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Biomedical subjects

A D Malcolm

Publications and source records attributed to A D Malcolm.

At least 37 records · Page 2Linked to original sources
PubMed1990

In situ and slot hybridization analysis of RNA in colorectal tumours and normal colon shows distinct distributions of mitochondrial sequences.

cDNA clones of mRNAs with an abnormal abundance in familial adenomatous polyposis were used to examine the levels and distribution of the mRNAs in tissues from 15 patients with colorectal cancer. Of 12 cloned sequences studied by slot hybridization, one was substantially reduced in tumours compared with normal tissue. Sequence analysis showed this to code for IgA. In situ hybridization was consistent with slot hybridization and immunohistochemistry. Two mitochondrially encoded sequences had distinct distributions detected by in situ hybridization but did not have detectable quantitative differences in whole tumour or mucosa extracts.

Adenocarcinoma↗
PubMed1990

Regional distribution of cholecystokinin messenger RNA in rat brain during development: quantitation and correlation with cholecystokinin immunoreactivity.

The regional distribution of CCK-octapeptide (CCK) and CCKmRNA were studied in parallel in rat brain during development in order to characterize changes in gene expression in different structures at different periods of development and to determine whether differential regulation of translation and peptide turnover occurs, by correlating peptide and mRNA levels. CCK was characterized by Northern blot analysis. Methods were developed for quantitation of CCKmRNA by in situ hybridization (ISH) and compared with results obtained by slot blot analysis. Analysis of CCKmRNA by ISH showed that CCKmRNA was abundant in cerebral cortex, hippocampus, dentate gyrus, thalamus, inferior colliculus and periaqueductal grey in keeping with the high concentrations of CCK-like immunoreactivity in cell bodies located in these regions. Quantitation of CCKmRNA by computerized densitometric image analysis by reference to [35S]brain paste standards gave a similar pattern to that obtained by slot blot analysis of regional RNA extracts. Distinctive patterns of CCK development were seen. In cerebral cortex and hippocampus, a marked increase in peptide and mRNA levels was found from day 1 which showed a steady increase up to adult levels. However, in the thalamus, the highest levels of mRNA and peptide were reached at a much earlier stage, generally by 14d. In contrast, inferior and superior colliculus showed a delayed onset of peptide expression.

Animals↗
PubMed1990

Comparison between the polymerase chain reaction and slot blot hybridization for the detection of HPV sequences in cervical scrapes.

Sixty-four women presenting with a single mildly abnormal smear were investigated for infection with human papillomavirus (HPV) type 16 using both slot blot hybridization and polymerase chain reaction (PCR) amplification. PCR was nearly three times more sensitive for the detection of HPV 16 DNA than slot blot hybridization. HPV 16 was not significantly associated with a risk of progression to CIN if PCR was used to screen for infection. Women who smoked were at significantly greater risk of progression to CIN than non-smokers.

Adult↗
PubMed1990

A prospective study of human papillomavirus infection of the cervix.

In a prospective study 42 women, diagnosed as having low grade cervical intraepithelial neoplasia (CIN), made a total of 281 clinic visits over a 45 month period. At each visit, they were subjected to cytological and colposcopical examination and samples were taken for human papillomavirus (HPV) DNA hybridization studies and for the detection of non-HPV infections. HPV types 16 and/or 18 were found in 25% of all the samples tested and these virus types were detected in five of six (83%) women whose lesions progressed compared to seven of 14 (50%) of those whose lesions regressed. The presence of HPV DNA was not a good prognostic indicator of progression since half of those whose disease regressed also harboured these viruses at some time. The recording of non-HPV infections almost 10 times more often in the women whose disease regressed than in those whose disease progressed could probably be accounted for by the former having a larger number of follow-up visits. Nevertheless, the significance of non-HPV infections also remains unclear.

Bacterial Infections↗
PubMed1990

Immunoglobulin V genes expression and mRNA sequencing in rheumatoid arthritis.

