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Biomedical subjects

G R Martin

Publications and source records attributed to G R Martin.

At least 217 records · Page 12Linked to original sources
PubMed1988

Platelet-derived growth factor enhances demineralized bone matrix-induced cartilage and bone formation.

Subcutaneous implantation of demineralized bone matrix induces the local formation of cartilage and bone. In this study we have investigated the influence of adding various growth factors to the implant. Cartilage formation was monitored by measuring collagen II mRNA levels, and bone formation in the implant was assessed from alkaline phosphatase activity and calcium content. Supplements of the platelet-derived growth factor to implants in older rats increased and production of mRNA for collagen II, alkaline phosphatase activity, and the calcium content of the implant, whereas the other growth factors tested were without effect. The data suggest that under some conditions bone induction is submaximal and can be increased by local supplement of platelet-derived growth factor (PDGF). The present observations may have important therapeutic implications in the treatment of nonunions of fractures and impaired bone formation in the aged.

Animals↗
PubMed1988

The glomerular distribution of type IV collagen and laminin in human membranous glomerulonephritis.

The glomerular distribution of type IV collagen and laminin, the major collagenous and noncollagenous components of the glomerular basement membrane, was studied by immunofluorescence microscopy in idiopathic and lupus membranous glomerulonephritis. Affinity-purified antibodies against type IV collagen reacted preferentially with the inner aspect and irregularly with the adjacent outer area of the thickened basement membrane. In contrast, laminin was detected along the inner aspect of the glomerular basement membrane, in subepithelial basement membrane protrusions ("spikes"), and in the newly formed basement membrane layer above the immune deposits. We conclude that type IV collagen and laminin do not codistribute in the newly formed matrix. This aberrant antigenic distribution may reflect a loss of coordinate biosynthesis or degradation of these matrix components by visceral epithelial cells.

Basement Membrane↗
PubMed1988

Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.

We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases. After less than 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.

Animals↗
PubMed1988

Alpha 1(IV) and alpha 2(IV) collagen genes are regulated by a bidirectional promoter and a shared enhancer.

Collagen IV is the major structural component of basement membranes and is a heterotrimer composed of two alpha 1(IV) and one alpha 2(IV) chains. Most collagen genes are dispersed in the human genome, such as the genes for collagen I, which are located on chromosomes 7 [alpha 1(I)] and 17 [alpha 2(I)]. In contrast, we have found that the murine alpha 1(IV) and alpha 2(IV) collagen chain genes exist in a head-to-head arrangement on opposite strands separated by 130 base pairs. By transfecting various portions of these genes into cells, we have found that transcription of the alpha 1(IV) and alpha 2(IV) genes is regulated by a bidirectional promoter located between the two genes working in concert with an enhancer located in the first intron of the alpha 1(IV) chain gene.

Amino Acid Sequence↗
PubMed1988

Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.

We have defined a signal responsible for the morphological differentiation of human umbilical vein and human dermal microvascular endothelial cells in vitro. We find that human umbilical vein endothelial cells deprived of growth factors undergo morphological differentiation with tube formation after 6-12 wk, and that human dermal microvascular endothelial cells differentiate after 1 wk of growth factor deprivation. Here, we report that morphological differentiation of both types of endothelial cells is markedly accelerated by culture on a reconstituted gel composed of basement membrane proteins. Under these conditions, tube formation begins in 1-2 h and is complete by 24 h. The tubes are maintained for greater than 2 wk. Little or no proliferation occurs under these conditions, although the cells, when trypsinized and replated on fibronectin-coated tissue culture dishes, resume division. Ultrastructurally, the tubes possess a lumen surrounded by endothelial cells attached to one another by junctional complexes. The cells possess Weibel-Palade bodies and factor VIII-related antigens, and take up acetylated low density lipoproteins. Tubule formation does not occur on tissue culture plastic coated with laminin or collagen IV, either alone or in combination, or on an agarose or a collagen I gel. However, endothelial cells cultured on a collagen I gel supplemented with laminin form tubules, while supplementation with collagen IV induces a lesser degree of tubule formation. Preincubation of endothelial cells with antibodies to laminin prevented tubule formation while antibodies to collagen IV were less inhibitory. Preincubation of endothelial cells with synthetic peptides derived from the laminin B1 chain that bind to the laminin cell surface receptor or incorporation of these peptides into the gel matrix blocked tubule formation, whereas control peptides did not. These observations indicate that endothelial cells can rapidly differentiate on a basement membrane-like matrix and that laminin is the principal factor in inducing this change.

