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Biomedical subjects

P M Wilson

Publications and source records attributed to P M Wilson.

At least 37 records · Page 2Linked to original sources
PubMed1994

Alpha 2-macroglobulin is present in and synthesized by the cornea.

PURPOSE: The purposes of this study were to determine whether the proteinase inhibitor alpha 2-macroglobulin is present in the cornea, and, if so, where it is located, and whether it is synthesized by the cornea, and, if so, where it is being synthesized. METHODS: alpha 2-Macroglobulin was immunolocalized using a double antibody technique and quantified by immunodot blot assays, and its identity was confirmed by Western blot analysis. Corneal synthesis of this inhibitor was determined by immunoprecipitation of extracts from corneas incubated in organ culture with 35S-methionine. mRNA was localized by in situ hybridization of 3H-labeled cDNA to the inhibitor. RESULTS: alpha 2-Macroglobulin was localized in the epithelial, endothelial, and stromal cells. It was also found in the stromal extracellular matrix. When extracts of the epithelium, stroma, and Descemet's membrane-endothelium were analyzed by Western blot, an immunoreactive band for this inhibitor was detected in all extracts. This band comigrated with the alpha 2-macroglobulin form isolated from plasma. Metabolically labeled inhibitor was immunoprecipitated from the stromal layer but not from the epithelial or endothelial layer. However, when examined by in situ hybridization, mRNA was localized to epithelial and endothelial cells in addition to stromal keratocytes. CONCLUSIONS: Because alpha 2-macroglobulin has the ability to inhibit a wide range of proteinases, it is probable that this inhibitor plays an important role in protecting the cornea from damage caused by proteinases. This includes proteinases synthesized by the cornea and those released from inflammatory cells and invading organisms.

Adolescent↗
PubMed1994

Corneal synthesis of alpha 1-proteinase inhibitor (alpha 1-antitrypsin).

PURPOSE: To determine if the cornea synthesizes alpha 1-proteinase inhibitor (alpha 1-antitrypsin). METHODS: Human corneas were placed in organ culture for 24 hours in the presence of 35S-methionine to radiolabel corneal proteins. Monoclonal antibodies were used to precipitate labeled alpha 1-proteinase inhibitor. The immunologically isolated inhibitor was electrophoresed on polyacrylamide gels and visualized by autoradiography or by staining for protein. Human corneas were also fixed with formalin and imbedded in paraffin. Sections were probed with 3H-labeled complementary DNA probes to the coding region of alpha 1-proteinase inhibitor. RESULTS: Metabolically labeled alpha 1-proteinase inhibitor was recovered from organ-cultured corneas and the cornea-conditioned medium. Specific messenger RNA was observed in the cornea by in situ hybridization most prominently in corneal epithelial cells. CONCLUSIONS: alpha 1-Proteinase inhibitor is synthesized and released by human corneal epithelial cells. These results indicate that the cornea has the ability to locally control degradation through synthesis of this inhibitor without total dependence on a supply of the inhibitor from the vascular system.

Adult↗
PubMed1993

The human VH3b gene subfamily is highly polymorphic.

We have previously shown that human antibody (Ab) directed against the capsular polysaccharide of the important bacterial pathogen, Haemophilus influenzae type b (Hib) is encoded by a small group of VH3 gene family members. The majority of anti-Hib PS Ab use members of the smaller VH3b subfamily. To examine directly the available human VH3 repertoire, we have used PCR to amplify and clone candidate germ-line VH3b H chain V region genes from two unrelated subjects from whom anti-Hib polysaccharide mAb had been previously obtained. A single functional VH3b germ-line gene was obtained from one subject. This gene is identical throughout the coding region to the previously identified gene 9.1. Twelve distinct VH3b germ-line sequences, 87.6-99.8% homologous to one another, were obtained from the second subject. One of these genes, LSG1.1, is also identical to the 9.1 germ-line gene, and a second, LSG6.1, is identical to a previously reported cDNA, M85. These germ-line VH3b genes are 82.7-94.1% homologous to rearranged anti-Hib PS VH3b segments obtained from these subjects. Our findings further demonstrate that considerable polymorphism of VH segments exists in the human population. Despite the presence of very highly homologous VH elements in the germ line, particular genes are highly conserved within the outbred human population.