Antibodies to exogenous antigen and to self antigen--that is, autoantibodies, are both encoded by the combining of V, D, and J genetic elements in the heavy chain, and V and J in the light chain immunoglobulins. The mechanism which generates autoantibodies does not seem to differ from that which generates the immune response to foreign antigen, and analysis of DNA from normal and autoimmune strains of mice appears similar with no appreciable defects in the immunoglobulin germline genes. Although there is restricted use of particular gene families in murine antibodies to exogenous antigen, the preferential use of particular V genes in murine autoantibodies remains controversial. Until recently, because of the obvious limitations on human experimentation, there was little information about the genetics of the human autoimmune response. Recent developments in molecular cloning techniques, however, some of which are discussed here, have shown that the germline arrangement of the human immunoglobulin variable genes differs from that found in the mouse. Furthermore, there is increasing evidence that human autoantibodies make restricted use of the V gene repertoire, and this indicates that the human autoimmune response is less polymorphic than the murine autoimmune response.

Animals↗
PubMed1989

Identification of a human ribosomal protein mRNA with increased expression in colorectal tumours.

A human ribosomal protein cDNA was selected from a normal colon cDNA library on the basis of overexpression in familial adenomatous polyposis. Nucleotide sequence analysis was used to identify this cDNA as corresponding to the human equivalent of the rat ribosomal protein L31 (HL31). We have quantified the expression of HL31 mRNA in colorectal tumours and found overexpression in 23 out of 23 cases. Our results indicate that HL31 is associated with a malfunction of normal growth regulatory mechanisms in these tumours, and suggest a role for HL31 in proliferation and neoplasia.

Adenocarcinoma↗
PubMed1989

Nucleic acid analysis by sandwich hybridization.

One of the most significant achievements of the biochemist during the past two decades is the use to which immunologically based assays have been put in clinical diagnosis (Hood et al.: Immunology, 1984). The problem faced and surmounted by immunologists in effecting the transition from research tool to routine clinical assay bears a remarkable similarity to that confronting the molecular biologist today; i.e., how can nucleic acid hybridization, a technique of obvious potential (Meinkoth and Wahl: Anal Biochem 138:267-284, 1984; Syvanen: Med Biol 64:313-324, 1986; Matthews and Kricka: Anal Biochem 169:1-25, 1988), be modified in order to fulfill all necessary parameters of a routine diagnostic assay? There are several such requirements, and the importance placed on each depends on the objectives of the assay: the technique must be sensitive, specific, and reproducible. Other advantages would be cost-effectiveness, ease of manipulation, and amenability to automation. Ideally, the signal detection should be based on a non-radioactive system, because of the instability of probes labelled with isotopes like 32p, and the potential hazards involved in their handling and disposal. The sandwich hybridization for the analysis of nucleic acid sequences was first used in 1977 (Dunn and Hassell: Cell 12:23-36, 1977), but its potential as a diagnostic assay was not realized until 1983, when it was applied to the detection of adenovirus DNA in nasopharyngeal aspirates from children with acute respiratory infection (Ranki et al: Gene 21:77-85, 1983). It has since been modified and used not only for the detection of microbial infection (Virtanen et al.: Lancet i:381-383, 1983; Ranki et al.: Cur Top Microbiol Immunol 104:307-318, 1983; Lehtomaki et al.: J Clin Microbiol 24:108-111, 1986; Virtanen et al.: J Clin Microbiol 20:1083-1088, 1984; Palva and Ranki: Clin Lab Med 5:475-490, 1985; Polsky-Cynkin et al.: Clin Chem 31:1438-1443, 1985; Parkkinen et al.: J Med Virol 20:279-288, 1986; Palva: FEMS Microbiol Lett 28:85-91, 1985; Palva et al: FEMS Microbiol Lett 23:83-89, 1984; Zolg et al.: Mol Biochem Parasitol 22:145-151, 1987; Palva: J Clin Microbiol 18:92-100, 1983), but also for the analysis of nucleotide sequence variations (Langdale and Malcolm: Gene 36:201-210, 1985). We will discuss the development of the sandwich technique and the advantages it conveys over the more conventional nucleic acid hybridization formats, together with new developments which will ensure that it earns a place alongside immunoassay in the diagnostic laboratory.

Animals↗
PubMed1988

DNA hybridisation of cervical scrapes: comparison with cytological findings in Papanicolaou smears.