Basement Membrane↗
PubMed1988

Low production of procollagen III by skin fibroblasts from patients with Ehlers-Danlos syndrome type IV is not caused by decreased levels of procollagen III mRNA.

The Ehlers-Danlos syndrome type IV represents a heterogeneous group of diseases, the molecular defect of which seems to reside in a defective synthesis and processing of collagen III. Here we present data concerning both protein and mRNA levels of collagens I and III in cell cultures established from skin of nine patients and six controls. All patients investigated were characterized by a reduced amount of procollagen III in fibroblast cultures. However, the levels of collagen III propeptides, measured by a radioimmunoassay in culture medium of fibroblasts from the patients, were either decreased or in the range of control levels. Using hybridization with cDNA probes specific for pro alpha 1 (I) and pro alpha 1 (III) collagen chains we determined the ratios of procollagens I and III mRNA. Although synthesis of procollagen III was markedly reduced in fibroblasts of all patients studied, no parallel decrease of procollagen III specific mRNA was found. However, two distinctive groups of patients were identified, one with an unaltered ratio of procollagen I/III mRNA and the other which had an even higher proportion of collagen III mRNA.

Cells, Cultured↗
PubMed1988

Differences in agonist dissociation constant estimates for 5-HT at 5-HT2-receptors: a problem of acute desensitization?

1. The agonist dissociation constant for 5-hydroxytryptamine (5-HT) was estimated in the guinea-pig isolated trachea by the method of receptor inactivation. The value obtained (pKA = 6.45) was significantly lower than estimates previously obtained in the rabbit aorta and rat jugular vein, although all three tissues are supposed to contain the same 5-HT2 class of receptor. 2. The antagonist dissociation constant for alpha,alpha-dimethyltryptamine was also estimated in the guinea-pig trachea. The pKB value (5.43) was not significantly different from previous estimates in the rabbit aorta and rat jugular vein, consistent with receptor homogeneity between the three tissues. 3. The effect-time profiles corresponding to individual 5-HT applications were more transient in the guinea-pig trachea than in the rabbit aorta. This difference could be accounted for using a simple model of acute receptor desensitization (Leff, 1986), assuming that the conversion of active agonist-receptor complexes into inactive ones was faster in the guinea-pig trachea than in the rabbit aorta. 4. Computer simulation of the desensitization model showed that the discrepancy of pKA estimates for 5-HT between the rabbit aorta and guinea-pig trachea could also be explained using the same rate constant difference that accounted for the difference in effect-time profiles. This analysis indicated that the estimate made in the trachea was erroneously low, whereas that made in the aorta was concluded to be correct. 5. The apparent association between transience of response and pKA estimates is discussed with particular attention to the reliability of agonist affinity estimates in receptor classification.

Animals↗
PubMed1988

Routine evaluation of BACTEC NR-16A and NR-17A media.

The purpose of this study was to determine whether the BACTEC NR-16A and NR-17A media were more effective than the BACTEC NR-6A and NR-7A media in recovering organisms from the blood of patients undergoing antimicrobial therapy. A total of 986 sets of four blood culture bottles were compared, giving 141, 174, 93, and 104 isolates with BACTEC NR-6A, NR-16A, NR-7A, and NR-17A, respectively. BACTEC NR-6A and NR-7A media recovered 234 isolates, whereas BACTEC NR-16A and NR-17A media recovered 278 isolates. The recovery rate of bacteria when aerobic resin media were used was better than that with conventional aerobic media (P less than 0.001). The mean detection times were 51.5 and 69.7 h with NR-16A and NR-6A, respectively (P less than 0.01), whereas they were 68.2 and 71.3 h with NR-17A and NR-7A, respectively (P greater than 0.05). The small number of anaerobes recovered precluded a statistical comparison of relative recovery for that group of organisms.