Amino Acid Sequence↗
PubMed1993

Restricted immunoglobulin VH usage and VDJ combinations in the human response to Haemophilus influenzae type b capsular polysaccharide. Nucleotide sequences of monospecific anti-Haemophilus antibodies and polyspecific antibodies cross-reacting with self antigens.

To examine the human antibody repertoire generated against a biologically significant antigen we have obtained sequences of heavy chain variable region genes (IgVH) from 15 monoclonal antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). All VH segments are members of the VH3 family and 9 of 15 are members of the smaller VH3b subfamily. Restriction is evident by the shared use of certain VDJ joints in independent hybridomas from different subjects. Two hybridomas generated from the same subject demonstrate identical heavy chain variable region gene sequences but differ in isotype and rearrange alternative light chain variable region genes (IgVL), suggesting that in a normal immune response, a single pre-B cell clone may use different light chain rearrangements and give rise to progeny capable of reacting with antigen. Using a polymerase chain reaction assay optimized to detect base pair differences among VH genes we demonstrate that at least a portion of expressed anti-Hib PS VH genes have undergone somatic mutation. Anti-Hib PS heavy chain genes are homologous to VH segments encoding autoantibodies and two hybridomas secrete anti-Hib PS antibody that cross-reacts with self antigens (double-stranded DNA and single-stranded DNA). Comparison of VH regions of self-reactive and monospecific anti-Hib PS Ab demonstrates no consistent structural feature correlating with fine antigen specificity. These data demonstrate significant restriction in VH usage and VDJ recombination in the anti-Hib PS response and confirm that autoantibodies may be elicited during normal immune responses.

Amino Acid Sequence↗
PubMed1993

Diversity of immunoglobulin light chain usage in the human immune response to Haemophilus influenzae type b capsular polysaccharide.

The response to the capsular polysaccharide of Haemophilus influenzae type b (Hib PS) has been used to determine the molecular basis of antibody gene diversity in humans. In contrast to the relatively restricted nature of anti-Hib PS heavy-chain variable region gene expression, a variety of light-chain variable region genes may encode this antibody (Ab) response. Light-chain variable region gene usage appears to determine the expression of certain Ab idiotypes and fine antigen specificity. To further define the role of light-chain variable region gene usage in important anti-Hib PS Ab subgroups, we have cloned and sequenced a number of immunoglobulin light-chain variable region genes (IgVL) from human monoclonal IgA anti-Hib PS Ab generated in response to Hib PS-protein conjugate vaccines. Three of these Ab are encoded by unusual variable segments. One kappa-Ab is encoded by the "predominant" V kappa II A2 germline gene but, in contrast to a previously reported A2-encoded IgVL sequence, differs from the A2 germline sequence. The IgVL sequence of a second Ab is the only sequence of a kappa-Ab that cross-reacts with the structurally related antigen Escherichia coli K100 polysaccharide reported to date. This IgVL is encoded by a V kappa III-segment most closely homologous to the Humhv328/L16 germline gene, whereas previous reports suggested V kappa III-encoded anti-Hib PS Ab might be exclusively encoded by the germline gene Humhv325/A27.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Making sexuality education and prevention programs relevant for African-American youth.

A demographic and cultural profile of African-American youth is presented. Culturally-based differences in decision-making are reviewed. Attitudes and cultural values related to identity and self-image, gender role socialization, contraception, marriage and parenthood, homosexuality, and HIV/AIDS are described. Recommendations and strategies to make sexuality education and pregnancy prevention programs relevant to African-American youth are offered.

