Isolation of Zn2+ as an Endogenous Agonist of GPR39 from Fetal Bovine Serum

We attempted to determine natural agonists of GPR39 in fetal bovine serum (FBS). FBS was conditioned to extract peptides and fractionated by two types of HPLC. The activity of each fraction was monitored by intracellular calcium mobilization. Then the purified active ingredient was analyzed by inductively coupled plasma mass spectrometry. In this fashion, Zn^{2 +} ion was identified as an agonist of GPR39, though no peptidergic molecules were found. The calcium-mobilizing activity of Zn^{2 +} was not abolished by pertussis toxin but was by a phospholipase C (PLC) inhibitor, U73122, indicating that the activity of GPR39 is mediated through the G_qα -PLC pathway. In addition, Zn^{2 +} also activated mouse and rat GPR39, showing that the function of GPR39 as a Zn^{2 +} receptor is conserved across species. This study is the first exploration of GPR39 agonists in FBS and indicates that GPR39 functions as a Gq-coupled Zn^{2 +}-sensing receptor.     

A new Zn2+-stimulated sphingomyelinase in fetal bovine serum

Fetal bovine serum contains a Zn2+-dependent sphingomyelinase with optimal activity at pH 5.5 in vitro. Activity could be demonstrated with a liposomal sphingomyelin substrate suspension but was stimulated up to 15-fold by Triton X-100. Under a variety of conditions tested, phosphatidylcholine, lysophosphatidylcholine, glycerophosphocholine, and p-nitrophenyl phosphate were not substrates for this activity. Several inhibitors of serum alkaline and acid phosphatases had no effect on the activity. The enzyme resembles the acid lysosomal sphingomyelinase in pH optimum and inhibition by AMP but differs in inhibition by EDTA, stimulation by Zn2+, and heat lability at 55 degrees C. It resembles the neutral, Mg2+-stimulated enzyme in inhibition by EDTA and heat lability but differs in metal ion requirement and pH optima. Of the sera tested, activity was highest in fetal bovine serum, with fetal bovine greater than newborn bovine greater than horse greater than human; more than 95% of the activity is in the lipoprotein-free infranatant of serum (d greater than 1.21). This activity appears to be a hitherto undescribed sphingomyelinase. Its biological functions are not known but may subserve a special role in sphingomyelin catabolism in the circulation, in blood vessel walls, or in the tissue(s) of origin.     

Molecular mechanism of Zn2+ agonism in the extracellular domain of GPR39

Ala substitution of potential metal-ion binding residues in the main ligand-binding pocket of the Zn2+-activated G protein-coupled receptor 39 (GPR39) receptor did not decrease Zn2+ potency. In contrast, Zn2+ stimulation was eliminated by combined substitution of His17 and His19, located in the N-terminal segment. Surprisingly, substitution of Asp313 located in extracellular loop 3 greatly increased ligand-independent signaling and apparently eliminated Zn2+-induced activation. It is proposed that Zn2+ acts as an agonist for GPR39, not in the classical manner by directly stabilizing an active conformation of the transmembrane domain, but instead by binding to His17 and His19 in the extracellular domain and potentially by diverting Asp313 from functioning as a tethered inverse agonist through engaging this residue in a tridentate metal-ion binding site.     

Proteomic and functional characterization of endogenous adiponectin purified from fetal bovine serum

Adiponectin is a plasma protein exclusively secreted from fat tissue. Many recent pharmacological studies suggest that recombinant adiponectin has multiple therapeutic potentials for obesity-related metabolic disorders, including type 2 diabetes, dyslipidemia, insulin resistance and atherosclerosis. However, the physiological relevance of these findings remains to be further established. In the present study, we have purified endogenous adiponectin from fetal bovine serum and characterized its post-translational modifications and physiological functions in animal models. Endogenous bovine serum adiponectin consists predominantly of full-length proteins that form multiple oligomeric complexes, including trimers, hexamers and higher molecular species. Two-dimensional gel electrophoresis revealed that bovine serum adiponectin exists as multiple post-translationally modified isoforms with distinct molecular weight and isoelectric point. Further analysis using mass spectrometry and Edman degradation sequencing demonstrated that five conserved lysine residues (Lys 28, 60, 63, 72 and 96) within the collagenous domain of bovine adiponectin are hydroxylated and glycosylated by a glucosyl alpha(1-2)galactosyl group. Injection of endogenous bovine adiponectin into C57 mice potently decreased circulating glucose levels and enhanced lipid clearance after a high fat meal. Chronic administration of this protein for a period of two weeks significantly increased insulin sensitivity and glucose tolerance, and depleted hepatic lipid accumulation in high-fat fed mice. These results provide direct evidence that endogenous bovine adiponectin is a physiological hormone that can regulate lipid and glucose metabolism.     

