Multiple nuclear dots antinuclear antibodies are not specific for primary biliary cirrhosis. Multiple nuclear dots antinuclear antibodies display a specific immunofluorescence pattern on HEp-2 cells. They have been reported to be strongly associated with primary biliary cirrhosis, especially when sicca syndrome was present. To determine whether multiple nuclear dots antinuclear antibodies are specific for primary biliary cirrhosis, we studied the clinical, biochemical, immunological and morphological features of 38 patients between December 1983 and September 1990 who had serum multiple nuclear dots antinuclear antibodies detected in an immunology laboratory of a large medical center. Sufficient information was reliable in 36 patients; the group included 31 women and 5 men (mean age = 57.6 +/- 14.5, range = 30 to 87). Fifteen patients (42%) had primary biliary cirrhosis, 5 patients (14%) had type 1 autoimmune chronic active hepatitis, 4 patients (11%) had liver disease of unknown cause and 12 patients (33%) had various immunological disorders but no liver disease. Two of the patients with primary biliary cirrhosis (13%) had clinical sicca syndrome. Our study demonstrates the following: (a) serum multiple nuclear dots antinuclear antibodies are not specific for liver disease because they can be observed in one third of patients with various immunological disorders without liver involvement, and (b) serum multiple nuclear dots antinuclear antibodies are not specific for PBC because they can also be observed in type 1 autoimmune chronic active hepatitis. Our results also suggest that patent sicca syndrome is abnormally present in patients with primary biliary cirrhosis and multiple nuclear dots antinuclear antibodies.


Glucocorticoid stimulates hepatitis B viral gene expression in cultured human hepatoma cells. Glucocorticoids have been shown to influence the severity of hepatitis B virus-related chronic hepatitis in human. However, very little is known about the effects of glucocorticoids on hepatitis B virus replication in vitro. In this report, we used a well-differentiated human hepatoma cell line, Hep3B, transfected with hepatitis B virus complementary DNA as a model to show that a glucocorticoid analog, dexamethasone, can directly stimulate the production of HBsAg and HBeAg. Elevation of 3.5-kb pregenomic RNA and all other viral RNAs in the transfected Hep3B cells after dexamethasone treatment supports the hypothesis that glucocorticoids directly stimulate hepatitis B virus gene expression in vitro. The concentration of dexamethasone for its half-maximal stimulatory activity toward HBsAg, HBeAg and all viral transcripts was approximately 10(-8) mol/L, close to the affinity of glucocorticoid receptors to [3H]-triamcinolone acetonide in Hep3B cells (approximately 10(-8) mol/L). Specific glucocorticoid antagonist RU38486 completely blocked dexamethasone-induced HBV gene expression, suggesting that the stimulatory effect of dexamethasone was mediated through specific glucocorticoid receptors.


Lipopolysaccharide treatment of rats alters antigen expression and oxidative metabolism in hepatic macrophages and endothelial cells. Endothelial cells and macrophages are located within the hepatic sinusoids. These two cell types play an important role in the clearance of bacterially derived lipopolysaccharide from the portal circulation. Our laboratory has previously demonstrated that treatment of rats with lipopolysaccharide results in the accumulation of macrophages in the liver that display properties of activated mononuclear phagocytes. This study was designed to analyze the effects of lipopolysaccharide on hepatic endothelial cells. Female Sprague-Dawley rats were treated with 5 mg/kg of lipopolysaccharide. Macrophages and endothelial cells were isolated from the rats 48 hr later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that lipopolysaccharide treatment of rats resulted in an increase in the number of both macrophages and endothelial cells recovered from the liver. Using specific monoclonal antibodies and flow cytometry, both macrophages and endothelial cells were found to express cell surface markers for Ia antigen, leukocyte common antigen, CD4 and the macrophage antigen, ED2. Macrophages expressed greater levels of these markers than endothelial cells. Flow cytometric analysis also revealed considerable subpopulation heterogeneity in the endothelial cells in antigen expression, physical characteristics and functional activity. Treatment of rats with lipopolysaccharide decreased expression of cell surface markers on the macrophages but not on the endothelial cells. This may be due to the distinct origin of these cells. To determine whether endothelial cells, like macrophages, were activated by lipopolysaccharide, we examined their ability to produce reactive oxygen intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)


