Characterization and regulation of the human ML1A melatonin receptor stably expressed in Chinese hamster ovary cells

Transforming growth factor-beta1 (TGF-beta1) inhibits theca-interstitial cell (TIC) androgen biosynthesis while enhancing progesterone production without altering P45017 alpha protein content. The purpose of the present study was to define the mechanism of TGF-beta 1 inhibition of ovarian androgen production by determining the effects of TGF-beta 1 on steroidogenic enzyme messenger RNA (mRNA) expression and 17 alpha-hydroxylase activity in TIC in vitro. TIC isolated from hypophysectomized immature rat ovaries by Percoll gradient centrifugation were cultured with and without LH and TGF-beta 1 up to 6 days. At various times, cytoplasmic mRNA was extracted from the TIC, and P450scc, 3 beta-HSD and P450(17 alpha) mRNA were measured by specific assays, using RT-PCR. Treatment with TGF-beta 1 alone (0.1-100 ng/ml) had no effect on mRNA expression at 2 days but increased P450scc and 3 beta-HDS mRNA at 4 days. TGF-beta did not alter the LH stimulation of P450scc and 3 beta-HSD mRNA up to 6 days but caused a modest (2.5-fold) increase in P450 (17 alpha) mRNA at 2 days. Specificity studies with inhibin-A (30 ng/ml), activin-A (100 ng/ml), and MIS (300 ng/ml) demonstrated that the effects of TGF-beta 1 were unique within this family of peptides. We next examined the effect of TGF-beta 1 on 17 alpha-hydroxylase activity. Kinetic analysis revealed that the 17 alpha-hydroxylase enzyme has an apparent Michaelis-Menten constant of 3.42 mumol/liter and maximum velocity of 0.23 pmol/min x mg protein. TGF-beta 1 inhibited 17 alpha-hydroxylase activity by a noncompetitive mechanism with an apparent inhibin constant (Ki) of 46.4 pM. The results of our studies demonstrate that TGF-beta 1 directly inhibits TIC androgen production by a noncompetitive mechanism. This novel mechanism may be important in preventing excessive androgen production in developing ovarian follicles without preventing differentiation of the TIC.     

Pharmacological characterization of the human vasopressin receptor subtypes stably expressed in Chinese hamster ovary cells

Three subtypes of human (h) arginine vasopressin (AVP) receptors, hV1A, hV1B and hV2, were stably expressed in Chinese hamster ovary (CHO) cells and characterized by [3H]-AVP binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. Scatchard analysis of saturation isotherms for the specific binding of [3H]-AVP to membranes, prepared from CHO cells transfected with hV1A, hV1B and hV2 receptors, yielded an apparent equilibrium dissociation constant (Kd) of 0.39, 0.25 and 1.21 nM and a maximum receptor density (Bmax) of 1580 fmol mg(-1) protein, 5230 fmol mg(-1) protein and 7020 fmol mg(-1) protein, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Pharmacological characterization of the transfected human AVP receptors was undertaken by measuring the relative ability of nonpeptide AVP receptor antagonists, YM087, OPC-21268, OPC-31260, SR 49059 and SR 121463A, to inhibit binding of [3H]-AVP. At hV1A receptors, the relative order of potency was SR49059>YM087>OPC-31260>SR 121463A> >OPC-21268 and at hV2 receptors, YM087=SR 121463A>OPC-31260>SR 49059> >OPC-21268. In contrast, the relative order of potency, at hV1B receptors, was SR 49059> >SR 121463A=YM087=OPC-31260=OPC-21268. In CHO cells expressing either hV1A or hV1B receptors, AVP caused a concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) with an EC50 value of 1.13 nM and 0.90 nM, respectively. In contrast, stimulation of CHO cells expressing hV2 receptors resulted in an accumulation of cyclic AMP with an EC50 value of 2.22 nM. The potency order of antagonists in inhibiting AVP-induced [Ca2+]i or cyclic AMP response was similar to that observed in radioligand binding assays. In conclusion, we have characterized the pharmacology of human cloned V1A, V1B and V2 receptors and used these to determine the affinity, selectivity and potency of nonpeptide AVP receptor antagonists. Thus they may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of AVP.     