Aerobiosis↗
PubMed1988

Altered mRNA expression of basement membrane components in a murine model of polycystic kidney disease.

Basement membranes surround the renal tubules and have been shown to limit their distension in vitro. Therefore, it has been postulated that a defect in a basement membrane component(s) underlies the pathogenesis of polycystic kidney disease. Here we have studied a murine model of congenital polycystic kidney disease and found by immunohistology, that the components of the peri-cyst basement membrane appeared to diminish with time. We also measured mRNA levels for collagen IV and laminin, and found a different pattern than in the normal mouse kidney. In normal kidneys, mRNA levels for the B1 and B2 chains of laminin were maximal at birth, and at 1 week for the alpha 1(IV) chain of collagen IV. With all three chains, the levels then rapidly declined. In contrast, mRNA for the alpha 1(IV) chain in congenital polycystic kidneys was half normal 1 week after birth and then increased. Laminin B1 and B2 chain mRNA's were 80% of normal at 1 week but were maintained at that level. As a control, beta-actin mRNA was examined and found to remain constant in both normal and diseased kidneys. In situ hybridization of cRNA probes for the alpha 1(IV) chain confirmed that cells associated with cysts were the principal source of expression of these basement membrane mRNAs. Thus, there exists an abnormal regulation of basement membrane gene expression in congenital polycystic kidney disease. The first stage is characterized by reduced levels of expression. In the second stage, the levels are abnormally high, perhaps representing a compensatory synthesis of basement membrane as cysts enlarge.

Animals↗
PubMed1987

YIGSR, a synthetic laminin pentapeptide, inhibits experimental metastasis formation.

The invasion of tumor cells through basement membranes is a critical step in the formation of metastases. The binding of the malignant cells to laminin in the basement membranes allows their attachment and activates their invasiveness. Recently a synthetic nonapeptide from the B1 chain sequence of laminin was identified as a major site for cell binding. A pentapeptide within the nonapeptide sequence was found to reduce the formation of lung colonies in mice injected with melanoma cells and also to inhibit the invasiveness of the cells in vitro.

Animals↗
PubMed1987

A pentapeptide from the laminin B1 chain mediates cell adhesion and binds the 67,000 laminin receptor.

Laminin promotes epithelial cell adhesion in part through a site of nine amino acids CDPGYIGSR on the B1 chain. Using smaller synthetic peptides from this sequence as well as various peptides with amino acid substitutions, we find that the minimum sequence necessary for efficient cell adhesion as well as receptor binding is YIGSR. The deletion of tyrosine or the substitution of arginine in the peptides resulted in a significant loss of activity. The presence of an amide group on the terminal arginine of either peptide increases activity significantly. YIGSR is active in promoting the adhesion of a variety of epithelial cells; however, it is inactive with chondrocytes, fibroblasts, and osteoblasts.

Animals↗
PubMed1987

Exaggerated spontaneous release of platelet-derived growth factor by alveolar macrophages from patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a fibrotic lung disease characterized by an increased number of mesenchymal cells in the alveolar walls. Alveolar macrophages constitutively express low levels of c-sis, the protooncogene coding for the B chain of platelet-derived growth factor, a protein with chemotactic and mitogenic activity toward mesenchymal cells. We therefore hypothesized that alveolar macrophages in patients with idiopathic pulmonary fibrosis may release increased amounts of platelet-derived growth factor, which might help to explain the accumulation of mesenchymal cells and the fibrosis of the lower respiratory tract in the disease. Evaluation of alveolar macrophages recovered from the lungs of patients with idiopathic pulmonary fibrosis demonstrated that these cells spontaneously released four times more platelet-derived growth factor than did alveolar macrophages recovered from normal persons (P less than 0.01). That the platelet-derived growth factor molecules were potentially active was shown by their chemotactic activity for smooth-muscle cells and their ability to act as a "competence" factor for fibroblast growth. These observations suggest the possibility that the accumulation of mesenchymal cells within the alveolar walls in patients with idiopathic pulmonary fibrosis may result partly from the exaggerated release of the potent mitogen platelet-derived growth factor by mononuclear phagocytes in the lower respiratory tract.