Novel Zn2+ Modulated GPR39 Receptor Agonists Do Not Drive Acute Insulin Secretion in Rodents

Type 2 diabetes (T2D) occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired β-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic β-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve β-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn²⁺ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO) mice and Zucker fatty rats. It is concluded that Zn²⁺ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.     

Iron-supplemented bovine serum as an alternative to fetal bovine serum in the CHO/HGPRT mutation assay

Iron-supplemented bovine calf serum (ICS) was found to be a viable alternative to fetal bovine serum (FBS) in the growth promotion and cloning efficiency of Chinese hamster ovary (CHO) cells that are used in the HGPRT mutation assay. Suspension cultures of CHO cells had an average generation time of 11.5 h in ICS and 13.6 h for cells maintained in FBS. This slight difference was due to lot variability on the part of FBS and could be eliminated by routine quality control measures. The average cloning efficiencies for CHO cells cloned in either ICS or FBS were 107% and 88%, respectively, and these values were not statistically different. No appreciable difference was noted in the spontaneous mutation rates of cells cloned in either ICS or FBS. Furthermore, the use of ICS in mutagenicity studies with genotoxic agents shows the serum to be at least equal or superior to FBS in the detection of both direct-acting mutagens and promutagens. These data suggest that ICS is an appropriate serum to be used in the CHO/HGPRT test system. Since ICS is more readily available and considerably less costly than FBS, a substantial reduction in the cost of the assay can be realized.     

5-Hydroxy-eicosapentaenoic acid is an endogenous GPR119 agonist and enhances glucose-dependent insulin secretion

GPR119 is one of the G-protein-coupled receptors expressed in pancreatic β-cells and intestinal endocrine cells. Since agonists to GPR119 stimulate glucose-dependent insulin secretion, GPR119 agonists are anticipated to promote anti-diabetic effects and control of glucose homeostasis. Here, we reported that an omega-3 unsaturated fatty acid metabolite, 5-hydroxy-eicosapentaenoic acid (5-HEPE), was a potent agonist for GPR119 and enhanced glucose-dependent insulin secretion. 5-HEPE stimulated cAMP accumulation in mouse MIN6 insulinoma cells and human HuTu80 intestinal adenocarcinoma cells. These effects were blunted by GPR119-specific siRNA. Recombinant GPR119 also responded to 5-HEPE as well as authentic agonists. Several previous reports have indicated the beneficial biological effects of omega-3 unsaturated fatty acids, and epidemiological studies have suggested that these fatty acids plays a protective role against diabetes. However, the molecular pharmacology and receptor identifications of omega-3 unsaturated fatty acids and their metabolites have not yet been well investigated. It is hoped that our findings will encourage novel investigations into the molecular relationships between omega-3 fatty acids and diabetes.     

Extraction from fetal bovine serum of erythrotropin, an erythroid cell-stimulating factor

A method is described for the rapid preparation of erythrotropin from commercially available fetal bovine serum. It consists of the reversed-phase extraction of fetal bovine serum using octadecylsilyl-silica cartridges followed by reversed-phase high-pressure liquid chromatography (HPLC). Further purification can be achieved by gel-permeation HPLC. Serum erythrotropin coelutes with erythrotropin I from fetal bovine intestine on reversed-phase HPLC. It has a molecular weight and an amino acid composition very similar to those reported for the intestinal erythrotropins. The easy preparation of serum erythrotropin with specific activities higher than commercially available erythropoietin preparations will facilitate the study of the physiological role of this factor in vivo and in vitro.     