Activation of phospholipase A2 by the nociceptin receptor expressed in Chinese hamster ovary cells

To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A2 (PLA2) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time- and dose-dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti-Ca2+-dependent cytosolic PLA2 (cPLA2) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time- and dose-dependent electrophoretic mobility shift of cPLA2, suggesting that phosphorylation of cPLA2 is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin-induced cPLA2 phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA2 through the action of PTX-sensitive G proteins and suggest that cPLA2 is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen-activated protein kinases.     

Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells

The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.     

Antiapoptotic signaling by the insulin receptor in Chinese hamster ovary cells

We have sought to determine whether insulin can promote cell survival and protect Chinese hamster ovary (CHO) cells from apoptosis induced by serum starvation. Low concentrations of insulin were antiapoptotic for cells overexpressing wild-type insulin receptors but not in cells transfected with kinase-defective insulin receptor mutants that lacked a functional ATP binding site. However, treatment with orthovanadate (50 microM), a widely used tyrosine phosphatase inhibitor, led a dramatic reduction in internucleosomal DNA fragmentation in both cell lines. Cells transfected with truncated receptor mutants in either the juxtamembrane or C-terminal domain were as responsive as cells overexpressing wild-type receptors in mediating insulin antiapoptotic protection. The mechanisms underlying insulin antiapoptotic protection were investigated using a variety of pharmacological tools known to inhibit distinct signaling pathways. The phosphatidylinositol-3' kinase inhibitors wortmannin and LY294002 had only a modest influence whereas blocking protein farnesylation with manumycin severely disrupted the antiapoptotic capacity of the insulin receptor. Of interest, cells gained antiapoptotic potential following inhibition of extracellular signal-regulated kinase activation with the pharmacological agent PD98059. Insulin induced MKK3/MKK6 phosphorylation and activation of p38 MAP kinase whose activity was inhibited with SB203580. However, the inhibition of p38 MAP kinase had no effect on the protection offered by insulin. We conclude that the antiapoptotic function of the insulin receptor requires intact receptor kinase activity and implicates a farnesylation-dependent pathway. Increase in cellular phosphotyrosine content, however, triggers antiapoptotic signal that may converge downstream of the insulin receptor.     

Functions of the N-glycans of rat leukemia inhibitory factor expressed in Chinese hamster ovary cells

A non-covalently coimmobilized bienzymic reactor of horseradish peroxidase (HRP) and cholesterol oxidase (COD), operating in a continuous organic flowing stream of 1X10-3 M p-anisidine in buffer-saturated (pH 7.0) toluene, has been employed for cholesterol determination in animal greases, such as pig, beef, and chicken fat, and codfish liver oil. The method provides a good linear relationship up to 1.8 X 10-3 M cholesterol and average recoveries of 99.5%, a high sensitivity, with a detection limit of 1 X 10-6 M of cholesterol and a good precision (an interday RSD of 1.8% for the determination of total cholesterol in a codfish oil sample). The method permits the direct spectrophotometric determination of free cholesterol present in animal grease samples without any pre-treatment, and the total cholesterol determination after a microwave-assisted saponification. The bienzymic reactor exhibits a good stability in the water-restricted environment, being possible to perform more than 180 analyses during a period of 10d with 1mg of HRP, and 1 mg of COD, equivalent to 220 purpurogallin units and 23 U, respectively.     

Biochemical characterization of the O-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells

Responses of individuals to the loss of a primary attachment object may be quite variable. In humans, it has been suggested that only about 25% of bereavements result in substantial psychological or medical morbidity (Hamburg et al. 1975). In nonhuman primates, which are used to model responses to separation and loss, a similar estimate of about 25% has also been obtained (McKinney 1985). In addition, there are wide-ranging species differences in vulnerability with regard to the nature and severity of the response to maternal separation and/or loss. All of these findings suggest that there are important processes, intrinsic and/or extrinsic to the individual, that contribute to the probability that a loss will produce a major behavioral or physiological response. We have been systematically examining some of the factors that may account for a portion of this variability in two species of macaques (bonnet monkeys Macaca radiata; and pigtail monkeys, M. nemestrina).     

