Biomedical subjects

J Mollenhauer

Publications and source records attributed to J Mollenhauer.

At least 37 records · Page 2Linked to original sources

PubMed2000

DMBT1 encodes a protein involved in the immune defense and in epithelial differentiation and is highly unstable in cancer.

The gene deleted in malignant brain tumors 1 (DMBT1) has been proposed as a candidate tumor suppressor for brain, gastrointestinal, and lung cancer. It codes for a protein of unknown function belonging to the superfamily of scavenger receptor cysteine-rich proteins. We aimed at getting insights into the functions of DMBT1 by expression analyses and studies with a monoclonal antibody against the protein. The DMBT1 mRNA is expressed throughout the immune system, and Western blot studies demonstrated that isoforms of DMBT1 are identical to the collectin-binding protein gp-340, a glycoprotein that is involved in the respiratory immune defense. Immunohistochemical analyses revealed that DMBT1 is produced by both tumor-associated macrophages and tumor cells and that it is deregulated in glioblastoma multiforme in comparison to normal brain tissue. Our data further suggest that the proteins CRP-ductin and hensin, both of which have been implicated in epithelial differentiation, are the DMBT1 orthologs in mice and rabbits, respectively. These findings and the spatial and temporal distribution of DMBT1 in fetal and adult epithelia suggest that DMBT1 further plays a role in epithelial development. Rearrangements of DMBT1 were found in 16 of 18 tumor cell lines, and hemizygous deletions were observed in a subset of normal individuals, indicating that the alterations in tumors may be a result of both pre-existing deletions uncovered by a loss of heterozygosity and secondary changes acquired during tumorigenesis. Thus, DMBT1 is a gene that is highly unstable in cancer and encodes for a protein with at least two different functions, one in the immune defense and a second one in epithelial differentiation.

Agglutinins↗

PubMed2000

Cell attachment, collagen binding, and receptor analysis on bovine articular chondrocytes.

The purpose of this study was to investigate collagen receptors on primary bovine articular chondrocytes from full-thickness and different layers of bovine articular cartilage. Cytometric studies with antibodies showed that approximately 56% of the chondrocytes from the superficial layer and 29% of the chondrocytes from the deep layer bound anti-annexin V. A similar tendency was found for alpha5 and beta1 integrin antibodies. Flow cytometric analysis initially detected annexin V on chondrocytes following isolation; the level of detection subsequently decreased by 24 hours, whereas that of alpha5 and beta1 integrins increased. Treatment of chondrocytes with collagenase at 24 hours restored the initially high epitope recognition of annexin V, indicating masking of annexin V by newly formed collagen matrix. There was little effect on detection levels for beta1 integrin. Contrary to the specific matrix receptor expression, chondrocytes from superficial and deep layers differed little in attachment to immobilized types I and II collagens. However, the attachment was more effectively inhibited with anti-annexin V than with integrin antibodies. Competition studies with preparations of soluble collagens revealed a preferential binding of bovine type-II collagen compared with bovine type-I collagen. Anti-annexin V antibodies inhibited binding of type-II collagen more effectively than anti-alpha5 or anti-beta1 integrin antibodies. Evidently, under the in vitro conditions of this study, annexin V is the quantitatively predominant type-II collagen receptor on bovine articular chondrocytes. This opens a discussion of the possibly dualistic metabolic/mechanical annexin V-integrin receptor elements.

Animals↗

PubMed2000

The antigenicity of fascia lata allografts.

OBJECTIVE: To determine whether commercially available fascia lata allograft material contains donor antigens. MATERIALS AND METHODS: Human leukocyte antigens (HLA) class I and II were assessed in: (i) freeze-dried fascia lata allografts; (ii) a Tutoplast fascia lata graft (Mentor Urology, Santa Barbara, CA, USA); (iii) an acellular dermal graft; and (iv) a successful donor fascia sacrocolpopexy graft one year after implantation, using a polymerase chain reaction sequence-specific primer-based assay. RESULTS: The donor for both the freeze-dried fascia lata and Tutoplast fascia lata was fully HLA-typed. At one year after implantation, antigens from the implanted sacrocolpopexy graft matched the host blood antigens. The antigenicity of the acellular dermal graft could not be ascertained because this material interfered with the assay. CONCLUSION: Donor fascia lata grafts prepared by freeze-drying or by the Tutoplast technique retain donor antigens. The significance of this antigenicity is unknown. All donor antigens are replaced by host antigens after implantation.

