PubMed Health⌕ Search

Biomedical subjects

J M Chirgwin

Publications and source records attributed to J M Chirgwin.

At least 37 records · Page 2Linked to original sources
PubMed1995

Chinese hamster rhodanese cDNA: activity of the expressed protein is not blocked by a C-terminal extension.

We describe a cDNA from Chinese hamster ovary cells which encodes a protein 91 and 96% identical to bovine and rat mitochondrial rhodaneses, respectively. Recombinant protein was expressed from the cDNA in Escherichia coli, purified to homogeneity, and found to have kinetic properties nearly indistinguishable from those of the bovine enzyme, the only cloned rhodanese previously verified by characterization of the recombinant protein. The carboxyl-terminus of the enzyme is characterized by a duplicated tripeptide, which can be proteolytically processed in vivo. We constructed a mutant in which the last 5 amino acids were replaced by 28 residues of unrelated sequence. This protein was expressed, purified, and found to have kinetic constants similar to those of the wild-type enzyme. The functionally verified Chinese hamster rhodanese cDNA encodes a protein of 297 residues and differs from the rat enzyme at 13 positions. None of these substitutions occurs at residues suggested to play essential roles in catalysis or structural stabilization.

Animals↗
PubMed1995

Role of glycosylation in the expression of human procathepsin D.

Human procathepsin D carries two N-linked glycosylation sites at asparagine residues 70 and 199, widely separated on the surface of the folded protein. We created monoglycosylated procathepsin D molecules by site-directed mutagenesis in vitro of the individual glycosylation sites. With only two exceptions, all 12 mutants of this type were expressed efficiently in mammalian cells. The expressed proteins were stable, targeted to the lysosome, and partially secreted into the medium. When both glycosylation sites were eliminated, however, the expressed proteins (9 different mutants) were stable but most were not secreted and targeted poorly to the lysosome. Mammalian fibroblasts appear to sort nascent procathepsin D efficiently only if it is N-glycosylated. Procathepsin D monoglycosylated at N70 is readily distinguished from the endogenous protein in transfected human cells and thus provides an excellent substrate for studying lysosomal targeting in an homologous system.

Animals↗
PubMed1995

Lysine residues in the C-terminal lobe and lysosomal targeting of procathepsin D.

A major pathway to the lysosome for soluble hydrolases involves the 6-phosphorylation of mannose residues. The initial step in this reaction is catalyzed by a phosphotransferase which recognizes lysosomal precursors. We constructed mutants of human procathepsin D whose targeting to the lysosome could be assayed directly in intact cells. Eight lysine residues were individually converted to glutamic acid on the surface of the carboxyl terminal lobe of the protein. Mutants with as many as four Lys to Glu mutations were normally targeted to the lysosome and processed to the mature form of the enzyme in transfected cells. We conclude that the C-terminal lobe of procathepsin D may not carry a determinant essential for lysosomal targeting in intact fibroblasts.

Animals↗
PubMed1994

Cloning and identification of annexin II as an autocrine/paracrine factor that increases osteoclast formation and bone resorption.

Autocrine products of osteoclasts such as interleukin-6 may play an important role in normal osteoclast formation and activity. To identify novel stimulatory factors for osteoclasts, we have prepared a mammalian cDNA expression library generated from highly purified human osteoclast-like multinucleated cells (MNC) formed in long term bone marrow cultures and screened this library for autocrine factors that enhance MNC formation. A candidate clone which stimulated MNC formation was isolated. Sequence analysis showed that this cDNA encoded annexin II (AXII). Purified recombinant AXII significantly increased MNC formation in human bone marrow cultures in the absence of 1,25-(OH)2 vitamin D3 and enhanced MNC formation in mouse bone marrow cultures treated with 10(-9) M 1,25-(OH)2 vitamin D3. The enhanced MNC formation in murine marrow cultures resulted in increased bone resorption. Treatment of fetal rat long bones with AXII and 1,25-(OH)2 vitamin D3 significantly increased bone resorption compared to 1,25-(OH)2 vitamin D3 alone. Reverse transcriptase polymerase chain reaction analysis demonstrated that AXII mRNA was expressed at high levels in RNA isolated from highly purified giant cells from osteoclastomas, human osteoclast-like MNC, and pagetic bone. Western blot analysis of conditioned media collected from human marrow cultures showed that AXII was present in the media. Furthermore, approximately 50% of total AXII produced by cells transfected with AXII cDNA was present in the conditioned media. These data suggest that the AXII is an autocrine factor that enhances osteoclast formation and bone resorption and demonstrate a previous unknown function for AXII.

