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Biomedical subjects

K Harper

Publications and source records attributed to K Harper.

At least 55 records · Page 3Linked to original sources
PubMed1988

Characterization of a functional recombinant rat liver aldehyde dehydrogenase: expression as a non-fusion protein in E. coli.

A cDNA encoding a rat liver inducible aldehyde dehydrogenase carried in a pUC8 plasmid is expressed in E. coli as a dimeric enzyme molecule functionally and physically identical to the authentic rat enzyme. The cDNA appears to be transcribed using the lac promoter, but is translated from an initiator codon 174 base pairs from the 5' end of the cDNA. The aldehyde dehydrogenase polypeptide is not produced as a fusion protein. This is the first example of the production by E. coli of a catalytically active, multimeric eukaryotic protein which is not a fusion protein.

Aldehyde Dehydrogenase↗
PubMed1988

Proximity of the CTLA-1 serine esterase and Tcr alpha loci in mouse and man.

The serine esterase CTLA-1 gene was shown by in situ hybridization to map to the D segment of mouse chromosome 14, the same localization as a member of the immunoglobulin superfamily, Tcr alpha. To further demonstrate the proximity of CTLA-1 and Tcr alpha, genetic linkage was tested in mouse using restriction fragment length polymorphisms and a backcross progeny, and no recombination was observed in the 100 backcross products studied. Recombination events between Tcr alpha/CTLA-1 and the markers Gdh-X and NP-1 show that the most probable order of these loci in the mouse 14D region is NP-1-Tcr alpha/Ctla-1-Gdh-X. In man, the human homologue of CTLA-1 was shown by in situ hybridization to map on chromosome 14, at 14q11-q12, where Tcr alpha also maps. Using the human cell line SUP-T1, bearing the inversion inv(14) (q11;q32), we further demonstrated the loci order in man to be centromere-NP-1-Tcr alpha-CTLA-1. To complement the cytogenetic and genetic mapping data, we tried to determine the physical distance between the two genes by pulsed field gel electrophoresis (PFGE). DNA prepared from various cell types, both mouse and human, were digested with a panel of rare cutter enzymes and hybridized first with CTLA-1, then with Tcr alpha probes. None of the bands identified hybridized with both Tcr alpha and CTLA-1 probes for either mouse or human cells. Although the physical mapping by PFGE is inconclusive, the cytogenetic and genetic data support close linkage of the Tcr alpha and CTLA-1 genes in both mouse and man, suggesting homology between the D region of mouse chromosome 14 and the q11-q12 region of human chromosome 14, encompassing the Tcr alpha and CTLA-1 loci. These findings also provide another example of proximity of genes coding for a member of the Ig super-family and a serine esterase.

Animals↗
PubMed1986

Duchenne muscular dystrophy in one of monozygotic twin girls.

Monozygotic twin girls are reported, one of whom has the typical clinical features of Duchenne muscular dystrophy despite a normal female karyotype. Although certain features of the biopsy were atypical, the clinical diagnosis was supported by persistent markedly raised blood creatine kinase levels and findings typical of DMD on electromyography and magnetic resonance spectroscopy. Analysis of an X linked DNA polymorphism in 16 independent somatic cell hybrids made between cells derived from each girl and a mouse line suggest that in one twin only the maternal X chromosome is active, whereas in the other the active X was paternally derived. More data are needed to exclude sampling error. These preliminary experimental results support the hypothesis that both girls are heterozygous for Duchenne muscular dystrophy. X inactivation, by chance, resulted in two contrasting cell masses with different active X chromosomes. This segregation was followed by, and may even have resulted in, twinning into a female pair, one normal and one with the full clinical features of the disease.

Biopsy↗
PubMed1986

Monoclonal antibodies specific for different regions of human apolipoprotein A-I. Characterization of an antibody that does not bind to a genetic variant of apoA-I (Glu----136 Lys).

Three monoclonal mouse hybridoma antibodies, designated 2AI, 4AI, and 5AI, specific for human plasma apolipoprotein A-I (apoA-I) were characterized. In an enzyme-linked immunosorbent assay (ELISA) each of the antibodies reacted with purified apoA-I and with A-I in normal human serum. Immunoblotting of apoA-I subjected to isoelectric focusing revealed that the three antibodies reacted with all the charge isomorphs of apoA-I and with proapoA-I. Using a solid phase competitive displacement assay, the antigenic determinant for antibody 5AI could be localized to cyanogen bromide fragment 3 of apoA-I (residues 113-148), while the epitope for antibody 4AI resided in cyanogen bromide fragment 4. Dot blot experiments and data obtained by the competitive displacement assay revealed that antibody 2AI reacts with high affinity with CNBr fragment 2 but that it also reacts with lower affinity with fragments 1 and 4. The antibody 5AI did not bind to a genetic variant of apoA-I (Glu----136 Lys), demonstrating that the substitution of a single amino acid in human apoA-I can cause the loss of an antigenic determinant.

