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Biomedical subjects

P J Hopkins

Publications and source records attributed to P J Hopkins.

13 recordsLinked to original sources

Characterization of the gene encoding carbonic anhydrase I from the pigtail macaque.

The structure of the gene encoding carbonic anhydrase I (CA I) was determined for the pigtail macaque Macaca nemestrina. When the deduced amino-acid sequence was compared with those of five other primates, four non-primate mammals and a turtle, seven residues were found to be unique and invariant to all of the CA I sequences. A scheme is presented for the probable evolutionary order of the six polymorphic nucleotide changes found in the coding regions of the CA I locus of pigtail macaques.

Amino Acid Sequence

Mutations in the promoter region of the gene for gp91-phox in X-linked chronic granulomatous disease with decreased expression of cytochrome b558.

We examined the molecular defect in two kindreds with "variant" X-linked chronic granulomatous disease (CGD). Western blots of neutrophil extracts showed decreased immunoreactive cytochrome b558 components gp91-phox and p22-phox. Analysis of mRNA demonstrated reduced gp91-phox transcripts, with relative preservation of an alternative mRNA species created by transcription initiation in the third exon of the gene. Single strand conformation polymorphism analysis of the 5' flanking region of the patients' gp91-phox genes revealed an electrophoretic abnormality not detected in 40 other gp91-phox genes. Genomic sequencing demonstrated a single base change associated with CGD in each kindred: in one, adenine to cytosine at base pair-57 and in the other, thymidine to cytosine at -55. These mutations are located between the "CCAAT" and "TATA" box consensus sequences involved in eukaryotic gene transcription. Gel shift assays revealed two specific DNA-protein complexes formed between phagocyte nuclear extracts and an oligonucleotide probe representing bases -31 to -68 of the gp91-phox promoter region; the faster-migrating complex could not be formed with oligonucleotides containing either of the promoter mutations. Thus, these promoter region mutations appear to be causally related to the loss of association of a DNA-binding protein and lead to diminished gp91-phox expression, abnormal transcription initiation, and the development of CGD.

Base Sequence

Mutation creates an open reading frame within the 5' untranslated region of macaque erythrocyte carbonic anhydrase (CA) I mRNA that suppresses CA I expression and supports the scanning model for translation.

A variant allele at the CA I locus that produces a deficiency of erythrocyte-specific CA I occurs as a widespread polymorphism in pigtail macaques from southeast Asia. Sequence analyses revealed a C----G substitution 12 nucleotides downstream of the cap site in the variant erythrocyte CA I mRNA. This mutation forms a new AUG start site and an open reading frame coding for 26 amino acids that terminates 6 nucleotides before the normal AUG initiation codon for CA I. It appears that the presence of this upstream open reading frame greatly diminishes reinitiation of translation from the normal start site, resulting in trace levels of CA I in erythrocytes. Preferential use of the first AUG codon supports the scanning model for translation initiation in eukaryotes.

Animals

Variation in coding exons of two electrophoretic alleles at the pigtail macaque carbonic anhydrase I locus as determined by direct, double-stranded sequencing of polymerase chain reaction (PCR) products.

Two, electrophoretically distinct, forms of carbonic anhydrase I (CA Ia and CA Ib) are found at high polymorphic frequencies in red cells of natural populations of pigtail macaques, Macaca nemestrina, from southeast Asia. By use of the polymerase chain reaction, exons of the CA I gene were amplified from homozygous (a/a, b/b) and heterozygous (a/b) animals. Direct sequencing of the amplified DNA from four animals revealed differences between the a and the b electrophoretic alleles ranging from three to six nucleotides, and from one to three differences within each allele. These results indicate a greater genetic variability at the CA I locus in this macaque species than previously realized.

Alleles

Chronic granulomatous disease: diagnosis and classification at the molecular level.

Chronic granulomatous disease (CGD) is caused by the failure of phagocytes to produce microbicidal derivatives of molecular oxygen, such as hydrogen peroxide. It is one of the best characterized of the phagocyte disorders and represents an important consideration in the differential diagnosis of recurrent infections. The clinical, biochemical, and molecular genetic aspects of CGD are reviewed in this context in this article.

Amino Acid Sequence

Three case reports of the metabolic and electroencephalographic changes during advanced Buddhist meditation techniques.

To examine the extent to which advanced meditative practices might alter body metabolism and the electroencephalogram (EEG), we investigated three Tibetan Buddhist monks living in the Rumtek monastery in Sikkim, India. In a study carried out in February 1988, we found that during the practice of several different meditative practices, resting metabolism (VO2) could be both raised (up to 61%) and lowered (down to 64%). The reduction from rest is the largest ever reported. On the EEG, marked asymmetry in alpha and beta activity between the hemispheres and increased beta activity were present. From these three case reports, we conclude that advanced meditative practices may yield different alterations in metabolism (there are also forms of meditation that increase metabolism) and that the decreases in metabolism can be striking.

Arousal

Origins and molecular evolution of the carbonic anhydrase isozymes.

Work on membrane-bound and subcellular forms of CA at the protein level, and the possibility of multiple forms of the mouse CA II gene at the DNA level, indicate that CA may represent an extensive multigene family. A method for classifying newly sequenced CA molecules, or genes encoding them, is discussed. Phylogenetic trees based on the existing sequence data are presented and discussed in terms of gene evolution. The active-site residues of CA II have been more conserved in evolution than those of CA I or CA III. After the gene duplications, CA III and CA I initially evolved more rapidly than CA II. Since the mammalian radiation, the CA II molecule as a whole has been accepting substitutions more frequently than CA I, which in turn is evolving more rapidly than CA III. These findings can be explained if external regions of CA I and CA III have been conserved in evolution owing to interactions with other molecules. Two such regions appear to be residues 18-37 in CA I and 231-250 in CA III. Spinach CA was purified and a small amount of sequence data collected. The difficulty in aligning it with animal CAs suggests that a plant CA may not be suitable to shed light on the active site and character of the ancestral eukaryote CA.

Amino Acid Sequence

Effect of remaining family members on fatness prediction.

As shown first by stepwise multiple correlations and then by family "sets" the probability that a parent or a child will be obese is a direct function of the fatness level of remaining family members. For a four-member nuclear family the probability that one member will be obese is well below chance expectancy (i.e., 12.6%) if the remaining three members are all lean and far higher (i.e., 40.7%) if the remaining family members are all obese. While mothers follow family line fatness expectancy, there is an excess of obese mothers in lean nuclear families, consistent with the inverse relationship between adult female fatness and socioeconomic status.

Adolescent

Differential fatness gain of low income boys and girls.

As shown in 564 girls and 553 boys followed for a period of 18 yr, long-term gain in both subscapular and triceps skinfold thickness was higher in children of lower family income level than those of higher family incomes. This differential fatness gain accounts for the socioeconomic "reversal" of fatness in the female shown in cross-sectional studies and newly extends the phenomenon to both sexes. The finding that low-income children show a greater long-term increase in fatness bears on the prevention and control of obesity.

Adipose Tissue