Biomedical subjects
Publications and source records attributed to J C Murray.
Epidermal cells from adult guinea pig skin attach and differentiate preferentially on substrates of type IV (basement membrane) collagen, compared to those of types I--III collagen. In contrast, guinea pig dermal fibroblasts attach equally well to all four collagen substrates. Fibronectin mediates the attachment of fibroblasts but not of epidermal cells to collagen.
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Established lines of fibroblasts have been shown to adhere to collagen substrates via a serum-derived glycoprotein. The attachment of various other cells to collagen types I-IV is examined here. Cells such as human skin fibroblasts, periosteum, hepatocytes, connective tissue cells, and monocytes required the serum glycoprotein and adhered equally well to all collagens, but attachment of chondrocytes, epidermal cells, and neutrophils was inhibited by the serum glycoprotein. Attachment of 2 tumorigenic cells, an osteosarcoma and a fibrosarcoma, was found to be unaffected by the serum glycoprotein. In addition, the fibrosarcoma and epidermal cells attached preferentially to type IV (basement membrane) collagen.
At 24 h after injection of 16-day chick embryos with [C-3H]pyridoxine hydrochloride, some of this label appears in the epiphysial cartilage. Over 35% of this radioactivity appears in the form of [G-3H]pyridoxal and a further 30% as other vitamin B-6 compounds. Partial purification of lysyl oxidase from the labelled epiphysial cartilage reveals a single peak of radioactivity coinciding with a single peak of enzyme activity. On dialysis against phosphate-buffered saline, 75% of this radioactivity is found to be non-diffusible. After incubation with isonicotinic acid hydrazide, a carbonyl reagent that appears to inhibit lysyl oxidase both in vivo and in vitro, a further 70% of the radioactivity is lost, with a roughly corresponding loss of enzyme activity. It is suggested that a form of vitamin B-6 is required as a cofactor of lysyl oxidase, and that this may have important implications in terms of connective-tissue metabolism.
Lysyl oxidase is the copper-dependent enzyme responsible for the normal cross-linking of both collagen and elastin which is necessary for their functional integrity. There is now strong evidence that this enzyme is vitamin-B6-dependent. The earliest visible lesion of atherosclerosis, commonly found in human neonatal coronary arteries and probably indicative of the location of future atherosclerotic plaques, is a focal splitting of the internal elastic lamina, the cause of which has hitherto remained unexplained. It is suggested that this lesion is the result of imperfect cross-linking of arterial elastin as well as collagen, and is caused by a maternal deficiency of vitamin B6 which is commonly found in pregnancy and which could thus impair the function of lysyl oxidase. Prophylactic supplementation of maternal diet with adequate vitamin B6 is therefore suggested.
Catechol analogs inhibit the activity of lysyl hydroxylase (peptidyllysine, 2-oxyglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4), a microsomal enzyme which catalyzes the transformation of certain lysyl residues in collagen to hydroxylysine. Chick embryo lysyl hydroxylase activity was measured by specific tritium release as tritiated water from an L-[4,5-3H]lysine-labelled unhydroxylated collagen substrate prepared from chick calvaria. Catechol analogs did not bind irreversibly to either enzyme or substrate, as full activity was restored with dialysis. Addition of excess cofactor, Fe2+, ascorbic acid, or alpha-ketoglutarate, did not affect inhibition. Kinetic analysis revealed that with respect to collagen substrate, catechol demonstrated a noncompetitive type of inhibition with a Ki of 15 muM.
Catechol analogs inhibit the formation of hydroxylysine-derived intermolecular collagen cross links in tissue cultures of chick embryo calvaria. Formation of intermolecular collagen cross links was measured following incorporation of [14C]lysine, reduction with sodium borohydride, and elution from an ion exchange column with a pyridine-formate gradient. Cultures grown in the presence of 10(-3) M catechol, 10(-3) M dopamine, 10(-3) M L-dopa, or 10(-3) M D,L-serine-(2,3,4-trihydroxybenzyl)-hydrazide demonstrated between 43 and 84% inhibition of hydroxylysine formation. Collagen biosynthesis was not diminished in these cultures as compared to controls without additions or with beta-aminopropionitrile when measured by collagenase digestion. The formation of hydroxylysine-derived intermolecular cross links was inhibited 34 to 93% for 5,5'-dihydroxylysinonorleucine and 7 to 71% for 5-hydroxylysinonorleucine. The catechol analogs also inhibit the activity of lysyl hydroxylase as measured by specific tritium release as triated water from an L-[4,5-3H]lysine-labeled unhydroxylated collagen substrate prepared from chick calvaria. Since catechol analogs inhibit the formation of hydroxylysine in a cell-free assay, these compounds must pass into the cells of calvaria in this culture system to inhibit intracellular hydroxylysine formation and subsequently to diminish the reducible intermolecular cross links of the newly synthesized collagen.