Animals↗
PubMed1994

Rapid secretion by a nonclassical pathway of overexpressed mammalian mitochondrial rhodanese.

Rhodanese is a small (33 kDa) monomeric sulfurtransferase which is synthesized on cytoplasmic ribosomes and imported into the mitochondrial matrix without cleavage of its amino terminus. When we transfected mammalian cell lines with rhodanese cDNA under the control of an efficient viral promoter, up to 40% of the overexpressed protein was secreted into the medium. This secretion was not the result of cell lysis, did not occur via the endoplasmic reticulum, and did not require the amino-terminal mitochondrial import signal. Addition of a carboxyl-terminal peptide extension did not block secretion, nor did a number of inhibitors of cellular sorting processes. Rhodanese polypeptide is known to associate with chaperonin proteins. In the absence of available mitochondrial import sites, such a complex in the cytoplasm of transfected cells could deliver unfolded rhodanese to export pores on the inner surface of the plasma membrane. This mechanism could contribute to the nonclassical secretion of cytoplasmically synthesized interleukins, growth factors, and lectins.

Amino Acid Sequence↗
PubMed1994

Mutations of noncatalytic sulfhydryl groups influence the stability, folding, and oxidative susceptibility of rhodanese.

Mutants of rhodanese (EC 2.8.1.1) which substitute serine residues for each of the 4 cysteine residues have been studied to determine the roles of cysteines in the structure and function of the enzyme. The proteins compared in these studies were: the wild-type, C63S, C247S, C254S, C263S, C254S/C263S, and C63S/C254S/C263S. These current studies show that cysteine 247 is the only cysteine required for the activity of the enzyme. Although the other sulfhydryl groups do not participate in sulfur transfer, mutations of the noncatalytic cysteines result in the destabilization of the native structure of the enzyme. All the active proteins had similar kinetic parameters. Mutants substituting cysteine 254, compared with the other species, were: (a) more resistant than wild-type to inactivation by dithiothreitol, (b) more readily reactivated following oxidative inactivation, and (c) found to adopt conformations that show increased exposure of hydrophobic surfaces following removal of the transferable sulfur. On the other hand, cysteine to serine substitutions had very little effect on: (a) the rates of oxidative inactivation, (b) the increased fluorescence following the removal of transferable sulfur, or (c) the effectiveness of spontaneous refolding after urea denaturation. Forms of rhodanese that were formerly considered to be irreversibly oxidized can be reactivated if the protein is denatured in urea before reductants are used. It is proposed that these forms differ from reversibly oxidized states due to the inaccessibility of intramolecular disulfides to reductants and not to the formation of higher oxidation states of the protein.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of the 5'-flanking region of the mouse tartrate-resistant acid phosphatase gene.