Adolescent↗
PubMed1992

Immunoglobulin light chain variable region gene sequences for human antibodies to Haemophilus influenzae type b capsular polysaccharide are dominated by a limited number of V kappa and V lambda segments and VJ combinations.

The immune repertoire to Haemophilus influenzae type b capsular polysaccharide (Hib PS) appears to be dominated by certain light chain variable region genes (IgVL). In order to examine the molecular basis underlying light chain bias, IgVL genes have been cloned from a panel of heterohybridomas secreting human anti-Hib PS (antibody) (anti-Hib PS Ab). One hybridoma, representative of the predominant serum clonotype of anti-Hib PS Ab in older children and adults following immunization or Hib infection, uses a V kappa II segment identical to the germline gene A2, and a JK3 segment. A second kappa hybridoma uses a member of the V kappa I family and a JK4 segment. Four lambda antibodies, all cross-reactive with the structurally related antigen Escherichia coli K100 PS, use V lambda VII segments which are 96-98% homologous to one another, and may originate from a single germline gene. Two additional lambda antibodies, not K100-cross-reactive, are encoded by members of the V lambda II family. All lambda antibodies use highly homologous J lambda 2 or J lambda 3 segments. The VJ joints of all lambda antibodies and the V kappa II-encoded antibody are notable for the presence of an arginine codon, suggesting an important role in antigen binding. Although more complex than heavy chain variable region gene usage, a significant portion of serum anti-Hib PS Ab is likely to be encoded by a limited number of V kappa and V lambda segments and VJ combinations, which may be selectively expressed during development, or following antigen exposure.

Adult↗
PubMed1990

Alpha-1 proteinase inhibitor levels in keratoconus.

The levels of alpha 1-proteinase inhibitor (alpha 1-antitrypsin) in keratoconus, normal human, and other diseased corneas were examined. Using an immunoperoxidase technique, the presence of this inhibitor was demonstrated in the epithelium, stroma and endothelium of all corneal sections. Compared with normal human controls, the staining intensity in the epithelium and stromal lamellae of keratoconus corneas was markedly reduced. Such a reduction was not seen in either scarred or other diseased corneas. Extracts of keratoconus and normal human corneas were subsequently analyzed for alpha 1-proteinase inhibitor by a dot blot assay using a monoclonal antibody against the inhibitor and a 125I-labelled secondary antibody. In agreement with the immunohistochemical findings, the alpha 1-proteinase inhibitor level found in the epithelium of keratoconus corneas was approximately one-fourth of that found in normal human controls. In addition, the stromal extracts of keratoconus corneas contained about one-sixth the inhibitor level of that in normal human extracts. These results lend further support to the hypothesis that degradation processes may be aberrant in keratoconus.

Adolescent↗
PubMed1989

Characterization of corneal proteoglycans under vitamin A deficiency.

Comparison of in vivo radiolabeled corneal proteoglycans from vitamin A deficient, pair-fed control and normal rabbits by chromatographic and enzymatic methods, reveals subtle but reproducible changes in the proteoglycans present in the vitamin A deficient corneas relative to those of pair-fed and normal control corneas. Although the total amounts of [3H]leucine and [35S]sulfate incorporated in vivo into the proteoglycans per cornea are similar in the three types of cornea, the proteoglycan affinity for DEAE-Sepharose is different, with more of the vitamin A deficient proteoglycans requiring a higher NaCl concentration for elution. Analysis of in vivo labeled rabbit corneal proteoglycans reveals that in vitamin A deficient animals, relative to pair-fed controls, there is (1) an increase in the proteoglycans digested by chondroitinase AC indicative of decreased epimerization of glucuronic acid to iduronic acid; (2) an increase in the amount of keratan sulfate proteoglycan with high affinity for an anion exchange resin, consistent with a greater negative charge; (3) differences in proteoglycan affinities for octyl-Sepharose, reflecting differences in hydrophobicity; (4) increased susceptibility to proteolysis by trypsin; (5) no significant difference in sulfation.