Fascia Lata↗

PubMed2000

The fate of rectus fascia suburethral slings.

OBJECTIVE: Autologous rectus fascia is commonly used to construct suburethral slings for the treatment of genuine stress incontinence. This fascia performs well and has not been associated with clinical problems related to its choice as a sling material. However, the histologic appearance of such slings after implantation has not been documented. STUDY DESIGN: At the time of revision of autologous rectus fascia suburethral slings in 5 patients, biopsy specimens of the slings were obtained and submitted for histologic examination. A specimen of rectus fascia before implantation was also obtained from a sixth patient who had no symptoms. RESULTS: After implantation autologous rectus fascia slings remain viable. There is fibroblast proliferation, neovascularization, and remodeling of the graft. No evidence of inflammatory reaction or of graft degeneration was detected. A linear orientation of connective tissue and fibroblasts was seen in some areas, whereas other areas had remodeled to form tissue similar to noninflammatory scar. CONCLUSION: Autologous rectus fascia slings undergo extensive remodeling after implantation.

Aged↗

PubMed2000

Urethral collagen morphologic characteristics among women with genuine stress incontinence.

OBJECTIVE: This was a study of the morphologic characteristics of urethral collagen in women with stress incontinence and continent control women. STUDY DESIGN: Urethral needle biopsy specimens were obtained from 31 women. Fifteen women were continent, and the other 16 had undergone full urogynecologic assessment for symptoms of urinary incontinence. Biopsy specimens were assessed under electron microscopy. Mean collagen fibril diameter was measured and collagen morphologic characteristics were assessed. RESULTS: The biopsy specimens from 30 women were included in the analysis. Collagen fibril diameter did not vary with continence status, the presence of pelvic organ prolapse, age, race or hormonal status. Alterations in collagen fibril morphologic characteristics were evident in the biopsy specimens from nine patients with incontinence. The alterations in collagen morphologic characteristics fell into three patterns, which for convenience were referred to as the obscured pattern, the dense pattern, and the degenerative pattern. CONCLUSION: Altered collagen morphologic characteristics are found in some patients with stress incontinence, and possible causes for those alterations are suggested by their appearances.

Adult↗

PubMed2000

Comprehensive allelotype and genetic anaysis of 466 human nervous system tumors.

Brain tumors pose a particular challenge to molecular oncology. Many different tumor entities develop in the nervous system and some of them appear to follow distinct pathogenic routes. Molecular genetic alterations have increasingly been reported in nervous system neoplasms. However, a considerable number of affected genes remain to be identified. We present here a comprehensive allelotype analysis of 466 nervous system tumors based on loss of heterozygosity (LOH) studies with 129 microsatellite markers that span the genome. Specific alterations of the EGFR, CDK4, CDKN2A, TP53, DMBT1, NF2, and PTEN genes were analyzed in addition. Our data point to several novel genetic loci associated with brain tumor development, demonstrate relationships between molecular changes and histopathological features, and further expand the concept of molecular tumor variants in neuro-oncology. This catalogue may provide a valuable framework for future studies to delineate molecular pathways in many types of human central nervous system tumors.

Alleles↗

PubMed2000

Human articular chondrocytes express osteogenic protein-1.

This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms. (J Histochem Cytochem 48:239-250, 2000)

Adolescent↗

PubMed2000

Detection and molecular mass determination of an HIV replication-enhancing female genital tract factor using a blot bioassay.

We previously reported that cervicovaginal lavages (CVL) contain a factor that enhances the replication of human immunodeficiency virus (HIV) by increasing virus transcription in T cells and monocytoid cells. This factor was named the HIV-inducing factor (HIF). To determine the molecular mass of HIF, we adapted a blot technique that involves nonreducing SDS-PAGE of CVL samples and electrophoretic transfer onto nitrocellulose paper followed by incubation of paper slices with HIV-infected monocytoid U1 cells. The slices with HIF bioactivity were detected by increased HIV production and measured by an HIV core protein (p24) ELISA. We refer to this technique as the "BioBlot" assay. The BioBlot assay successfully detected bioactivity of HIF anchored onto nitrocellulose and determined that HIF has a molecular mass of about 14 kDa. Paper slices with HIF-negative CVL samples as well as nitrocellulose paper samples without CVL did not enhance HIV production. This finding suggested that SDS-PAGE and nitrocellulose binding do not functionally alter the bioactive domain(s) of HIF structure. In addition to the detection of HIF bioactivity anchored to nitrocellulose and HIF molecular mass determination, the BioBlot technique offers an alternative, rapid method for other applications. These include the study of receptor-ligand interactions of mucosal proteins, direct bioactivity testing and molecular mass determination of secretory substances.