Biological Assay↗
PubMed1987

A rapid in vitro assay for quantitating the invasive potential of tumor cells.

We have reconstituted a matrix of basement membrane onto a filter in a Boyden chamber and assessed the ability of various malignant and nonmalignant cells to penetrate through the coated filter. Cells from all the malignant cell lines tested were able to cross the matrix in 5-6 h, whereas human fibroblasts as well as mouse 3T3 and 10T1/2 cell lines, which are not tumorigenic, were not invasive. In addition, normal primary prostate epithelial cells and benign prostatic hyperplasia cells were not invasive when tested in this assay, whereas malignant prostate carcinoma cells were highly invasive. Parallel experiments with these prostatic cells using the intrasplenic assay for metastasis detection in the nude mouse confirmed the benign behavior of the former cells and the metastatic phenotype of the latter ones. These results suggest that this in vitro test allows the rapid and quantitative assessment of invasiveness and a means to screen for drugs which alter the invasive phenotype of tumor cells.

Animals↗
PubMed1987

Effects of activity on ascending aortic velocity in children with valvar aortic stenosis.

The peak velocity of aortic blood flow was measured in 12 normal children and 20 children with valvar aortic stenosis (AS) by continuous-wave Doppler ultrasound (CWD). Measurements were made at rest, after 2 forms of exercise and, in the 11 children who underwent cardiac catheterization, while under sedation. The exercise was both formal cycle ergometry and a 200-meter jog. The peak velocity in normal children was 1.5 +/- 0.2 m/s at rest, increasing to 2.1 +/- 0.4 m/s (p less than 0.01) after running and to 1.9 +/- 0.4 m/s (p less than 0.01) after cycling. Peak velocity in children with AS was 3.5 +/- 0.8 m/s at rest, increasing to 4.4 +/- 0.8 m/s (p less than 0.01) after running and to 4.5 +/- 0.5 m/s (p less than 0.01) after cycling. The increase in peak velocity was greater in children with AS than in normal children. Measurements of left ventricular (LV) aortic pressure differences by cardiac catheterization were compared to those made by CWD, using the modified Bernoulli equation. The best correlation of the CWD prediction of LV aortic pressure differences was achieved when the children were sedated. The peak velocity of blood flow in the ascending aorta varied with level of activity, and this variability must be considered when using the Bernoulli equation to predict LV aortic pressure differences. CWD prediction of the LV aortic pressure difference was best when the child was sedated (r = 0.98). It is therefore prudent to consider the velocity measurement proximal to the AS to calculate LV aortic pressure differences more accurately unless the child is sedated.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1987

Cell-free translation products of basement membrane RNA from the EHS tumor.

The biosynthetic products of the Engelbreth-Holm-Swarm (EHS) tumor and the cell-free translation products of EHS tumor cell RNA were characterized. Six distinct gene products (three laminin polypeptides, entactin/nidogen, and two collagen IV chains) comprising the basement membrane matrix were identified by a combination of proteolytic digestion and immunologic techniques. Analysis of the cell-free translation products using EHS tumor RNA precipitated by anti-laminin serum confirms earlier evidence that there are at least two B chains encoded by different genes. The anti-laminin serum also recognized entactin/nidogen, which was further identified by specific immunoprecipitation with anti-entactin serum. Radiolabeled laminin A chains, synthesized by the EHS tumor in organ culture, were also identified by the anti-laminin serum but were not detected among the cell-free translation products of EHS tumor RNA. Pulse-chase studies of EHS tumor in organ culture as well as in vitro translation of EHS tumor RNA suggest that the precursor forms of alpha 1(IV) and alpha 2(IV) collagen chains are nearly identical in size, with apparent molecular weights of 170,000. The mRNAs encoding these two polypeptides migrate differently on sucrose gradients. It is likely that glycosylation and hydroxylation of collagen IV account for the major differences in molecular weight of mature alpha 1(IV) and alpha 2(IV) chains in the EHS tumor matrix.

Animals↗