Effect of amino acid and serum deprivation on the regulation of RNA synthesis in cultured Chinese hamster ovary cells

In a proline-requiring Chinese hamster ovary cell line, if both proline and serum are removed from the culture medium, net RNA synthesis is reduced to about 12 % of the unstarved control. This reduction in RNA synthesis is comparable to the stringent regulation of RNA in bacteria. A beta-globulin carbohydrate containing (3.5 % $\text{w}\text{w}$) protein factor was isolated and partially purified from fetal calf serum. The isolated serum factor is able to replace whole serum in stimulating cellular RNA synthesis and has an RNAase inhibitory effect in vitro. The effect of proline starvation and serum factor deprivation on RNA synthesis are independent and additive; each regulates about half of the total RNA synthesized. The regulation appears to affect the synthesis of all species of cytoplasmic RNA.     

Measurement of human leukocyte microsomal HMG-CoA reductase activity

Methods were developed for determination of microsomal HMG-CoA reductase activity from freshly isolated human lymphocytes, monocytes, and granulocytes or cultured human lymphoid cells. Reductase activity in monocytes is approximately twice that in lymphocytes or granulocytes. The activity in cultured cells is approximately 34-fold greater than that in freshly isolated cells. Assay conditions were such as to preclude formation of HMG-CoA cleavage products. Leukocyte reductase activity was inhibited by dichloroacetate, a noncompetitive inhibitor of rat liver reductase and a serum cholesterol-lowering agent in man. Measurement of microsomal reductase activity from freshly isolated leukocytes may prove useful in assessing in vivo regulation of cholesterol synthesis in man.     

Multiple controls for the synthesis of muscle-specific proteins in BC3H1 cells

The regulation of the synthesis of muscle-specific proteins has been examined in BC3H1 cells, a smooth muscle-like cell line isolated by Schubert et al. (J. Cell Biol., 1974, 61: 398-413.). The synthesis of both creatine kinase and the acetylcholine receptor appear to be under dual control, a positive control due to cell-cell contact which increases the rate of synthesis of this protein, and a negative signal, elicited by serum components, that decreases the rate of synthesis of these proteins. Induction of muscle-specific proteins in BC3H1 cells is a reversible process and can be arrested after partial induction has taken place by the addition of serum or high-molecular-weight protein fraction from serum to these cells. The high-molecular-weight protein fraction from serum is not by itself mitogenic for Bc3H1 cells and cannot be replaced by a variety of known hormones (mitogenic factors).     

Classification of Chitosanases by Hydrolytic Specificity toward N 1,N 4-Diacetylchitohexaose

The immediate response of protein degradation to food intake and the factors for its regulation in rat skeletal muscle were examined. The concentration of N ^τ-methylhistidine (MeHis) in serum and the rates of MeHis release from isolated soleus and extensor digitorum longus muscles were reduced in the period from 3 to 6h after refeeding, indicating that the rate of myofibrillar protein degradation in the rat decreased immediately after refeeding. Changes in the serum concentration of insulin and corticosterone were not synchronized with those in the myofibrillar protein degradation. When rats were fed on a protein-free diet, no reduction of serum MeHis concentration or of the rate of MeHis release from isolated muscles after refeeding was apparent. Furthermore, there was a tendency toward suppressing myofibrillar protein degradation with a higher protein content of the diet. These results suggest that the suppression of myofibrillar protein degradation by food intake was regulated by dietary proteins.     

Localization of the human dopamine beta hydroxylase (DBH) gene to chromosome 9q34

A human cDNA clone for dopamine beta hydroxylase (DBH) has been isolated from a phaeochromocytoma library. In situ hybridization of this probe to replication-banded chromosomes has localized the gene to chromosome 9q34. The structural gene for the enzyme is therefore close to the ABO blood group locus. This suggests that the previously described activity variation in levels of serum DBH may reflect alterations in either the structure or regulation of the DBH coding sequences. Both biochemical and genetic evidence therefore indicate independence of DBH from the pterin-dependent aromatic amino acid hydroxylases of the neurotransmitter pathways.     