Biological Assay↗

PubMed1999

The genomic structure of the DMBT1 gene: evidence for a region with susceptibility to genomic instability.

Increasing evidence has accumulated for an involvement of the inactivation of tumour suppressor genes at chromosome 10q in the carcinogenesis of brain tumours, melanomas, and carcinomas of the lung, the prostate, the pancreas, and the endometrium. The gene DMBT1 (Deleted in Malignant Brain Tumours 1) is located at chromosome 10q25.3-q26.1, within one of the putative intervals for tumour suppressor genes. DMBT1 is a member of the scavenger-receptor cysteine-rich (SRCR) superfamily and displays homozygous deletions or lack of expression in glioblastoma multiforme, medulloblastoma, and in gastrointestinal and lung cancers. Based on these properties, DMBT1 has been proposed to be a candidate tumour suppressor gene. We have determined the genomic sequence of DMBT1 to allow analyses of mutations. The gene has at least 54 exons that span a genomic region of about 80 kb. We have identified a putative exon with coding potential for a transmembrane domain. Our data further suggest that alternative splicing gives rise to isoforms of DMBT1 with a differential utilization of SRCR domains and SRCR interspersed domains. The major part of the gene harbours locus specific repeats. These repeats may point to the DMBT1 locus as a region susceptible to chromosomal instability.

Agglutinins↗

PubMed1999

Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D.

Surfactant protein D (SP-D) is an oligomeric C type lectin that promotes phagocytosis by binding to microbial surface carbohydrates. A 340-kDa glycoprotein (gp-340) has been shown to bind SP-D in the presence of calcium but does so independently of carbohydrate recognition. This protein exists both in a soluble form and in association with the membranes of alveolar macrophages. The primary structure of gp-340 has been established by molecular cloning, which yielded a 7,686-bp cDNA sequence encoding a polypeptide chain of 2, 413 amino acids. The domain organization features 13 scavenger receptor cysteine-rich (SRCR) domains, each separated by an SRCR-interspersed domain, except for SRCRs 4 and 5, which are contiguous. The 13 SRCR domains are followed by two C1r/C1s Uegf Bmp1 domains separated by a 14th SRCR domain and a zona pellucida domain. gp-340 seems to be an alternative spliced form of DMBT1. Reverse transcription-PCR analysis showed that the main sites of synthesis of gp-340 are lung, trachea, salivary gland, small intestine, and stomach. Immunohistochemistry revealed strong staining for gp-340 in alveolar and other tissue macrophages. Immunostaining of the macrophage membrane was either uniform or focal in a way that suggested capping, whereas other macrophages showed strong intracellular staining within the phagosome/phagolysosome compartments. In some macrophages, SP-D and gp-340 were located in the same cellular compartment. Immunoreactive gp-340 was also found in epithelial cells of the small intestine and in the ducts of salivary glands. The distribution of gp-340 in macrophages is compatible with a role as an opsonin receptor for SP-D.

Amino Acid Sequence↗

PubMed1999

Functional failure of fascia lata allografts.

OBJECTIVES: Fascia lata allografts are commonly used in urogynecologic procedures. Functional failure of several grafts has occurred, and such failure has been recognized as a materials problem in 12 patients. STUDY DESIGN: Twelve patients with failure of an initial urogynecologic procedure performed with irradiated and freeze-dried donor fascia lata grafts underwent reoperation. Portions of the implanted fascia lata grafts could be retrieved in 7 cases. Graft specimens underwent histologic processing followed by hematoxylin and eosin staining. RESULTS: Histopathologic analyses of the retrieved material demonstrated several ongoing processes in the failed grafts. A few grafts showed areas of ideal remodeling. Most grafts, however, showed areas of disorganized remodeling and areas of graft degeneration. Evidence of immune reaction to the graft was observed in some cases. CONCLUSION: The high materials failure rate associated with the use of irradiated and freeze-dried donor fascia lata grafts suggests that such tissue should not be used for urogynecologic procedures.

Fascia Lata↗

PubMed1999

Failure of allograft suburethral slings.