Little information is available on the molecular mechanisms controlling osteoclastic bone resorption. We used tartrate-resistant acid phosphatase (TRAP) to begin to investigate the regulation of bone resorption at the molecular level. TRAP is expressed at high levels in osteoclasts and may play an important role in the bone resorptive process. Therefore, we isolated the murine TRAP gene from a mouse spleen genomic library and characterized its promoter. A restriction map was generated for the 17 kb TRAP insert. A 2 kb SmaI fragment, containing the 5'-flanking region, was subcloned and the nucleotide sequence determined. Sequence analysis of the SmaI fragment revealed the presence of numerous candidate transcription factor binding sequences, including those for AP1 and H-APF-1. The H-APF-1 site matches the consensus sequence for the IL-6-regulated transcription factor. An intron was identified at -1 to -393 bp relative to the ATG. The presence of an intron was confirmed by PCR analysis of RNA isolated from murine osteoclasts. Primer extension analysis indicated the presence of a transcription initiation site at -552 bp from the ATG. The region from -1846 to 2bp relative to the ATG initiation codon drove the transient expression of a luciferase reporter gene when transfected into HRE H9 rabbit endometrial cells. PMA treatment of HRE H9 cells enhanced luciferase transcription approximately threefold. These data suggest that the TRAP promoter is complex and contains multiple regulatory elements. The availability of the TRAP promoter may also permit production of transgenic mice, which can be used to develop previously unavailable osteoclast cell lines.

Acid Phosphatase↗
PubMed1993

Tartrate-resistant acid phosphatase gene expression as a facile reporter gene for screening transfection efficiency in mammalian cell cultures.

The efficiency of DNA transfection into mammalian cell cultures has been monitored using a variety of reporter assays. However, the common procedures are expensive, time-consuming and usually cannot identify the transfected cell population directly. In the present communication we describe a simple, inexpensive and efficient method to directly identify DNA transfection in mammalian cells using tartrate-resistant acid phosphatase (TRAP) gene expression. The method involves the transfection of a plasmid (pCT3), which contains TRAP cDNA driven by a CMV promoter, into mammalian cells. The cells can then be stained for TRAP activity, and the transfection efficiency can be determined by simply counting the positively transfected cells in a defined area with a microscope. This method permits screening of mammalian cells for transfection efficiency in multi-well plates. After waiting 30-40 minutes to allow the TRAP assay to saturate, wells can be scored in 1-2 minutes with little difficulty in detecting the transfected cells.

Acid Phosphatase↗
PubMed1992

Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese.

Recombinant bovine rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been purified to homogeneity from Escherichia coli BL21(DE3) by cation-exchange chromatography. Recombinant and bovine liver rhodanese coelectrophorese under denaturing conditions, with an apparent subunit molecular weight of 33,000. The amino terminal seven residues of the recombinant protein are identical to those of the bovine enzyme, indicating that E. coli also removes the N-terminal methionine. The Km for thiosulfate is the same for the two proteins. The specific activity of the recombinant enzyme is 12% higher (816 IU/mg) than that of the bovine enzyme (730 IU/mg). The two proteins are indistinguishable as to their ultraviolet absorbance and their intrinsic fluorescence. The ability of the two proteins to refold from 8 M urea to enzymatically active species was similar both for unassisted refolding, and when folding was assisted either by the detergent, lauryl maltoside or by the E. coli chaperonin system composed of cpn60 and cpn10. Bovine rhodanese is known to have multiple electrophoretic forms under native conditions. In contrast, the recombinant protein has only one form, which comigrates with the least negatively charged of the bovine liver isoforms. This is consistent with the retention of the carboxy terminal residues in the recombinant protein that are frequently removed from the bovine liver protein.

Animals↗
PubMed1992

Chinese hamster ovarian cells transfected with human parathyroid hormone-related protein cDNA cause hypercalcemia in nude mice.