Animals↗
PubMed1989

Laminar and non-laminar patterns of acetylcholinesterase activity in the marsupial lateral geniculate nucleus.

Acetylcholinesterase (AChE) distribution in the dorsal lateral geniculate nucleus (LGd) of 3 polyprotodont and 3 diprotodont marsupials has been compared with the cytoarchitecture and, as appropriate, with retinal terminal bands (RTBs) as demonstrated by tracers injected into the vitreous body. In this series those polyprotodont marsupials showing only partial segregation of retinal input (Virginia opossum and Tasmanian devil), display the least cell laminar differentiation. In them AChE staining is mainly diffuse but stronger in areas of greatest retinal terminal overlap. Segregation of visual input increases progressively in the remaining polyprotodont (eastern quoll) and in the diprotodonts (Tasmanian bettong, Tasmanian potoroo and brush-tailed possum), culminating in the last-named, in which segregation is most complete. Related to this development varying numbers of cell laminae/sublaminae appear and retinal terminal laminae/sublaminae (bands) are revealed. AChE activity can be correlated with both specific cell laminae/sublaminae and retinal terminal laminae/sublaminae in these complex LGds. Greatest AChE staining in every case is related to laminae/RTBs located in the medial part of the lateral (alpha) segment of LGd. Cytoarchitecturally, the ventral lateral geniculate nucleus (LGv), unlike the heterogeneous LGd, is remarkably homogeneous in the series of animals studied and shows well-stained AChE patches relating to areas receiving significant retinal input.

Acetylcholinesterase↗
PubMed1989

Features of Coxsackie B virus (CBV) infection in children with prolonged physical and psychological morbidity.

A retrospective study of the clinical features in 39 children who were investigated for evidence of Coxsackie B virus (CBV) infection is reported. Eighteen children were found to have serological evidence of infection. An extensive range of features was elicited in both seropositive and seronegative patients, most children complaining of abnormalities referable to muscle and, in particular, of weakness and easy fatiguability. Children with evidence of CBV infection were significantly more likely to belong to social classes I and II, to have relatives with serological evidence of CBV infection, and to show certain dysphoric features as well as to complain of sore throats. The relationship between CBV infection and 'myalgic encephalomyelitis' or 'post-viral syndrome' is discussed, and it is suggested that these descriptions are inappropriate given our current knowledge, and inadequately describe the clinical features seen in the children under study. An alternative description, 'fatigue-dysphoria syndrome' is proposed.

Child↗
PubMed1989

Localization and quantification of alpha-1-proteinase inhibitor in the human cornea.

alpha-1-Proteinase inhibitor, formerly called alpha-1-antitrypsin, was detected in human corneal epithelium, stroma, Descemet's membrane and endothelium using an indirect immunolocalization technique. The average alpha-1-proteinase inhibitor levels detected by an immunodot blot assay in the epithelium, stroma and Descemet's membrane-endothelium extracts per total human cornea were 29.5 micrograms, 54.3 micrograms and 3.5 micrograms, respectively. Immunolocalization on Western blots of SDS polyacrylamide electrophoresis gels revealed that all three layers contained a molecule with a molecular weight equal to the native alpha-1-proteinase inhibitor. Additionally, in the epithelial and stromal extracts minor bands at 75 kD and 110 kD were noted which are possibly due to complexes with proteases. The 110 kD band alternatively may be a dimer of the inhibitor. The epithelial extract contained bands at 40 kd and less than 30 kD indicating the presence of proteolysis products. alpha-1-Proteinase inhibitor probably plays a major role in the protection of the cornea from proteases released by inflammatory cells.

Adult↗
PubMed1985

Pyrophosphate:fructose 6-phosphate 1-phosphotransferase and glycolysis in non-photosynthetic tissues of higher plants.