Homogentisic acid inhibits the in vitro activity of chick embryo lysyl hydroxylase, a microsomal enzyme which catalyzes the transformation of certain lysyl residues in collagen to hydroxylysine. Chick embryo lysyl hydroxylase activity was measured as specific tritium release as tritium water from a [4,5-(3)H]lysine-labeled unhydroxylated collagen substrate prepared from chick calvaria. Kinetic studies revealed a linear, noncompetitive type of inhibition with respect to collagen substrate with a Ki of 120-180 muM. The inhibition by homogentisic acid was reversible in that enzyme activity could be restored after dialysis of preincubated mixtures of homogentisic acid with enzyme or substrate. The inhibition by homogentisic acid was competitive with respect to ascorbic acid, and the addition of reducing agents, such as ascorbic acid or 1,4-dithiothreitol, protected lysyl hydroxylase activity from homogentisic acid inhibition.In organ cultures of embryonic chick calvaria, biosynthesis of hydroxylysine-derived intermolecular collagen cross-links was inhibited in a dose-dependent manner by 0.5-5 mM homogentisic acid. Because homogentisic acid inhibits the formation of hydroxylysine in a cell-free assay and in organ cultures, this compound must pass into the cells of calvaria to inhibit intracellular hydroxylysine formation and subsequently to diminish the reducible intermolecular cross-links of the newly synthesized collagen. We propose that the inhibition of lysyl hydroxylase and the resulting hydroxylsine-deficient, structurally modified collagen may be clinically significant in the defective connective tissue found in alkaptonuric patients.
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Widened arterial pulse pressure is often found with conditions having exaggerated diastolic runoff flow, such as aortic insufficiency and arteriovenous malformation. We report a 17-year-old boy with metastatic testicular choriocarcinoma and a widened pulse pressure. Ultrasonography and Doppler analysis revealed significant runoff flow into multiple large hepatic metastases. After chemotherapy, there was diminution in his metastases, a narrowing of the pulse pressure, and no evidence of runoff flow in the liver. These cardiovascular findings have not been previously described in association with metastatic vascular malignancies.
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OBJECTIVES: Endothelial monocyte-activating polypeptide-2 (EMAP-2) is a novel protein that demonstrates potent proinflammatory activity in vivo and in vitro. The purpose of this study was to investigate the expression of EMAP-2 in normal pregnancy and in pregnancies complicated with preeclampsia and to test the hypothesis that EMAP-2 is a causative agent of the endothelial activation of preeclampsia. METHODS: Expression of EMAP-2 in the placenta was investigated using reverse transcriptase-polymerase chain reaction to detect mRNA, and immunohistochemistry with an EMAP-2-specific polyclonal antiserum was carried out to detect protein. Levels of circulating EMAP-2 protein were measured in the blood of nonpregnant, normal pregnant, and preeclamptic individuals using a specific enzyme-linked immunoabsorbent assay and the molecular forms present assessed by Western blotting. RESULTS: EMAP-2 is transcribed and translated by the placenta troughout pregnancy and in preeclampsia and can be detected in the plasma of nonpregnant and pregnant individuals. Levels of circulating EMAP-2 antigen are raised in pregnancy compared with nonpregnant controls; however, levels in patients with preeclampsia are identical to those in normal pregnant individuals. CONCLUSION: While circulating levels of EMAP-2 are increased in pregnancy, there is no evidence that EMAP-2 is involved directly in the pathogenesis of preeclampsia.
Progressive pseudorheumatoid dysplasia is an autosomal recessive skeletal dysplasia with radiographic changes in the spine similar to Spondyleopiphyseal dysplasia tarda and clinical, though not radiographic resemblance to rheumatoid arthritis. About two-thirds of the reported patients are of Arabic and Mediterranean origin which reflects the relative high incidence in this population. We performed homozygosity mapping utilising the DNA pooling approach to map progressive pseudorheumatoid dysplasia to a chromosomal region on the long arm of chromosome 6. We examined a possible candidate gene in the same region of linkage, namely COL10A1, for alterations in this disorder. We did not identify any mutations in our family, but did not totally exclude COL10A1 gene from being the disease-causing gene.
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Autoimmune hemolytic anemia (AIHA) is a hematologic disorder that is rarely seen in infants and young children. Most cases are associated with viral or bacterial infection, but the immunologic events leading to hemolysis are poorly understood. We describe two infants with severe cytomegalovirus (CMV)-associated warm antibody AIHA. One case was immunohematologically analyzed and showed suggestive evidence that endogenous anti-CMV IgG antibodies were the pathogenic antibodies leading to hemolysis, implicating a possible causal relationship between AIHA and CMV infection. Both patients were ultimately treated with intravenous CMV immune globulin, with subsequent improvement. These cases suggest that investigation for the presence of CMV in infantile AIHA is warranted and that CMV immune globulin should be considered as a therapeutic option.