Properties of a Zn2+-glycerophosphocholine cholinephosphodiesterase from bovine brain membranes

A Zn²⁺-glycerophosphocholine cholinephosphodiesterase was purified with a specific activity of 4.6 μmole/min·mg protein from bovine brain membranes by procedures involving PI-PLC solubilization, concanavalin A affinity chromatography, CM-sephadex chromatography and Sephadex G-150 chromatography. Based on molecular weight determination gel chromatography and SDS polyacrylamide gel electrophoresis, the phosphodiesterase activity appears to be a dimeric protein (110 kDa) composed of two subunits with a molecular weight of approximately 54 kDa. The Km value for p-nitrophenylphosphocholine and the optimum pH were found to be 16 μM and pH 10.5, respectively. The phosphodiesterase was inhibited by Cu²⁺, but not the other divalent metal ions. The activity of the apoenzyme was remarkably activated by Co²⁺ or Zn²⁺, but not Mn²⁺ or Mg²⁺. In addition, the inactivation of the enzyme in glycine buffer was prevented by Mn²⁺ or Zn²⁺, but not Co²⁺ or Mg². In a separate experiment, comparing properties of the purified and membrane-bound phosphodiesterases, the forms of two enzymes were quite similar except in stability. Both enzymes were more stable at pH 7.4 than pH 5 or 10. However, the membrane-bound enzyme was more stable than the soluble enzyme at all three pHs. These data suggest that the activity of the phosphodiesterase may be stabilized in-vivo.     

Effectiveness of autologous serum as an alternative to fetal bovine serum in adipose-derived stem cell engineering

In cell culture, medium supplemented with fetal bovine serum is commonly used, and it is widely known that fetal bovine serum supplies an adequate environment for culture and differentiation of stem cells. Nevertheless, the use of xenogeneic serum can cause several problems. We compared the effects of four different concentrations of autologous serum (1, 2, 5, and 10 %) on expansion and adipogenic differentiation of adipose-derived stem cells using 10 % fetal bovine serum as a control. The stem cells were grafted on nude mice and the in vivo differentiation capacity was evaluated. The isolation of adipose-derived stem cells was successful irrespective of the culture medium. The proliferation potential was statistically significant at passage 2, as follows: 10 % autologous serum >10 % fetal bovine serum = 5 % autologous serum >2 % autologous serum = 1 % autologous serum. The differentiation capacity appeared statistically significant at passage 4, as follows: 10 % fetal bovine serum >10 % autologous serum = 5 % autologous serum >2 % autologous serum = 1 % autologous serum. Ten percent autologous serum and 10 % fetal bovine serum had greater differentiation capacity than 1 and 2 % autologous serum in vivo, and no significant difference was observed between the groups at ≥5 % concentration at 14 weeks. In conclusion, 10 % autologous serum was at least as effective as 10 % fetal bovine serum with respect to the number of adipose-derived stem cells at the end of both isolation and expansion, whereas 1 and 2 % autologous serum was inferior.     

Complete genome sequence of a bovine viral diarrhea virus 2 from commercial fetal bovine serum

We isolated a bovine viral diarrhea virus (BVDV) from commercial fetal bovine serum and designated it HLJ-10. The complete genome is 12,284 nucleotides (nt); the open reading frame is 11,694 nt, coding 3,898 amino acids. Phylogenetic analysis indicated that this strain belongs to BVDV group 2.     

Isolation and characterization of a specific endogenous Na+,K+-ATPase inhibitor from bovine adrenal