Isolation of angiotensin converting enzyme (ACE) binding protein from human serum with an ACE affinity column.

Immobilized angiotensin-converting enzyme (ACE) was utilized as an affinity ligand to isolate a naturally occurring ACE binding protein from normal human serum. The enzyme was isolated from solubilized bovine lung membrane preparations by lisinopril affinity chromatography. It had an estimated molecular weight of 180 000 and was recognized by the anti-ACE antibody for the rabbit testicular ACE in immunoblots. ACE was immobilized onto epoxy Sepharose as well as Affi-Gel 15. Immobilized ACE on Affi-Gel 15 had higher catalytic activity (0.176 U/mL) compared with the enzyme immobilized on epoxy Sepharose (0.00005 U/mL). Immobilized ACE served as the affinity ligand for the identification of the ACE binding protein in human serum with an estimated molecular weight of 14 000 as observed by SDS polyacrylamide gel electrophoresis. The identification and further characterization of ACE binding proteins in serum and tissues may facilitate the greater understanding of the endogenous regulation of this key enzyme, which is involved in blood pressure homeostasis.     

Gsα stimulation of mammalian adenylate cyclases regulated by their hexahelical membrane anchors

Mammalian adenylate cyclases (ACs) are pseudoheterodimers with dissimilar hexahelical membrane-anchors, isoform-specifically conserved for more than half a billion years. We exchanged both membrane anchors of the AC isoform 2 by the quorum-sensing receptor from Vibrio harveyi, CqsS, which has a ligand, Cholera-Autoinducer-1 (CAI-1). In the chimera, AC activity was stimulated by Gsα, CAI-1 had no effect. Surprisingly, CAI-1 inhibited Gsα stimulation. We report that Gsα stimulation of human AC isoforms 2, 3, 5, and 9 expressed in Sf9 cells is inhibited by serum as is AC activity in membranes isolated from rat brain cortex. AC2 activation by forskolin or forskolin/Gsα was similarly inhibited. Obviously, serum contains as yet unidentified factors affecting AC activity. The data establish a linkage in ACs, in which the membrane anchors, as receptors, transduce extracellular signals to the cytosolic catalytic dimer. A mechanistic three state model of AC regulation is presented compatible with all known regulatory inputs into mammalian ACs. The data allow designating the membrane anchors of mammalian ACs as orphan receptors, and establish a new level of AC regulation.     

Characterization of contractile function and expression of muscarinic receptors,G proteins and adenylate cyclase in cultured tracheal smooth muscle of Swine

Smooth muscle cells lose their contractile function and phenotype very rapidly when placed in culture. During organ culture of smooth muscle strips, phenotype is lost more slowly. In the present studies, we established an organ culture model to study contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase in different serum concentrations in tracheal smooth muscle from swine. The results show that contractile function and the amounts of M(3) receptors, G proteins and adenylyl cyclase were maintained for up to 5 days in culture. The expression of M(2) receptors was significantly decreased in culture when compared to freshly isolated muscles. Maximal isometric tension was significantly increased in cultured muscles compared with freshly isolated muscles. Different serum concentrations did not significantly affect contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase. In conclusion, our studies suggest that cultured smooth muscle might be used as a model to study the regulation of contractile function of smooth muscle by various signal transduction pathways.     

Isolation and analysis of murine serum amyloid P component cDNA clones

In contrast to other animals, the biosynthesis of serum amyloid P component in mice is regulated as an acute-phase protein. As a first step in studying the regulation and biosynthesis of serum amyloid P component in the mouse, cDNA clones have been isolated from a liver cDNA library and sequenced. The largest of these clones was 960 bp in length, and contained an open reading frame encoding a protein of 224 amino acids. Comparison of the mouse cDNA sequence to that published for humans (Mantzouranis, E. C., S. B. Dowton, A. S. Whitehead, M. D. Edge, G. A. P. Bruns, and H. R. Colten, 1985. J. Biol. Chem. 260:7752.) revealed 74% identity for nucleotides in the translated region. Northern-blot analysis demonstrated that murine serum amyloid P component synthesis in the liver is directed by a 1.2-kb mRNA that is elevated in high responder (C57BL/6J) mice after thioglycollate-induced inflammation.     