BACKGROUND: Parathyroid hormone-related protein (PTH-rP) has been implicated as a causative factor in the humoral hypercalcemia of malignancy. Animal models of hypercalcemia of malignancy that have traditionally utilized human or animal tumors or injections or infusions of hypercalcemic factors have limitations that may prevent exact delineation of the biologic effects of tumor-produced PTH-rP. EXPERIMENTAL DESIGN: To assess the effects of tumor-produced PTH-rP in vivo, we have transfected Chinese hamster ovarian (CHO) cells with cDNA encoding human preproPTH-rP-(1-141) which then stably express only PTH-rP. We inoculated these tumor cells into nude mice, and compared tumor-bearing nude mice with similar tumor-bearing nude mice inoculated with non-transfected CHO cells using standard parameters of calcium homeostasis. RESULTS: The nude mice carrying tumors expressing PTH-rP became hypercalcemic over 12 to 18 days. Blood ionized calcium values correlated positively with plasma PTH-rP concentration and tumor volume. Plasma PTH-rP concentrations in hypercalcemic mice were similar to those reported in humans with hypercalcemia of malignancy. Bone histology and histomorphometry from hypercalcemic nude mice demonstrated increased bone resorption when compared with mice bearing nontransfected CHO tumors. CONCLUSIONS: We have produced an animal model of tumor-produced PTH-rP by transfecting CHO cells with the cDNA for PTH-rP and inoculating these tumor cells into nude mice. Hypercalcemia in this model is mediated in part by the effects of PTH-rP to increase osteoclastic bone resorption. The model is advantageous because PTH-rP alone is secreted in a prolonged, constitutive fashion with pharmacokinetics similar to naturally occurring tumors. It should prove to be a useful model to study the effects of tumor-produced PTH-rP in vivo.

Animals↗
PubMed1991

Expression of cloned bovine adrenal rhodanese.

A cDNA for the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) has been cloned from a bovine adrenal library. An initiator methionine codon precedes the amino-terminal amino acid found in the isolated protein. Rhodanese is synthesized in the cytoplasm and transferred to the mitochondrial matrix. Thus, any amino-terminal sequence required for organelle import is retained in the mature protein. Furthermore, the DNA sequence shows that there are three additional amino acids, Gly-Lys-Ala, at the carboxyl terminus that are not found by protein sequencing. Additionally, comparison of the published amino acid sequence with that encoded by the open reading frame revealed three differences in the amino acid sequence. Comparison of the bovine and chicken liver sequences shows an overall level of 70% sequence homology, but there is complete identity of all residues that have been implicated in the function of the enzyme. When two mammalian cells, cos-7 and 293 cells, were transiently transfected with a plasmid containing the rhodanese coding region, rhodanese activity in lysates increased approximately 20-fold. Fluorograms of denaturing polyacrylamide gels detected a large increase in a polypeptide that co-migrated with the native protein and reacted with anti-rhodanese antibodies. Nondenaturing gels showed two active species that co-migrated with the two major electrophoretic forms purified by current procedures. Escherichia coli, transformed with a plasmid containing the rhodanese coding region, showed a 15-fold increase in rhodanese activity over baseline values. When the E. coli recombinant protein was analyzed on a nondenaturing gel, only one species was observed that co-electrophoresed with the more electropositive variant seen in purified bovine liver rhodanese. This single variant could be converted by carboxypeptidase B digestion to a form of the enzyme that co-migrated with the more electronegative species isolated from bovine liver. Thus, two major, enzymatically active electrophoretic variants, commonly observed in mammalian cells, can be accounted for by carboxyl-terminal processing without recourse to other post-translational modifications.

Adrenal Glands↗
PubMed1990

Cathepsin D and prognosis in breast cancer.