Animals↗
PubMed1986

Prevalence of positive epicutaneous tests among infants, children, and adolescents.

We studied 314 otherwise healthy children younger than 18 years of age with epicutaneous testing, and found that 20% of those tested had at least one positive epicutaneous test result. Neomycin, nickel, and potassium dichromate were the most prevalent allergens detected in our study group. We found that sensitization occurred at less than 5 years of age for all but fragrance allergens. Aluminum chambers and the American Academy of Dermatology Patch Test Kit were found to be safe for use in infants, children, and adolescents.

Adolescent↗
PubMed1985

First trimester prenatal diagnosis and detection of carriers of haemophilia A using the linked DNA probe DX13.

Although the use of a gene specific deoxyribonucleic acid (DNA) probe is the method of choice for detecting carriers of genes for rare genetic disorders, there will always be families in which such probes cannot be used because key subjects are not informative for restriction fragment length polymorphisms in or around the gene. In these cases closely linked DNA markers have to be used. An X chromosome specific DNA probe, DX13, which is closely linked to the haemophilia A locus on the X chromosome, was used for early prenatal diagnosis in two cases and to detect carriers in a series of nine possible heterozygote women. The first reported crossover between DX13 and the factor VIII:C locus was observed in this study. There are complexities inherent in using any linked DNA probe for assignment of genes, but such techniques are clinically important.

Female↗
PubMed1985

Linkage studies of X-linked mental retardation: high frequency of recombination in the telomeric region of the human X chromosome (fragile site/linkage/recombination/X chromosome).

One of the commonest forms of X-linked mental retardation is associated with a fragile site at Xq27 on the human X chromosome which can be visualised structurally after culturing cells in folate-deficient media. Unusually, the mutation can be transmitted through a phenotypically normal male. There is already some evidence that the gene loci for G6PD and factor IX are linked to this mental retardation locus. We have followed the inheritance of a DNA sequence 52A, in fragile site families that are also informative for factor IX. We demonstrate that these probes are localised at Xq27/Xq28-Xqter, close physically to the fragile site. We did not find close linkage between 52A, factor IX, and the fragile site in the families studied despite 52A and factor IX showing linkage in normal families. We discuss the importance of these data for the genetic mapping of this region of the human X chromosome and the implication for the use of these DNA probes for clinical diagnosis.

Animals↗
PubMed1984

A clinically useful DNA probe closely linked to haemophilia A.

Genetic linkage studies were done in families segregating for haemophilia A with an X-chromosome-specific probe, DX13, which has been localised to band Xq28. When DNA is digested with the restriction enzyme Bg1 II, this probe recognises a restriction fragment length polymorphism, for which 50% of females are heterozygous. No crossovers were seen between the haemophilia A locus and the DX13 locus (lod score = 5.4 at recombination fraction [theta] 0.0; 95% confidence limits of theta, 0-12%). This indicates that the DX13 probe is closely linked to the haemophilia A locus and is likely to prove useful in carrier detection and prenatal diagnosis.

Alleles↗
PubMed1984

Use of a chromosome 21 cloned DNA probe for the analysis of non-disjunction in Down syndrome.

A recombinant clone was isolated containing a sequence which occurs only on human chromosome 21 and defines a two-allele restriction fragment length polymorphism showing Mendelian inheritance. Forty seven percent of the London population are heterozygous for the polymorphism. The chromosomal location of the DNA sequence homologous to the probe was confirmed using rodent-human somatic cell hybrids. DNA from persons with Down syndrome and from their parents was analysed. It was possible to confirm trisomy 21 by dosage hybridisation to Southern blots, and to determine the origin of the supernumerary chromosome. The technique will be of use for determination of the paternal or maternal origin of nondisjunction in cases of Down syndrome which are not informative using existing markers.

Adult↗
PubMed1984

Pathogenic free-living amebae. Immunocytologic demonstration and species identification.

This report describes methods for preparation, immunologic marking, and staining of amebae in formalin-fixed brain tissue, and in exudates, discharges and body fluids. The suggested procedures provide slide preparations of unknown specimens together with known amebae from cultures as controls, all on a single slide for each antiserum. This allows for cytologic studies of host cells, morphology of the amebae present, and specific immune marking of the amebae by use of immune sera and protein A Staphylococcus. Such preparations may also be used for immunoperoxidase and immunofluorescence. The need for the utilization of direct microscopic examination by the methods suggested here, or comparable ones, is discussed.

Agglutination Tests↗
PubMed1983

Identification of an autosomal locus affecting steroid sulfatase activity among inbred strains of mice.