Apoptosis in batch cultures of Chinese hamster ovary cells

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the extent of death in these cultures, we first tried to determine the mode of death. We found that more than 80% of the cells in a standard serum-free batch culture of CHO cells in suspension died via apoptosis--as evidenced by condensed chromatin and the appearance of a characteristic DNA ladder. Furthermore, when protein synthesis was inhibited using cycloheximide, the cells underwent rapid apoptosis indicating that death proteins were present in greater abundance than survival proteins in our CHO cells. Cell lysate from CHO cells showed evidence of cysteine protease (caspase) activity. Caspases of the Interleukin-1-beta-Converting Enzyme (ICE) family, e.g., CPP32, Mch-1, etc., have been implicated in the apoptotic process. Surprisingly, a caspase peptide inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoro-methyl-ketone (z-VAD.fmk), was unable to substantially extend the life of a serum-free batch culture of CHO cells. In addition, z-VAD.fmk was only marginally able to extend viability in response to withdrawal of growth and survival factors, insulin and transferrin. In both these instances, z-VAD.fmk was able to prevent cleavage of caspase substrates, but not protect cells from death. However, we found that bcl-2 expression was able to significantly extend viabilities in CHO batch culture. Bcl-2 expression also substantially extended the viability of cultures in response to insulin and transferrin withdrawal. These results provide interesting insights into the pathways of death in a CHO cell.     

Pharmacological properties of acquired excitotoxicity in Chinese hamster ovary cells transfected with N-methyl-D-aspartate receptor subunits

The cytotoxicity induced by the transient expression of functional N-methyl-D-aspartate (NMDA) receptors has been examined with the use of a luciferase reporter assay in Chinese hamster ovary cells. Various NMDA receptor antagonists, in a dose-dependent manner, prevented a loss of luciferase activity 24 to 48 hr post-transfection of either the NR1/NR2A or NR1/ NR2B subunit receptor configurations, likely correlating to the time required to express functionally these receptors. Both glutamate and NMDA were potently cytotoxic to transfected cells previously protected by antagonists. The novel ifenprodil analog (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP101,606-27) protected cells expressing NR1/NR2B, but not those cells expressing either NR1/NR2A or, putatively, NR1/NR2A/NR2B. Decreased cytotoxicity was observed when a mutated NR1 subunit (N616R) with reduced Ca++ permeability was used in coexpression studies with NR2A or NR2B. In contrast to our results with NR1/NR2A or NR1/NR2B, cells expressing NR1/NR2C did not perish. Our studies suggest that expression of functional NMDA receptors in non-neuronal cells leads to a form of excitotoxicity similar to that observed in mammalian neurons in vitro.     

Proton and chloride currents in Chinese hamster ovary cells

Despite being one of the most frequent neoplasms occurring in the endocrine system, thyroid carcinoma is, nevertheless, a relatively rare event (0.5-1.5% of all malignant tumours in man); the differentiated forms are the most prevalent and are characterized by a high mean survival rate, whereas the very aggressive forms are rare and prognosis is unfavourable. Diagnostic evaluation of carcinomatous lesions, particularly in the early stages, may give rise to considerable difficulties at a clinical level due to the differentiation of the benign lesions, which are a frequent finding. The traditional clinico-semeiological and instrumental parameters, which, in the past, were used in the assessment of suspected malignancy, should not be considered as markers of malignancy; however, exposure to ionizing radiations during childhood may have a well defined role of risk. Following the recent progress in genetic and molecular studies, it is now possible to exploit genetic-molecular tumor markers and, at present, thyroid medullary carcinoma may be identified also in the absence of clinical evidence, particularly the familial form, thus allowing suitable prophylaxis in those subjects with specific genetic impairment (e.g. preventive thyroidectomy in infancy). Since no discriminating clinico-semeiological parameters are available, considering the aspecificity of scintigraphic findings and the lack of reliability of echographic imaging in providing data which enable us to distinguish a rare neoplastic pattern from the more frequent finding of a benign thyroid mass, fine-needle aspiration (FNA) cytology may today be considered the technique of choice in the screening of the thyroid nodule. Our experience in over 12,000 nodular lesions since 1982, has confirmed that the cytological examination is the most discriminating investigation, diagnostic reliability being far greater than that of traditional techniques. Considering the high frequency of thyroid nodule disease which rarely harbours a carcinomatous lesion, a very scrupulous diagnostic algorithm is mandatory. The FNA cytology, together with morphofunctional and immunological examinations, as well as dynamic exploration of the thyroid hypothalamo-pituitary axis, which allows a nosographic picture of the thyroid nodule disease, provides a more discriminating appraisal for the surgical approach to a single, solitary or prominent nodule.     