We investigated the possibility that cathepsin D, an estrogen-induced lysosomal protease, might have value as a prognostic factor in breast cancer by studying frozen tissue specimens from 397 patients. We measured the 34-kd mature form of the enzyme by Western blot assay and densitometry. Among 199 patients with node-negative disease, but not among 198 with node-positive disease, high levels of cathepsin D proved to be a significant predictor of reduced disease-free survival (median follow-up, 64 months), either as a continuous variable (log cathepsin D; P = 0.018) or as a dichotomous variable with an optimized cutoff point (P = 0.0001). Results were similar for overall survival (P = 0.009 and 0.0001, respectively). Relating the level of cathepsin D to other prognostic factors in the patients with node-negative disease, we found an association with aneuploidy but none with estrogen or progesterone receptors, tumor size, or the age of the patient. In multivariate analyses, a high level of cathepsin D was the most important independent factor in predicting shorter disease-free and overall survival in patients with node-negative disease. As compared with the risk in women with low levels of cathepsin D, the relative risk of tumor recurrence was 2.6 (95 percent confidence interval, 1.6 to 4.4) and the relative risk of death was 3.9 (95 percent confidence interval, 2.1 to 7.3) among those with high levels of cathepsin D. For disease-free survival, cathepsin D status was predictive of outcome primarily among those with aneuploid tumors; the actuarial five-year recurrence rates of aneuploid tumors were 60 percent among women with high levels of cathepsin D and 29 percent among those with low levels, as compared with 22 percent for all diploid tumors. We conclude that cathepsin D may be an independent predictor of early recurrence and death in node-negative breast cancer.

Biomarkers, Tumor↗
PubMed1990

Molecular biology for nonmolecular biologists.

Molecular biology offers various powerful tools for understanding the etiology of diabetes mellitus. These methods can be understood by nonmolecular biologists. Topics briefly introduced include 1) control of gene expression, 2) cloning of genes, 3) how gene promoters work, 4) transgenic mice, 5) mutagenesis in vitro, 6) human molecular genetics, and 7) the new powerful polymerase chain-reaction technique, which greatly simplifies many of the other methods.

Animals↗
PubMed1989

Proinsulin I and II gene expression in inbred mouse strains.

Mice and rats express two nonidentical insulins from a pair of unlinked genes. We have applied a nuclease protection assay, which can sensitively quantify each of the mouse insulin mRNAs, to the resolution of the following questions concerning their expression. First, it has not been established whether alterations in expression of one or both of these genes cause differing total insulin biosynthetic capacity noted between several inbred mouse strains. These studies showed that the relative abundance of mRNAs encoding mouse insulins I and II was identical in four separate mouse strains. In spontaneously obese, hyperinsulinemic (db/db)C57BL/KsJ mice, both proinsulin I and proinsulin II mRNAs were increased relative to the levels in normal (+/db) C57BL/KsJ mice, but again the ratio of the two mRNAs did not differ. The ratio was nearly identical to that for the orthologous mRNAs in rats, indicating that the mechanisms which regulate insulin mRNAs in rodents are conserved in both genes in several mouse strains and between rodent species. This finding suggests that differences between mouse strains in insulin biosynthetic capacity result from differences in the glucose sensing/signalling mechanism at a point before coordinate gene transcription. Second, low levels of insulin synthesis have been suggested as an explanation for relatively high levels of insulin in several nonpancreatic tissues. We showed that the ribonuclease protection assay, sufficiently sensitive to measure 1/2000th the amount of insulin mRNA present in pancreas, was unable to detect insulin mRNA in salivary gland. This result indicates that the high levels of radioimmunoassayable insulin detected in salivary glands are not the result of insulin synthesis in situ.

Animals↗
PubMed1987

46 kd mannose 6-phosphate receptor: cloning, expression, and homology to the 215 kd mannose 6-phosphate receptor.

We have isolated cDNA clones encoding the entire sequence of the bovine 46 kd cation-dependent mannose 6-phosphate (CD Man-6-P) receptor. Translation of CD Man-6-P receptor mRNA in Xenopus laevis oocytes results in a protein that binds specifically to phosphomannan-Sepharose, thus demonstrating that our cDNA clones encode a functional receptor. The deduced 279 amino acid sequence reveals a single polypeptide chain that contains a putative signal sequence and a transmembrane domain. Trypsin digestion of microsomal membranes containing the receptor and the location of the five potential N-linked glycosylation sites indicate that the receptor is a transmembrane protein with an extracytoplasmic amino terminus. This extracytoplasmic domain is homologous to the approximately 145 amino acid long repeating domains present in the 215 kd cation-independent Man-6-P receptor.

Amino Acid Sequence↗