We have found an activity variant for testicular and liver steroid sulfatase among inbred strains of mice that is not X-linked. C57BL/6J, SM/J and SWR/J testicular extracts hydrolyze 3H-dehydroepiandrosterone sulfate twice as rapidly as do A/J extracts. The C3H/HeJ and DBA/2J strains were intermediate. The Km values for C57BL/6J and A/J are 2.29 +/- 0.10 and 1.01 +/- 0.02 microM, respectively. The F1 values in both directions were intermediate, which argues against X-linkage of this trait. F2 values show scattered high-intermediate-low values compatible with assay variation superimposed on the segregation of codominant alleles. When assayed for both testicular and liver steroid sulfatase, nine recombinant inbred lines between A/J and C57BL/6J segregate to near the parental strain values. Thus, this activity variation for steroid sulfatase appears to be determined by a single gene, which is not X-linked. Sex and steroidal hormone differences in liver steroid sulfatase activity were not present in the A/J strain, but females of the C57BL/6J and some recombinant inbred lines had higher levels. Electrophoretic studies only disclosed a variant in the SM/J strain, which seems to be secondary to the well-known neuraminidase variation in SM/J.

Animals↗
PubMed1981

Genetic aspects of the effects of methylmercury in mice: the incidence of cleft palate and concentrations of adenosine 3':5' cyclic monophosphate in tongue and palatal shelf.

Concentrations of adenosine 3':5' cyclic monophosphate (cAMP) were measured in the tongues and palates of 14.5-day-old fetuses from control and methylmercury-treated mothers of four inbred lines of mice which represent the four possible combinations of two H-2 alleles and two residual genetic backgrounds. The incidence of cleft palate in fetuses from control and methylmercury-treated mothers was also examined. The H-2 alleles significantly affected the degree of reduction of cAMP concentration in palates seen in fetuses from mothers treated with methylmercury. Neither the H-2 allele nor the residual genetic background played a role in the effect of methylmercury on cAMP concentrations in fetal tongues. The magnitude of increase in the incidence of cleft palate with methylmercury treatment was approximately the same for all lines. Thus, methylmercury-induced cleft palate may not be mediated by the reduction of cAMP. Finally, fetuses with cleft lip had increased palatal cAMP levels, whether or not they were from control or methylmercury treated mothers.

Animals↗
PubMed1980

Surface coagglutination with formalinized, stained protein A staphylococci in the immunologic study of three pathogenic amebae.

The indirect immunofluorescence and, to a lesser extent, the immunoperoxidase methods have become the principal means of conducting immunological studies upon amebae. These procedures require the conjugation of fluorescein or enzyme with antiglobulin reagents and fixation of the amebae. The present report describes a somewhat similar methodology wherein formalinized, stained protein A staphylococci combined with specific antibody have been found to coagglutinate upon a portion of the amebic surface of living trophozoites in a fashion similar to direct immunofluorescence. The prepared staphylococci without attached antibody have also been found to coagglutinate in an identical fashion after the specific antibody has been deposited upon the amebic surface. This is comparable to indirect immunofluorescence reaction. A comparison of this procedure with the immunofluorescence method of identification and with measurement of titer of serum antibody is presented, which indicates that the new technique compares favorably with the immunofluorescence results.

Agglutination Tests↗
PubMed1979

Immunological studies of the sperm-specific phosphoglycerate kinase-2 of mice.

Antiserum was prepared to 500-fold purified PGK-2A from mouse testes. Depsite the apparent high purity of the immunizing antigen, double diffusion analyses showed two precipitin lines, one of which can be shown to correspond to PGK-2. An immunoabsorbent column of liver homogenate coupled to cyanogen bromid-activated Sepharose was used to remove the contaminating antibody. Both immuno-inactivation and double diffusion analyses demonstrated that the PGK-2 antigen appeared in testes between days 30 and 34 of postnatal development. The antisera prepared against highly purified PGK-2A did not disclose any immunological differences between PGK-2A, PGK-2B, and PGK-2C either by double diffusion analyses or quantitative immunoprecipitation. Antisera to PGK-2 did not inhibit in vitro fertilization of zona-free hamsters ova by mouse spermatozoa.

Animals↗
PubMed1978

Comparative evaluation of a modified zinc sulfate flotation technique.

A modified zinc sulfate flotation technique using Formalinized fecal specimens (F-ZnSO4) was compared to the Formalin-ether (FE) concentration method for the laboratory diagnosis of intestinal parasites. Many laboratories have difficulty storing, using, and disposing of either and need a procedure for concentrating fecal specimens which does not require ether. Comparative data were obtained for the recovery of protozoan cysts and helminth eggs and larvae from feces preserved in Formalin less than and longer than 1 month. Whereas the FE method was found generally to be more efficient, F-ZnSO4 was apparently more effective for the recovery of some species of parasites. F-ZnSO4 was not satisfactory for recovery of schistosome eggs. We conclude that, except for schistosomes, F-ZnSO4 compares favorably to the FE method for detecting infections of clinical significance.

Eukaryota↗