Spin assay as a general method for studying plasma protein binding. Bilirubin-albumin binding.

A mutant of the Chinese Hamster Ovary cell line, CHO-K1, has been isolated which is resistant to killing by 25-hydroxy cholesterol in the absence of cholesterol. There is no effect on acetate incorporation into cholesterol by 25-hydroxy cholesterol in the mutant under conditions in which incorporation is inhibited in the parent cell. The mutant also appears to be defective in the regulation of cholesterol synthesis by serum choleserol.     

Oxidative stress-induced dysregulation of arteriolar wall shear stress and blood pressure in hyperhomocysteinemia is prevented by chronic vitamin C treatment

We aimed to test the hypothesis that an enhanced level of reactive oxygen species (ROS) is primarily responsible for the impairment of nitric oxide (NO)-mediated regulation of arteriolar wall shear stress (WSS) in hyperhomocysteinemia (HHcy). Thus flow/WSS-induced dilations of pressurized gracilis muscle arterioles (basal diameter: approximately 170 microm) isolated from control (serum Hcy: 6 +/- 1 microM), methionine diet-induced HHcy rats (4 wk, serum Hcy: 30 +/- 6 microM), and HHcy rats treated with vitamin C, a known antioxidant (4 wk, 150 mg. kg body wt-1.day-1; serum Hcy: 32 +/- 10 microM), were investigated. In vessels of HHcy rats, increases in intraluminal flow/WSS-induced dilations were converted to constrictions. Constrictions were unaffected by inhibition of NO synthesis by N omega-nitro-L-arginine methyl ester (L-NAME). Vitamin C treatment of HHcy rats reversed the WSS-induced arteriolar constrictions to L-NAME-sensitive dilations but did not affect control responses. Similar changes in responses were obtained for the calcium ionophore A-23187. In addition, diastolic and mean arterial blood pressure and serum 8-isoprostane levels (a marker of in vivo oxidative stress) were significantly elevated in rats with HHcy, changes that were normalized by vitamin C treatment. Taken together, our data show that in chronic HHcy long-term vitamin C treatment, by decreasing oxidative stress in vivo, enhanced NO bioavailability, restored the regulation of shear stress in arterioles, and normalized systemic blood pressure. Thus our study provides evidence that oxidative stress is an important in vivo mechanism that is primarily responsible for the development of endothelial dysregulation of WSS in HHcy.     

Social cues can play a role in timing onset of the breeding season of the ewe

Female Suffolk sheep were pinealectomized around the vernal equinox to eliminate the major environmental input to the reproductive system (photoperiod) and then either isolated from, or maintained with, pineal-intact gonad-intact sheep. The ewes were ovariectomized and treated with constant-release oestradiol implants and reproductive state was monitored by measuring serum LH concentrations. Pinealectomized ewes that were isolated from the normal flock showed a 2 1/2-month delay in onset of the seasonal rise in LH values compared with that of pineal-intact controls (18 November vs 5 September). On the other hand, pinealectomized ewes that were maintained with the flock showed an onset of the seasonal rise in LH that was not delayed. These results suggest a timekeeping role for social cues for timing onset of the breeding season in an animal that normally relies on photoperiodic signals for temporal regulation of the seasonal reproductive cycle.     

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.     

An anorectic proteoglycan of membrane origin

The endogenous substance(s) involved in the regulation of food intake has been isolated from serum, urine and feces. In the present study, a similar type of anorexigenic proteoglycan was isolated from human rat erythrocyte membranes and rat liver membranes. Membranes were suspended in 2.0% deoxycholate and allowed to stand at 25 degrees C for 30 min. The suspension was treated with 5% TCA, supernatant was collected, dialyzed and concentrated. TCA-soluble proteins were fractionated on Sephadex G-150. The active second peak fractions were further purified on DEAE-Sephadex A-25. Biologically active substance reduced the appetite in rats significantly when given intraperitoneally. The proteoglycan (50 kDa) consisted of 70-85% carbohydrate. Similar properties of plasma and membrane anorectic substance further indicated its membrane origin. We believe that this anorectic proteoglycan is anchored to cell membranes and released into the blood circulation to regulate the food intake.     

