Regulation of tissue inhibitor of metalloproteinases-3 gene expression by transforming growth factor-beta and dexamethasone in bovine and human articular chondrocytes.

Physiological and pathological degradation of cartilage extracellular matrix (ECM) is regulated by the balance between tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs). We examined the potential of chondrocytes from normal bovine or human osteoarthritic (OA) cartilage to express RNA for the new inhibitor TIMP-3 and studied its regulation by an inducer of matrix synthesis, transforming growth factor-beta (TGF-beta). Freshly released chondrocytes constitutively expressed three transcripts of TIMP-3 that are induced by serum factors.

Structure of the human retinoic acid receptor beta 1 gene.

We report the isolation and characterization of genomic sequences coding for the 5' end of human retinoic acid receptor beta 1, a fetal RAR isoform which is also expressed in small cell lung cancers. Primer extension analysis revealed a principal transcription start site with a secondary site 23 bp further upstream in both SCLC and fetal tissues. The sequences isolated were CpG-rich between -60 and the 3' end of the first exon but there were no features like a TATA-box or an Inr element.

Proton gradient formation in early endosomes from proximal tubules.

Heavy endosomes were isolated from proximal tubules using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. Two small GTPases (Rab4 and Rab5) known to be specifically present in early endosomes were identified in our preparations. Endosomal acidification was followed fluorimetrically using acridine orange.

In vivo hydroxylation of the neurotoxin, 1-methyl-4-phenylpyridinium, and the effect of monoamine oxidase inhibitors: electrospray-MS analysis of intra-striatal microdialysates.

The neurotoxin 1-methyl-4-phenylpyridinium (MPP+) has been shown to increase hydroxyl radical formation in the striatum. The production of hydroxyl radicals correlates with the MPP(+)-driven dopamine release which presumably leads to increased metabolism via monoamine oxidase or increased dopamine autoxidation. Both processes result in enhanced production of hydrogen peroxide, which in the presence of iron(II) ions decomposes to the hydroxyl radical.

Isolation of heavy endosomes from dog proximal tubules in suspension.

During the preparation of a suspension of dog kidney proximal tubules by collagenase treatment, an uptake of FITC-albumin was demonstrated. This process is attributed to the activation of receptor-mediated endocytosis leading to the appearance of FITC-albumin into intracellular vesicular structures. The isolation of brush border membrane vesicles (BBMV) from the dog kidney proximal tubules in suspension by the magnesium precipitation technique leads to the copurification of a large population of endosomes.

Functional implications of mutations within polyomavirus large T antigen Rb-binding domain: effects on pRb and p107 binding in vitro and immortalization activity in vivo.

In this study, we have extensively modified the Rb-binding domain of polyomavirus large T antigen. Mutant polyomavirus large T antigens were tested for their ability to bind pRb and p107 in vitro and assayed for their capacity to immortalize primary rat embryo fibroblasts in vivo. Polyomavirus large T antigen bound pRb and p107 through a common region located between amino acids 141 to 158, containing the consensus Rb-binding sequence D/N-L-X-C-X-E.

CD31 (PECAM-1) is a differentiation antigen lost during human CD4 T-cell maturation into Th1 or Th2 effector cells.

The CD31 antigen (PECAM-1) has been reported to be a stable marker for a human CD4 T-cell subpopulation unable to produce interleukin-4 (IL-4). We show here that CD31 expression is not stable inasmuch as CD4 T-cell lines and clones derived from cell-sorted neonatal CD31+ cells lose CD31 upon repetitive cycles of stimulation and IL-2 expansion. Moreover, various cytokines (IL-1 alpha, IL-4, IL-6, transforming growth factor-beta) fail to reinduce CD31 on CD31- clones.

Decreased Fas antigen receptor expression in testicular tumor cell lines derived from polyomavirus large T-antigen transgenic mice.

MT-PVLT-10 transgenic mice express large T-antigen of polyomavirus under the control of the mouse metallothionein-1 promoter and mates of this transgenic line develop testicular tumors at advanced ages. The differential display technique was employed to compare mRNA expression from immortalized cell lines derived from normal or adenomatous testis from MT-PVLT-10 transgenic males. Using this technique, a complementary DNA fragment corresponding to the mouse Fas antigen receptor was recovered from normal testicular cells but not from tumor cells.

Overexpression of her-2/neu in human prostate cancer and benign hyperplasia.

Overexpression of the neu oncoprotein has been described in several tumor models including breast and prostate cancer. Overexpression of neu has been reported to have prognostic significance in certain tumors but controversy continues regarding the role and frequency of neu overexpression in prostatic cancer. The objectives of the study were twofold.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA is constitutively expressed in bovine, human normal, and osteoarthritic articular chondrocytes.

Tissue inhibitors of metalloproteinases (TIMPs) inhibit the extracellular matrix (ECM) metalloproteinases (MMPs). To determine the source of TIMPs in synovial fluids of patients with osteoarthritis (OA), the ability of chondrocytes to express TIMP-2 and its regulation by agents found in inflammed joints was investigated. The constitutive TIMP-2 mRNA expression was demonstrated in chondrocytes from normal bovine, human OA and normal cartilage.

Influence of alkyltransferase activity and chromosomal locus on mutational hotspots in Chinese hamster ovary cells.

