larger family size translate into higher poverty rates, particularly among children. That, in turn, may affect children's ability to realize their full educational potential. Still, Hispanics, particularly men, have high labor force participation rates and will become an expanding part of the future labor force. As such, they will be expected to contribute to the retirement benefits of an ever aging U.S. population.

Adolescent↗
PubMed1996

Characterization of rotaviral RNA isolated from children with gastroenteritis in Poland.

Rotaviruses were detected in 37% (331/895) of stool specimens collected from children with gastroenteritis attending three hospitals in Warsaw between January 1981 and March 1994. An analysis of rotavirus electrophoretypes circulating in Warsaw and Cracow, Poland, over 6 years showed that all 176 electrophoretypes encountered were characteristic of group A rotaviruses. Based on variations in the migration pattern of RNA genome segments when passed through polyacrylamide gels, 10 different electrophoretypes were identified: 86% were long patterns and 14% were short patterns. Mixed infections were observed in two cases.

Adolescent↗
PubMed1996

Inter-relationships among subgroups, serotypes, and electropherotypes of rotaviruses isolated from humans.

In an epidemiological study of human rotavirus (HRV) infections in metro Jeddah, Saudi Arabia, the relationships among subgroups, serotypes, and RNA electropherotypes of the rotavirus isolates were investigated. Of the 523 rotavirus-positive stool specimens, 245 were examined for subgroup, serotype, and electropherotype. Of these, 84 isolates were analyzed for their subgroup and RNA electropherotype specificites, 12 (14.3%) were of subgroup 1, 69 (82.1%) were of subgroup II, and 3 (3.6%) were a mixture of subgroup I and II. Of the subgroup 1 specimens, 5 (41.7%) showed long electrophoretic migration patterns and 7 (58.3%) showed short patterns. In subgroup II specimens, 66 (95.7%) were of long patterns and 3 (4.3%) of short patterns. The relationship between HRVS serotypes and electropherotypes was also determined for the same 245 rotavirus specimens. Of these, 36 (14.7%) exhibited short RNA patterns and 209 (85.3%) exhibited long patterns. The short pattern specimens consisted of serotype 1 (8.3%), serotype 2 (63.9%), serotype 3 and serotype 4 (2.8%) each. The long pattern specimens consisted of serotype 1 (60.3%), serotype 2 (1.4%), serotype 3 (7.2%) and serotype 4 (17.7%). Among the previous 245 specimens, subgroup specificities were available for 51 specimens. All subgroup I were of serotype 2, and all subgroup II were of serotype 1, 3 or 4. RNAs of either subgroup showed both long and short electropherotypes. No relationship could be established between subgroups or serotypes and a particular electropherotype. It seems unlikely that electropherotyping of human rotavirus (HRV) can be used for identifying the subgroups or serotypes of strains.

Child, Preschool↗