Shrinkage-induced protein tyrosine phosphorylation in Chinese hamster ovary cells

To investigate the signal transduction of osmotic stress, we examined hypertonicity-induced tyrosine phosphorylations in Chinese hamster ovary cells. Hyperosmosis elicited characteristic phosphotyrosine accumulation in at least 3 proteins (approximately 42, approximately 85, and approximately 120 kDa). The most prominent response occurred in the 85-kDa band (p85) whose phosphorylation was rapid, sustained, apparent already at mild hypertonicity (350 mosM), proportional to the extracellular osmotic concentration, and reversible. Hyperosmotic environment could not induce tyrosine phosphorylation if cell shrinkage was prevented by nystatin and appropriately composed media. Conversely, isotonic shrinkage caused strong tyrosine phosphorylation. Thus, the initial signal is a decrease in cell volume and not an increase in the intra- or extracellular osmotic concentration, or a rise in cytosolic K+ and Cl- levels. Tyrosine phosphorylation of p85 was not due to the hypertonicity-induced protein kinase C-dependent stimulation of the extracellular signal-regulated protein kinase, nor to the activation of stress-activated protein kinases. Tonicity-responsive proteins interacted with Grb2-glutathione S-transferase fusion proteins: the 120-kDa protein complexed with the SH2 and both SH3 domains, whereas p85 associated with the SH2 and the N-terminal SH3 domains of the adapter. Tyrosine phosphorylation of p85 is a sensitive indicator of reduced intracellular hydration and might signify a hitherto unrecognized, early volume-dependent signaling event.     

In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells

Coagulation factor IX is a serine protease for which high-level expression of biologically active protein in heterologous cells is limited due to inefficient proteolytic removal of the propeptide as well as vitamin K-dependent carboxylation of multiple amino-terminal glutamic acid residues. We have overexpressed the vitamin K-dependent gamma-carboxylase cDNA and monitored its ability to improve factor IX processing in Chinese hamster ovary (CHO) cells. From amino acid sequence analysis of bovine liver vitamin K-dependent gamma-carboxylase, degenerate oligonucleotides were used to isolate a 3.5-kbp bovine cDNA that encoded a 758-residue open reading frame. Expression of the cDNA in COS-1 and CHO cells yielded 17- and 16-fold increases in the in vitro gamma-carboxylase activity of microsomal preparations, respectively. Anti-serum raised against a predicted peptide sequence reacted with a 94-kDa polypeptide in the partially purified bovine liver preparation as well as in stably transfected CHO cells. The amount of antibody reactivity correlated with the increased ability to carboxylate a peptide substrate in vitro. These results strongly support the conclusion that the cDNA encodes the vitamin K-dependent gamma-carboxylase. Transient transfection of the gamma-carboxylase expression vector into factor IX-expressing CHO cells did not improve the specific procoagulant activity of secreted factor IX. In contrast, transfection of an expression vector encoding the propeptide processing enzyme PACE (paired basic amino acid cleaving enzyme) did improve the specific activity of secreted factor IX by 3-fold. These results demonstrate that the ability of CHO cells to modify glutamic acid residues to gamma-carboxyglutamic acid in secreted factor IX is not limited by the expression of the vitamin K-dependent gamma-carboxylase alone.     