High-density mutational spectra have been established for exon 3 of the gene encoding adenine phosphoribosyltransferase (APRT) of the Chinese hamster ovary (CHO) cell line derivative D422 and closely related and/or modified lines by using the mutagen ethyl methanesulfonate (EMS). The total number of selectable sites (GC-->AT transitions yielding a selectable APRT- phenotype) was estimated at 31 based on our own accumulated data base of 136 sequenced exon 3 mutations and on literature reports. D422 and two other APRT hemizygous lines each yielded very similar spectra and showed two populations of mutable sites: (i) 24 "baseline" sites that followed the Poisson distribution and therefore were equally susceptible to mutation and (ii) two hotspots, one comprising a cluster at nucleotides 1293-1309 and the other at nucleotide 1365.

Structure of the gene coding for the mouse TRiC-P5 subunit of the cytosolic chaperonin TRiC.

TRiC is a cytoplasmic chaperonin involved in actin and tubulin folding. It is formed by six to nine different but related proteins of 52 to 65 kDa arranged in two hetero-oligomeric rings. We have cloned the gene coding for the mouse TRiC-P5 subunit (also called CCT gamma) using a XbaI-DraIII fragment of the mTRiC5 cDNA.

Maturation of neonatal human CD4 T cells: III. Role of B7 co-stimulation at priming.

We previously reported that human naive CD4 T cells differentiate into effector cells producing type 1 (IL-2, IFN-gamma) and type 2 (IL-4, IL-5, IL-10) cytokines after priming with anti-CD3 mAb presented on irradiated CD32-transfected mouse L fibroblasts, in the absence of exogenous cytokine. Here we first show that the CD32 L fibroblasts act not only by cross-linking anti-CD3 mAb but also by providing a B7-mediated co-stimulation signal which is required for the activation of naive T cells. Using a selected anti-CD3 mAb (64.

The cytosolic chaperonin subunit TRiC-P5 begins to be expressed at the two-cell stage in mouse embryos.

The cytosolic chaperonin TRiC is a large protein complex involved in the folding of newly synthesized actin and tubulin. The fertilization of the mouse oocyte is followed by a remodelling of the actin and tubulin filaments. The TRiC subunit TCP1 is expressed only from the 4-cell stage on, even though actin and tubulin are synthesized in the previous stages.

Identification and characterization of human mitochondrial methenyltetrahydrofolate synthetase activity.

We present evidence for the presence of the folate metabolism enzyme methenyltetrahydrofolate synthetase (MTHFS) in mitochondria. MTHFS activity was identified in the matrix of mitochondria purified from human liver biopsies. Mitochondrial and cytoplasmic MTHFS specific activities are similar, 85% of the total cellular MTHFS activity is in the cytoplasm and both native enzymes have similar molecular weights (approximately 25 kDa).

Absence of human papillomavirus sequences in ovarian pathologies.

The role of human papillomaviruses (HPVs) in ovarian epithelium (either normal or neoplastic) remains controversial. We have investigated by PCR the presence of HPV DNA in fresh tissue from ovarian neoplasms and in primary cell cultures established from either normal, benign or malignant ovarian surface epithelia. None of the fresh samples and primary ovarian cultures contained HPV DNA sequences.

Lamin A precursor is localized to intranuclear foci.

Lamin A is synthesized in the cytoplasm as a precursor bearing a carboxyl-terminal CaaX box or isoprenylation signal. This precursor is post-translationally processed through multiple steps: isoprenylation with a farnesyl residue on the cysteine of the CaaX box, proteolytic removal of the last three amino acids, carboxymethylation of the cysteine residue and, finally, proteolytic removal of 15 amino acids from the carboxyl terminus. This last step gives rise to mature lamin A from which the isoprenylated terminus has been removed.

Primary cultures of normal and tumoral human ovarian epithelium: a powerful tool for basic molecular studies.

To begin delineating the cellular and molecular events that are important in ovarian carcinogenesis, we have developed a simple and rapid method for the establishment of primary cultures derived from benign tumors, malignant tumors, and ascites of the ovary that are representative of the original clinical material from which they are derived. From 23 ovarian epithelial ascites collected, 13 were successfully established in culture and cells survived an average of 7 to 8 passages. From 65 solid epithelial ovarian tumors (benign and malignant) 36 were cultured for an average of 6 passages for cultures derived from benign tumors and 11 or 12 passages in the case of malignant tumors.

TRiC-P5, a novel TCP1-related protein, is localized in the cytoplasm and in the nuclear matrix.

We have recently reported the cloning of a novel protein, TRiC-P5, with significant homology with protein 1 of the t-complex (TCP1). In the present study, the cellular localization of TRiC-P5 in Raji cells has been determined using an antiserum raised against a 18.5 kDa fusion protein.

Assignment of the human homologue of the mTRiC-P5 gene (TRIC5) to band 1q23 by fluorescence in situ hybridization.

The TCP1 ring complex (TRiC) is a molecular chaperone involved in actin and tubulin folding. Little is known about the components of this complex. The first component identified was TCP1, a protein coded by a gene in the t-complex locus on mouse chromosome 17.

Establishment and characterization of testicular cell lines from MT-PVLT-10 transgenic mice.

Males of the MT-PVLT-10 transgenic mouse line, which expresses the polyomavirus large T-antigen under the control of the metallothionein promoter, develop testicular adenomas and display seminal vesicle enlargement. Histological analysis of adenomatous testis indicates a predominance of Leydig cells, with few normal Sertoli cells or seminiferous tubules remaining. Primary cell cultures established from the testes of control nontransgenic animals (all ages) and young MT-PVLT-10 animals (before the appearance of any phenotype) quickly entered crisis and died.