Bactericidal activity of testicular macrophages

The purpose of these studies was to determine if testicular macrophages are capable of bactericidal activity. Testicular macrophages were isolated from adult Wistar rats and studied in vitro. Studies were designed to determine if these cells could kill pathogenic gram-negative organisms and if these cells secreted lysozyme, an enzyme involved with the lysis of the cell wall of gram-positive bacteria. The regulation of lysozyme secretion by hormones and lipopolysaccharide was also studied. The secretion of this enzyme by testicular macrophages was also compared to enzyme secretion by macrophages isolated from other tissues. We also studied the secretion of superoxide anion, which is known to be involved in cytotoxic reactions. It was found that testicular macrophages were capable of killing up to approximately 38% of a virulent encapsulated strain of Klebsiella pneumoniae within 1 h. This process was in part dependent upon the presence of immune serum generated against these organisms but could not be mimicked by control serum or immune serum tested in the absence of macrophages. Testicular macrophages secreted lysozyme in culture for at least 8 days; however, macrophages from the peritoneal cavity and lung secreted significantly more lysozyme under the same conditions. Lipopolysaccharide suppressed lysozyme secretion in a dose-dependent manner, whereas neither follicle-stimulating hormone, testosterone, nor leuteininzing hormone had an effect on lysozyme secretion. Finally, testicular macrophages secreted superoxide anion in a manner similar to peritoneal macrophages. These studies indicate that testicular macrophages have the capability to mount an appropriate defense against pathogenic bacteria by opsonization-dependent phagocytosis, the secretion of lysozyme, and the production of super oxide anion.     

Primary cultures of rat pancreatic acinar cells in serum-free medium

Summary Rat pancreatic acinar cells were isolated and cultured in Ham's F12 medium with 15% bovine calf serum. Caerulein, insulin, somatostatin, and dexamethasone (DEX) had no effect on intracellular or secreted amylase in these cultured cells. A serum-free medium, using Waymouth's MB 752/1 supplemented with albumin, epidermal growth factor (EGF), DEX, and HEPES, was then developed to avoid serum factors that might mask hormonal effects. In this SF medium, pancreatic acinar, cells maintained the morphological and ultrastructural characteristics of freshly isolated cells and secreted amylase in response to the secretagogue, carbamyl choline. Insulin, at a concentration of 1 μg/ml, significantly increased intracellular and secreted amylase activity after 3 d. This model cell system can be used to study the regulation of the synthesis of amylase and other pancreatic enzymes in vitro.     

Stimulant-free preculture in heterologous serum-supplemented medium induces unresponsiveness of T cells to subsequent mitogenic stimulation

Quite often freshly isolated lymphocytes are kept in culture before experimentation for 1 or more days without any stimulus. Most of the time, culture is supplemented with fetal bovine serum (FBS) which is heterologous to all species except bovine. In the present study, we found that freshly isolated murine T cells show a good proliferative response to concanavalin A (Con A) and phorbol ester (PMA)/ionomycin in FBS medium, without any detectable background proliferation. However, the cells kept in the same culture without any stimulus for prolonged period of time (referred to as preculture in this report) showed reduced response to Con A and PMA/ionomycin in a time-dependent manner. Almost a complete loss of response to Con A was observed within 1 day of preculture. However, loss of response to PMA/ionomycin was observed only after 2 days of preculture. Interestingly, similar preculture in autologous mouse serum-supplemented media did not cause any loss of the response to these mitogens. The loss of responsiveness of T cells during preculture in heterologous serum was irreversible. The heterologous serum-induced unresponsiveness of T cells to these mitogens was also prevented by adding Calphostin C, a specific protein kinase C (PKC) inhibitor, during preculture in heterologous serum. These results showed that prolonged stimulant-free preculture in heterologous serum induces irreversible unresponsiveness of T cells to mitogens through the down regulation of T cell receptor signaling pathway, which can be prevented by autologous serum or a PKC inhibitor.