One hundred and sixty four cervical scrapes were taken over 26 months from 143 women aged 17 to 53 years, using an Ayre spatula. A smear was prepared from each sample for Papanicolaou staining and for cytological examination for evidence of human papillomavirus (HPV) infection. The cells remaining on the spatula were harvested for DNA analysis. These samples were probed for HPV 6, 16, and 18 and the results of DNA hybridisation and cytology were compared. Cytological changes of HPV (anucleate keratinised and keratinised squamous cells, koilocytic change and dyskeratosis, or multinucleation, or both) were detected in 103 (72%) of the samples where HPV DNA was detected; koilocytic changes were present in only 67 (48%). It is concluded that screening for a range of viral changes is a more sensitive method of detecting HPV infection than screening for koilocytic change alone.

Adolescent↗
PubMed1988

Sexual transmission of human papillomaviruses in heterosexual and male homosexual couples, studied by DNA hybridisation.

The prevalence of human papillomavirus (HPV) 6, 11, 16, and 18 in 36 heterosexual couples and seven male homosexual couples with genital warts was investigated for evidence of sexual transmission of genital HPV. The prevalence of virus type and number of copies of viral genome equivalents/cell in the lesions were assessed, and the factors influencing transmission analysed. Our results show that HPV 6 and, to a lesser extent, HPV 11 were the types most readily transmitted, and that transmission appears to depend on the copy number and the duration and frequency of exposure.

DNA, Viral↗
PubMed1987

Effects of ppGpp on transcription by DNA-dependent RNA polymerase from Escherichia coli: circular dichroism, absorption and specific transcription studies.

Concrete evidence is presented for conformational changes elicited in RNA polymerase upon binding ppGpp by circular dichroism measurements. In the presence of 100 microM ppGpp, the molar ellipticity of RNA polymerase at 220 nm is reduced by 14% from the initial value of - 11,100 deg X cm2 X dmol-1 at 25 degrees C. In vitro transcription on templates containing the beta-lactamase promoter and colicin E1 promoter on poly[d(A-T)] is inhibited by ppGpp. None of these templates had GC-rich nucleotide sequence near the transcription initiation site, and yet they were influenced by ppGpp. Comparison of the effect on the synthesis of mRNAs for beta-lactamase and colicin E1 and the synthesis of the proteins themselves indicates that the effect of ppGpp is at the level of transcription for the former case and involves coupled transcription-translation for the latter case. Difference absorption, polyacrylamide gel electrophoresis, and nitrocellulose filter-binding studies show that the binding of ppGpp to RNA polymerase does not impair the extent of the interaction between enzyme and DNA. Kinetic studies suggest that ppGpp affects transcription initiation on beta-lactamase promoter. On poly[d(A-T)], ppGpp affects the rate of open complex formation and is a mixed inhibitor with respect to the incorporation of nucleotides. Our results are consistent with the idea that ppGpp acts as a regulator by binding at a site different from the active site and changes the RNA polymerase conformation, causing altered transcriptional behavior on different DNA templates.

Circular Dichroism↗
PubMed1987

Prevalence of HPV DNA and viral copy numbers in cervical scrapes from women with normal and abnormal cervices.

Cervical scrapes were obtained from 215 women with cytologically normal cervices and 74 women with cervical intraepithelial neoplasia (CIN) and probed for HPV 6, 11, 16, and 18 by dot-blot hybridization. Viral copy numbers were determined by densitometric scanning of autoradiographs. The prevalence of HPV DNA in women with normal smears was as follows: 23 per cent of women attending a Family Planning Clinic (FPC), 16 per cent of women attending a Sexually Transmitted Diseases (STD) clinic, and 48 per cent of women attending a laser follow-up clinic after treatment of CIN. Ten per cent of women with normal cervices harboured HPV 16. Viral copy numbers ranged from 1300 to 52,800 genome equivalents per cell. Twenty-seven HPV-positive women with normal smears (including four patients infected with HPV 16) were followed for up to 3 years. None developed CIN irrespective of copy number. Our results show that HPV is present in normal cervices in the absence of CIN. Copy number has no relation to the development of CIN and factors other than the amount of HPV DNA may trigger the neoplastic process.

Adolescent↗