In order to identify a specific endogenous Na+,K+-ATPase inhibitor which could possibly be related to salt-dependent hypertension, we looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na+,K+-ATPase; (ii) competitive displacing activity against [3H]ouabain binding to the enzyme; (iii) inhibitory activity for 86Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C18 open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na+,K+-ATPase such as unsaturated free fatty acids, lysophosphatidylcholines, vanadate, dihydroxyeicosatrienoic acid, dehydroepiandrosterone sulfate, dopamine, lignan, ascorbic acid, etc. This substance was further purified by using an additional five steps of high-performance liquid chromatography with five different types of columns. Molecular mass was estimated as below 350 by fast atom bombardment mass spectroscopy and ultrafiltration. Heat treatment at 250 degrees C for 2 h and acid treatment with 6 N HCl at 115 degrees C for 21 h almost completely destroyed the inhibitory activity of the purified substance for Na+ pump activity. Additionally, alkaline treatment with 0.2 N NaOH at 23 degrees C for 2 h destroyed approximately 70% of the inhibitory activity, whereas boiling for 10 min and various enzyme digestion did not destroy the activity. The dose dependency for the four types of the activities for this substance paralleled those of ouabain, spanning 2 orders of magnitude in concentration range. The inhibitory potencies of the purified substance for Na+,K+-ATPase, Na+ pump, and ouabain binding activities were diminished with increasing K+ concentration, exhibiting a characteristic typical of cardiac glycosides. This substance had no effect on the Ca2+-ATPase activity or the Ca2+ loading rate into the vesicle prepared from skeletal muscle sarcoplasmic reticulum. These results strongly suggest that this water-soluble nonpeptidic Na+,K+-ATPase inhibitor may be a specific endogenous regulator for the ATPase.     

Isolation and identification of germ cells from fetal bovine ovaries

Gonadal cell suspensions were made from bovine fetuses of 35–55-, 56–80-, and 80–130-day age groups corresponding to the periods predominated by primordial germ cells (PGCs), oogonia, and meiotic cells, respectively. Germ cells identified on morphological criteria prior to their isolation from suspensions were compared histochemically and morphologically with cells in cryosections, impression smears, and semithin sections of similar gonads. Oocytes were distinguished by their chromosomal configurations in cell spreads. In suspensions from 35–55-day fetuses, cells considered to be PGCs stood out by their size, large nucleus, intracytoplasmic vesicles, and occasional blebbing. The somatic cells were smaller and contained little cytoplasm and few vesicles. In bovine gonads, in contrast to murine gonads, alkaline phosphatase (AP) activity was not specific enough to identify germ cells once they had entered the gonad. In ovaries from the 56–80-day age group, cells similar to PGCs, but slightly larger and with more cytoplasmic vesicles, were identified as oogonia. The cytoplasmic vesicles stained positively for lipid. In ovaries of 80–130-day fetuses, oogonia, oocytes, degenerating germ cells, and multinucleate germ cells were recognized. Degenerating germ cells exhibited a variety of morphological characteristics and were consistently positive for acid-phosphatase activity. Binucleate germ cells appeared around day 85 of gestation, while multinucleate germ cells were seen from day 95. It was concluded that bovine mitotic germ cells can be isolated from gonadal cell suspensions and that the best time to recover them is between 50 and 70 days of gestation. © 1994 Wiley-Liss, Inc.     

Goat serum: an alternative to fetal bovine serum in biomedical research

Serum is frequently added to the defined basal medium as a source of certain nutritional and macromolecular growth factors essential for cell growth. Although a number of synthetic media have been prepared serum continues to be used in cell culture by many investigators. The best supplementation to a basal medium is fetal bovine serum (FBS) that is most frequently used for all types of cell cultures. During last four decades National Institute of Virology, Pune, has been working on isolation and identification of viruses from clinical specimens, employing tissue culture. Initially FBS was used for this purpose. However, due to its prohibitive cost and uncertain supply an alternative was sought. Commercially available sera from newborn calf, sheep, horse, human and serum obtained from goat blood (available from local abattoir) were tried. Goat serum (GS) was found to be suitable for most of the cell lines and primary cultures. Primary cultures from guinea pig embryo, monkey kidney, chick embryo, mouse peritoneal macrophages, and established cell lines were prepared and grown in growth media supplemented with GS. These cultures were studied for their morphology and growth in comparison with cultures grown in FBS containing media, and were used for mass cultivation of cells, quantitation and susceptibility of various virus strains, studies on effects of different nutrients and natural substances on cellular metabolism and virus replication, epitope analysis of various strains of Japanese encephalitis (JE) virus, strain differentiation studies, studies on antibody dependent plaque enhancement, assay of murine migration inhibition factor. Monoclonal antibodies against JE virus adapted to GS were characterised for their retention of functionalities. The results were comparable to those of cell cultures grown in FBS containing media. Similar results on chromosome studies were obtained from patient's whole blood cultures prepared in GS and FBS containing growth media. Organ cultures from mammalian, reptile and avian hosts; successfully grown in GS supplemented growth media, were used for different virological studies. Growth media supplemented with GS were used for in vitro cultivation of malarial parasites. Thus since the last three decades many scientists are using GS in place of FBS, in various fields of biomedical research. The present article reviews an account of the same.     