The activity of pyrophosphate:fructose-6-phosphate 1-phosphotransferase [PFK (PPi); EC 2.7.1.90] in extracts of the storage tissues of leek (Allium porrum), beetroot (Beta vulgaris) and roots of darnel (Lolium temulentum) exceeded 0.15 mumol/min per g fresh wt. As net flux from fructose 1,6-bisphosphate to fructose 6-phosphate in these tissues is unlikely, it is suggested that PFK (PPi) does not contribute to gluconeogenesis or starch synthesis. The maximum catalytic activities of PFK (PPi) in apex, stele and cortex of the root of pea (Pisum sativum) and in the developing and the thermogenic club of the spadix of cuckoo-pint (Arum maculatum) were measured and compared with those of phosphofructokinase, and to estimates of the rates of carbohydrate oxidation. PPi and fructose 2,6-bisphosphate in Arum clubs were measured. The above measurements are consistent with a glycolytic role for PFK (PPi) in tissues where there is marked biosynthesis, but not in the thermogenic club of Arum. The possibility that PFK (PPi) is a means of synthesizing pyrophosphate is discussed.

Fructosediphosphates↗
PubMed1985

A photographic perspective on the origins, form, course and relations of the acetylcholinesterase-containing fibres of the dorsal tegmental pathway in the rat brain.

The dorsal tegmental pathway in the rat brain has been studied using acetylcholinesterase (AChe) staining alone, after lesions, and combined with the horseradish-peroxidase (HRP) tracing method. This paper characterises in photographs, diagrams and text the origins, form, extent and relations of its visible AChe-staining fibres in 3 planes. This record should provide a template for further investigations. The pathway largely takes origin from ChAT-containing pedunculopontine (PPTg) and laterodorsal (LDT) nuclei; some non-cholinergic cell groups may also contribute, notably locus coeruleus (LC). It takes the form of a horizontally disposed fan which radiates from the pontomesencephalic area to the forebrain. Its lateral portion is bunched and consists mainly of cholinergic fibres whereas the cholinergic status of its fully unfurled intermediate and partly unfurled medial contingents (which mainly accompany the central tegmental tract) is more doubtful. The changing form and relations of PPTg and LDT are adumbrated including that of the microcellular nucleus (MI) to the former and of Barrington's detrusor nucleus (B) which is unstained, to the latter. Functional overlapping between non-cholinergic and cholinergic nuclei in the peribrachial region are noted and some correlations adduced.

Acetylcholinesterase↗
PubMed1983

Role and location of NAD malic enzyme in thermogenic tissues of Araceae.

This work was done to discover how those nonphotosynthetic tissues of the Araceae that become thermogenic release, as CO2, carbon recently fixed by phosphoenolpyruvate carboxylase. Extracts of clubs of the spadix of Arum maculatum showed no activity for phosphoenolpyruvate carboxykinase and low activities of NADP malic enzyme. NAD malic enzyme activity in the above extracts and in those of thermogenic tissues of other Araceae was appreciable. Analysis of homogenates of clubs of Typhonium giraldii by differential centrifugation and sucrose gradients showed that NAD malic enzyme was confined to mitochondria. Centrifugation of mitochondria after freezing and thawing left all the NAD malic enzyme in the supernatant. NAD malic enzyme in isolated, intact mitochondria was completely latent, and was completely protected from exogenous trypsin. The responses of this latency and protection to different concentrations of Triton X-100 suggested that none of the NAD malic enzyme was accessible from either the outside or the intermembrane space of the mitochondria. Treatment of excised clubs of A. maculatum with 2-N-butylmalonate largely prevented the development of the rapid respiration responsible for thermogenesis, and severely inhibited dark fixation of 14CO2. The conclusion is that in mature clubs of the Araceae phosphoenolpyruvate is converted to malate in the cytosol by phosphoenolpyruvate carboxylase and NAD malate dehydrogenase, and that this malate then enters the mitochondrial matrix where it is converted to pyruvate by NAD malic enzyme.

Carbon Dioxide↗