OBJECTIVES: To report our experience of using freeze-dried irradiated fascia lata allografts for suburethral sling procedures. PATIENTS AND METHODS: Between December 1996 and September 1998, 35 patients (mean age 60.25 years, range 37-79) underwent suburethral sling placement with fascia allograft. These patients were reviewed, with the findings at the time of any surgical re-exploration. Eleven (31%) had undergone prior surgery for genuine stress incontinence and 32 (91%) had a preoperative diagnosis of intrinsic sphincter deficiency. RESULTS: On re-operation for persistent or recurrent stress incontinence, the allograft was present but grossly degenerated in two (6%) patients and completely absent in five (14%) patients. Histology of a retrieved graft fragment showed both fibroblast proliferation and degeneration within the graft. CONCLUSION: The use of freeze-dried, irradiated fascia lata for suburethral sling procedures was associated with a material failure rate of >/=20%. We caution against its use in this setting.

Adult↗

PubMed1999

Expression of anchorin CII (cartilage annexin V) in human young, normal adult, and osteoarthritic cartilage.

In its tissue-specific function as a collagen receptor of chondrocytes, cartilage annexin V (anchorin CII) occupies a key position in the organization of the cell-extracellular matrix (ECM) junction for the tissue. The general role of annexin V (Anx V) in other tissues suggests involvement in cellular secretory processes and in regulation of apoptosis. Immunohistochemical analysis of Anx V in growth plate cartilage, confirmed by in situ hybridization, suggests that Anx V is prominently expressed and forms a major constituent of growth plate chondrocytes. Anx V epitopes are also located in the pericellular matrix of hypertrophic cartilage. In adult articular cartilage the expression is downregulated, with the highest levels of immunostaining found in the upper third of the articular cartilage layers and almost no antigen found in the deep layers. Osteoarthritic (OA) cartilage is characterized by a significant upregulation of message and protein throughout the entire depth of the tissue, an accumulation of cytoplasmic annexin V epitopes, and a release of epitopes into the pericellular and interterritorial matrix, in part co-localized with granular structures. Therefore, Anx V expression and tissue distribution may serve as a histological marker for metabolic alterations and for changes in the cellular phenotype associated with OA.

Adult↗

PubMed1998

Culture of chondrocytes in alginate gel: variations in conditions of gelation influence the structure of the alginate gel, and the arrangement and morphology of proliferating chondrocytes.

Sodium alginate, which gels in the presence of calcium ions, is commonly used for culture of anchorage-independent cells, such as chondrocytes. Normally, the gel appears microscopically homogeneous but, depending on the conditions of gelation, it may contain a varying number of small channels that extend inward from the surface. We have examined the influence of these channels on the morphology of cultured chondrocytes entrapped in alginate beads. Growth-plate or articular chondrocytes cultured in alginate normally proliferate and form rounded cell clusters but, in alginate beads containing numerous channels, many chondrocytes become aligned and form columns similar to those in the growth plate in vivo. As the pattern of cellular growth and morphology in alginate is profoundly influenced by the presence of channels in the gel, further studies were conducted to determine what specific conditions of gelation affect their formation. The channels are especially numerous when both the alginate and the gelling solutions lack sodium ions or other monovalent cations. The channels are cavities in the gel formed by particulate blocking of the rapid diffusion of calcium ions from the gelling solution into the boundary of the calcium alginate solution, and hence they extend inward from cells at the surface of the alginate gel. An understanding of the conditions under which these channels develop makes it possible either to avoid their formation or, alternatively, to enhance the number of channels in order to encourage proliferating cells to grow in radial columns, rather than in a less organized pattern characteristic of most culture systems.

Alginates↗

PubMed1998

Molecular analysis of childhood primitive neuroectodermal tumors defines markers associated with poor outcome.

PURPOSE: The diagnostic and prognostic significance of well-defined molecular markers was investigated in childhood primitive neuroectodermal tumors (PNET). MATERIALS AND METHODS: Using microsatellite analysis, Southern blot analysis, and fluorescence in situ hybridization (FISH), 30 primary tumors and six CSF metastasis specimens were analyzed for loss of heterozygosity (LOH) of chromosomes 1q31, 6q, 9q22, 10q, 11, 16q22, and 17p13.1 and/or high-level amplification of the c-myc gene. Experimental data were compared with clinical stage and outcome. RESULTS: LOH of chromosome 17p13.1 was found most frequently (14 of 30 tumors, six of six CSF metastasis specimens); LOH of chromosomes 10q, 16q22, 11, 6, 9q22, and 1q31 was observed in 20.6%, 20%, 14.3%, 12%, 10%, and 0%, respectively. Eight of 32 tumors and CSF specimens showed amplification of c-myc. All tumors with amplification of c-myc were resistant to therapy and had a fatal outcome (mean survival time, 9.3 months). Tumors that displayed LOH of chromosome 17p were associated with metastatic disease. The prognosis of these tumors was worse only when associated with amplification of c-myc. Three of three patients with LOH of 9q22 relapsed. CONCLUSION: In our study, amplification of c-myc was a poor-prognosis marker in PNET. LOH of chromosome 17p was associated with metastatic disease. Molecular analysis of primary tumors using these markers may be useful for stratification of children with PNET in future prospective studies. The other aberrations investigated were not of significant prognostic value, but may provide an entry point for future large-scale molecular studies.