Stabilizing effect of Zn2+ in native bovine rhodopsin

Single-molecule force spectroscopy (SMFS) is a powerful tool to dissect molecular interactions that govern the stability and function of proteins. We applied SMFS to understand the effect of Zn2+ on the molecular interactions underlying the structure of rhodopsin. Force-distance curves obtained from SMFS assays revealed the strength and location of molecular interactions that stabilize structural segments within this receptor. The inclusion of ZnCl2 in SMFS assay buffer increased the stability of most structural segments. This effect was not mimicked by CaCl2, CdCl2, or CoCl2. Thus, Zn2+ stabilizes the structure of rhodopsin in a specific manner.     

Isolation and characterization of viruses from fetal calf serum

Summary Ten lots of specially procured fetal calf serum collected under sterile conditions and not filtered and 16 lots of commercial fetal calf serum were tested for both human and bovine viral contamination. The presence of viruses was evaluated by observing for cytopathogenic effect (CPE), hemadsorption with guinea pig erythrocytes, and interference with cytopathogenic challenge viruses in both embryonic bovine trachea (EBTr) and human diploid lung (HDL) cells. Isolates were characterized by their cytopathogenicity, morphology, serology, and ability to propagate and produce cPE in a variety of bovine and nonbovine cells. One isolate was unequivocally identified as bovine herpes virus 1, and the other was presumptively identified as a bovine virus-diarrhea virus. This study was supported by The National Institutes of Health under Contract PH 43-66-539.     

Ca2+ and Zn2+-binding properties of nitrated S-100b protein from bovine brain.

The single tyrosine residue in S-100b protein was nitrated by treatment with tetranitromethane in 0.1 M-Tris/HCl buffer, pH 8.0, containing 2 mM-EDTA. The nitrated protein did not differ significantly in secondary structure from its native unmodified counterpart, as revealed by far-u.v. c.d. measurements. The effect of Ca2+ on the modified protein was different from that on the native protein, e.g. addition of Ca2+ resulted in a loss of helical content from 55 to 47% with the native protein whereas Ca2+ had no significant effect on the gross conformation of the nitrated derivative. Near-u.v. c.d. studies also indicated a very minimal effect on the tyrosine residue and this was also reflected in the u.v.-absorption difference spectrum. Polyacrylamide-gel electrophoresis in the absence of SDS showed the nitrated S-100b to move faster in the presence of EDTA compared with the calcium-bound state, suggesting that the modified protein does bind Ca2+ although it does not undergo a major conformational change in response to Ca2+ addition. In contradistinction, Zn2+ binding was not influenced by nitration, as demonstrated by aromatic c.d. and u.v.-difference spectroscopy. It is clear from this study that the single tyrosine residue in S-100b is critical to sense the Ca2+-induced conformational changes in the protein.     

Isolation of fatty acid amide as an angiogenic principle from bovine mesentery

Erucamide (13-docosenamide) was found to be the major bovine mesentery angiogenic lipid as assessed by chorioallantoic membrane (CAM) assay. Two micrograms of this lipid caused angiogenesis in the assay. Angiogenic activity of this naturally occurring lipid was also found by rat corneal micropocket and mouse dorsal air-sac assays. Specificity of the chemical structure which elicited activity was low, however. The mechanism of angiogenic activity is unknown and this lipid does not promote proliferation of endothelial cells or induce inflammatory effects.     

Isolation of thyroglobulin from the bovine thyroid free of endogenous proteolytic activity

Thyroglobulin was isolated from the cattle thyroid gland by chromatography on Sephadex G-200 and Sepharose 4B. It was found homogeneous according to disc electrophoresis (pH 8.6) and analytical ultracentrifugation. However, gel filtration of thyroglobulin incubated at 37 degrees through Sepharose 6B revealed that it contained proteinases of the serine type. The conditions for the proteinases' inhibition were found. The possibility of obtaining thyroglobulin free of the proteinases by means of affinity chromatography is demonstrated.