Characterization of a diamine exporter in Chinese hamster ovary cells and identification of specific polyamine substrates

Export of the diamine putrescine was studied using inside-out plasma membrane vesicles prepared from Chinese hamster cells. Putrescine uptake into vesicles was a saturable and an ATP- and antizyme-independent process. Excess amounts of a series of diamines or monoacetyl spermidine, but not monoacetyl putrescine, spermidine, or spermine, inhibited putrescine transport. Putrescine uptake into vesicles prepared at pH 7.4 was suppressed at pH 5, compared with pH 7.4; was stimulated approximately 2.5-fold at pH 7.4 in vesicles prepared at pH 6.25, compared with vesicles prepared at pH 7.4; and was not inhibited by valinomycin in the presence of potassium ions. Reserpine and verapamil blocked [3H]putrescine uptake into inverted vesicles. Verapamil treatment caused an increase in intracellular contents of putrescine, cadaverine, and N8-acetylspermidine, in unstressed proliferating cells, or of N1-acetylspermidine, in cells subjected to heat shock to induce acetylation of spermidine at N1. These data indicate that putrescine export in Chinese hamster cells is mediated by a non-electrogenic antiporter capable of using protons as the counter ion. Physiological substrates for this exporter include putrescine, cadaverine, and monoacetyl spermidine and have the general structure NH3+-(CH2)n-NH2 + R at acidic or neutral pH.     

Modulation of membrane drug permeability in Chinese hamster ovary cells

The kinetics of colchicine uptake into Chinese hamster ovary cells have been investigated and found to be consistent with an unmediated diffusion mode. A variety of compounds such as local anesthetics and non-ionic detergents as well as drugs such as vinblastine, vincristine, daunomycin and actinomycin D potentiate the rate of colchicine uptake into these cells and into colchicine resistant mutants. In all cases, higher concentrations of these compounds were required to stimulate colchicine uptake in the colchicine resistant mutants than in the cells of the parental line. This stimulation was observed also in the uptake of puromycin, a structurally and functionally different drug. These stimulatory agents did not, however, cause the cells to become nonspecifically leaky since the uptake of 2-deoxy-D-glucose was unaffected. In addition, the activation energy of colchicine uptake was unaltered in the presence of stimulating agents, implying that they were not causing colchicine to enter the cells via a different mechanism. The results are compatible with the view that these compounds are membrane-active, and are able to stimulate an increased rate of unmediated diffusion of colchicine into the cells. It appears that a mechanism for the regulation of passive permeability is modified in the resistant mutants.     

Flow cytometric analyses of antibody binding to Chinese hamster ovary cells expressing human thyrotropin receptor

To develop a method that can be used to directly detect binding of antibodies to TSH receptor (TSHr), we employed Chinese hamster ovary (CHO) cells permanently transfected with a human TSHr complementary DNA (CHOR). These cells showed increased cAMP production when treated with either human TSH or thyroid-stimulating antibodies and decreased TSH-mediated cAMP production when treated with stimulation-blocking antibodies. We employed flow cytometry and rabbit antibodies against the extracellular domain of the TSHr (ETSHr) to test whether these cells can be used to directly detect and quantitate the binding of anti-TSHr antibodies. Rabbit anti-ETSHr bound specifically to CHOR cells, and the binding could be blocked with purified ETSHr. To test the feasibility of using these cells for epitope mapping, we tested the binding of rabbit antibodies raised against several synthetic TSHr peptides. Rabbit antipeptide 92 (amino acids 12-30) and 91 (amino acids 32-46) showed little or no binding to the CHOR cells. In contrast, antibodies raised against peptides 93 (amino acids 316-330), 95 (aa 325-345), 3A (aa 357-372), 367 (aa 367-386), and 1B (aa 362-376) showed significant binding to the CHOR cells. The specificity of binding of antipeptide antibodies was demonstrated by a complete inhibition of binding by corresponding peptides. When TSH-binding inhibitory Ig-positive sera from 15 patients with hyperthyroidism were tested, 8 of them showed specific binding to the CHOR cells compared to their relative binding to normal CHO cells; sera from all normal individuals tested did not exhibit specific binding to CHOR cells. These studies showed the usefulness of CHOR cells and flow cytometry in epitope mapping using sera with known specificities and the potential usefulness of the technique to detect anti-TSHr antibodies in patient sera.     

Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells

Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fc alpha receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.     

Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells

Thirty-six isolates, from man or swine, of Pasteurella multocida subsp. multocida producing (n = 13) or not producing (n = 23) the dermonecrotic toxin (DNT) were studied by numerical analysis, capsular typing and ribotyping. Toxigenic strains were also characterised by restriction fragment length polymorphism (RFLP) of the toxA gene and pulsed-field gel electrophoresis (PFGE). Numerical analysis differentiated the Pasteurella species and subspecies, but did not discriminate between toxigenic and nontoxigenic strains. RFLP demonstrated that toxA was located in a conserved part of the chromosome of all toxigenic strains. Ribotyping provided evidence of a close association between DNT production and one of the six EcoRI ribotypes designated as E2. In contrast, PFGE provided evidence for significant DNA polymorphism amongst the toxigenic strains. Results of phenotypic and genotypic studies suggested that toxigenic strains do not form a clone within the subspecies multocida. No difference was found between toxigenic strains of porcine or human origin by biochemical characterisation, capsular serotyping or genomic typing methods.     

Ca2+ triggers massive exocytosis in Chinese hamster ovary cells

We have tracked the cell surface area of CHO cells by measuring the membrane capacitance, Cm. An increase in cytosolic [Ca2+], [Ca2+]i, increased the cell surface area by 20-30%. At micromolar [Ca2+]i the increase occurred in minutes, while at 20 microM or higher [Ca2+]i it occurred in seconds and was transient. GTPgammaS caused a 3% increase even at 0.1 microM [Ca2+]i. We conclude that CHO cells, previously thought capable only of constitutive exocytosis, can perform Ca2+-triggered exocytosis that is both massive and rapid. Ca2+-triggered exocytosis was also observed in 3T3 fibroblasts. Our findings add evidence to the view that Ca induces exocytosis in cells other than known secretory cells.     

Inhibin interferes with activin signaling at the level of the activin receptor complex in Chinese hamster ovary cells

To gain more insight in the mechanism of action of inhibin, we studied the effect of inhibin on activin signaling in Chinese hamster ovary cells. Inhibin specifically counteracted activin-induced expression of a plasminogen activator inhibitor 1 promoter element (3TP) and of the junB gene, but was ineffective when the responses were induced by transforming growth factor-beta. This indicates that inhibin acts only on the activin-specific part of these signaling cascades. Using a constitutively active activin type IB receptor we determined whether inhibin acted at the level of the activin-receptor complex or downstream of it. The mutant activin receptor stimulated the expression of the 3TP promoter in the absence of activin. This stimulation was insensitive to inhibin, indicating that inhibin acts exclusively at or upstream of this activin type I receptor. In addition, competition studies using labeled activin showed that inhibin displaced activin from the activin type II receptors, especially from the activin type IIB receptor, but not from the type I receptors. In conclusion, these data show that in Chinese hamster ovary cells inhibin acts directly at the activin receptor complex, most likely through displacement of activin from the activin type II receptor.     

Evidence of high levels of methylglyoxal in cultured Chinese hamster ovary cells

Methylglyoxal is an alpha-ketoaldehyde and dicarbonyl formed in cells as a side product of normal metabolism. Endogenously produced dicarbonyls, such as methylglyoxal, are involved in numerous pathogenic processes in vivo, including carcinogenesis and advanced glycation end-product formation; advanced glycation end-products are contributors to the pathophysiology of aging and chronic diabetes. Despite recent advances in understanding of the systemic effects of methylglyoxal, the full significance of this compound remains unknown. Herein we provide evidence that the majority of the methylglyoxal present in vivo is bound to biological ligands. The basis for our finding is an experimental approach that provides a measure of the bound methylglyoxal present in living systems, in this instance Chinese hamster ovary cells; with our approach, as much as 310 microM methylglyoxal was detected, 100- to 1,000-fold more than observed previously in biological systems. Several artifacts were considered before concluding that the methylglyoxal was associated with cellular structures, including phosphate elimination from triose phosphates, carbohydrate degradation under the assay conditions, and interference from the derivatizing agent used as part of the assay procedure. A major source of the recovered methylglyoxal is most probably modified cellular proteins. With methylglyoxal at about 300 microM, 0.02% of cellular amino acid residues could be modified. As few as one or two "hits" with methylglyoxal per protein molecule have previously been reported to be sufficient to cause protein endocytosis and subsequent degradation. Thus, 5-10% of cellular proteins may be modified to physiologically significant levels.