Adolescent↗

PubMed1997

Absence of cell-surface annexin V is accompanied by defective collagen matrix binding in the Swarm rat chondrosarcoma.

Annexin V has been characterized as a major collagen type II binding cell-surface component of normal chondrocytes and is also called anchorin CII in chondrogenic populations. Herein we present evidence that in vitro cultured Swarm rat chondrosarcoma cells are not capable of binding collagen type II in significant quantities to their surfaces, as compared to normal rat chondrocytes. This finding coincides with a deficiency of annexin V on the surface of these cells. A small quantity of an intracellular polypeptide could be detected which is immunologically cross-reactive with annexin V but displayed a mobility in SDS-PAGE of less than 34 kD compared to the M(r) 36 kD of intact rat annexin V. By immunohistochemistry the protein could be localized in the cytoplasm of in vitro and in vivo grown tumor cells. By reverse transcription-polymerase chain reaction and Northern blot analysis, a regular-sized mRNA for annexin V could be detected in the chondrosarcoma cells that is expressed in only slightly lower quantities than in normal chondrocytes. Taken together, the data suggest a modified processing or turnover for annexin V in the chondrosarcoma excluding it from being a functionally active collagen type II binding protein. The findings support the hypothesis of cell-surface annexin V as a key component for the formation of the pericellular matrix of chondrocytes.

Amino Acid Sequence↗

PubMed1997

Effects of recombinant human osteogenic protein 1 on the production of proteoglycan, prostaglandin E2, and interleukin-1 receptor antagonist by human articular chondrocytes cultured in the presence of interleukin-1beta.

OBJECTIVE: Recombinant human osteogenic protein 1 (OP-1) is an effective stimulator of human cartilage 35S-proteoglycan synthesis. The present study was conducted to determine whether stimulation of human articular chondrocytes with OP-1 can help overcome interleukin-1beta (IL-1beta)-induced suppression of 35S-proteoglycan synthesis. METHODS: Human articular chondrocytes in alginate beads were maintained for 3 days in the absence (control) or presence of IL-1beta at 0.1-100 pg/ml with or without OP-1 at 50 ng/ml, in medium containing 10% fetal bovine serum (FBS). Incorporation of 35S-sulfate into proteoglycans was quantified during the last 4 hours of culture and reported as counts per minute per microg DNA. Release of interleukin-1 receptor antagonist (IL-1Ra) and prostaglandin E2 into the medium was monitored by immunoassay. RESULTS: IL-1beta at 10 pg/ml caused a 60% decrease in 35S-proteoglycan synthesis. This could be blocked by including 500 ng/ml IL-1Ra in the medium. The presence of 50 ng/ml OP-1 in the IL-1beta-containing medium was effective in restoring 35S-proteoglycan synthesis to the level of that found in cultures not treated with IL-1beta. The restorative effects of OP-1 and IL-1Ra were cumulative. The rate of release of prostaglandin E2 and IL-1Ra into the medium was not affected by the presence of OP-1. CONCLUSION: Treatment of human articular chondrocytes with OP-1 cultured in the presence of FBS is effective in overcoming the down-regulation of proteoglycan synthesis induced by low doses of IL-1beta.

Adolescent↗

PubMed1997

Annexins: what are they good for?

Annexins comprise a unique family of calcium- and phospholipid-binding proteins. At least one of the twenty members thus far described from this family can be found expressed in nearly every eukaryotic cell type. As common as these proteins may be, no one clear function for all has been established. Historically, individual members of this family have been given various names describing their ability to associate with a host of intra- and extracellular proteins and with cellular lipid membranes. The collection of reviews in this issue of CMLS represents an effort to offer a coordinated view of the research activities in the field and to extract structural and functional commonalities.

Animals↗