Endotoxins inhibit endocytotic catabolism of low-density lipoproteins in Hep G2 cells. The effects of endotoxins on the uptake and degradation of low-density lipoproteins in Hep G2, a well-differentiated human hepatoma cell line, were studied. The results showed that incubation of Hep G2 cells with 125I-labeled low-density lipoprotein in the presence of endotoxins caused decreased uptake and degradation of 125I-labeled low-density lipoprotein. The inhibitory effects of endotoxins on the uptake and degradation of 125I-labeled low-density lipoprotein were dose and time dependent. With a monoclonal low-density lipoprotein receptor antibody, it was found that endotoxins interfered with both low-density lipoprotein receptor-mediated and non-low-density lipoprotein receptor-mediated uptake. If, however, the cells were pretreated with endotoxins for 1 or 24 hr and then incubated with new medium without endotoxins, no inhibitory effect on the subsequent uptake and degradation of 125I-labeled low-density lipoprotein occurred. Endotoxins had no toxic effects on Hep G2 cells as judged by [3H]thymidine incorporation and by determination of cell growth. Also, endotoxins did not under our experimental conditions induce oxidative modification of low-density lipoprotein. Furthermore, reisolated low-density lipoprotein that had previously been incubated with endotoxin was catabolized to a lower extent by Hep G2 cells than was control low-density lipoprotein. We speculate that the inhibitory effect of endotoxins on cellular low-density lipoprotein catabolism is due to the formation of endotoxin-low-density lipoprotein complexes, which interfere with the binding of low-density lipoprotein to the cell surface.


Isoenzymes of carbohydrate metabolism in primary cultures of hepatocytes from thioacetamide-induced rat liver necrosis: responses to growth factors. Hepatocytes isolated from the liver of rats after a necrotizing dose of thioacetamide (6.6 mmol/kg) were used to study the postnecrotic process of liver regeneration. Flow cytometry analysis revealed populations of dedifferentiated hepatocytes exhibiting physical properties (size and fluorescence emission at 530 nm) similar to those found in fetal (22 days old) liver cells. The percentage of these cells increased progressively from 24 to 48 and 72 hr after thioacetamide administration. In primary cultures of hepatocytes the effects of phorbol 12-myristate 13-acetate, bombesin and insulin were investigated on the 6-phosphofructo 2-kinase/fructose 2,6 bisphosphate system. Bombesin and insulin stimulated 6-phosphofructo 2-kinase activity and fructose 2,6-bisphosphate content both in control and in thioacetamide-treated hepatocytes. However, phorbol 12-myristate 13-acetate stimulated 6-phosphofructo 2-kinase activity and increased fructose 2,6-bisphosphate concentration in thioacetamide-treated liver cells, whereas no similar response was found in hepatocytes from control rats. The response of postnecrotic thioacetamide-treated hepatocytes to phorbol 12-myristate 13-acetate was similar to that obtained from 22-day-old fetal liver cells, which reveals that different methods might control fructose 2,6-bisphosphate content and therefore the mechanisms of glycolysis and gluconeogenesis at this regulatory step. The lack of response to glucagon of glycogen phosphorylase a and 6-phosphofructo 2-kinase from thioacetamide-treated hepatocytes may indicate that the expression of specific enzymes of carbohydrate metabolism undergoes transitions to less-differentiated isoenzymatic forms. Moreover, the isoenzyme pattern of hexokinases elicits a complete disturbance in glucokinase and hexokinases activities.(ABSTRACT TRUNCATED AT 250 WORDS)


Role of cellular superoxide dismutase against reactive oxygen metabolite-induced cell damage in cultured rat hepatocytes. Reactive oxygen metabolites have been reported to be important in the pathogenesis of ischemia/reperfusion-induced and alcohol- and drug-induced liver injuries. We investigated the role of superoxide dismutase, cellular and extracellular, in preventing reactive oxygen metabolite-induced cytotoxicity in cultured rate hepatocytes. Cells were exposed to reactive oxygen metabolites enzymatically generated by hypoxanthine-xanthine oxidase. Cytotoxicity was quantified by measuring 51Cr release from prelabeled cells and lactate dehydrogenase release. Reactive oxygen metabolites caused dose-dependent cytotoxicity. Good correlation was found between the values for 51Cr and lactate dehydrogenase release. Reactive oxygen metabolite-induced cell damage was reduced by catalase but not by superoxide dismutase. Cellular superoxide dismutase and catalase activities were not increased after incubation with exogenous superoxide dismutase and catalase for up to 5 hr. Pretreatment with diethyldithiocarbamate inhibited cellular superoxide dismutase activity without inhibiting other antioxidants such as catalase, glutathione, glutathione reductase and glutathione peroxidase and sensitized cells to reactive oxygen metabolite-induced cytotoxicity. We conclude that hydrogen peroxide is an important mediator in hypoxanthine-xanthine oxidase-induced cell damage and that superoxide dismutase plays a critical role in cellular antioxidant defenses against hypoxanthine-xanthine oxidase-induced cytotoxicity in cultured rat hepatocytes in vitro.


Lack of association between cytomegalovirus infection, HLA matching and the vanishing bile duct syndrome after liver transplantation. In this study we evaluated the association between cytomegalovirus infection alone or in relation to human leukocyte antigen matching and the development of vanishing bile duct syndrome, a form of chronic hepatic allograft rejection. A total of 81 consecutive liver transplant recipients were studied. Cytomegalovirus infection developed in 46 recipients (57%), and vanishing bile duct syndrome occurred in 9 recipients (11%). Cytomegalovirus infection developed in only five of the nine patients with vanishing bile duct syndrome. Univariate analysis of pretransplant recipient/donor cytomegalovirus serological tests and human leukocyte antigen typing showed they were not significant risk factors for the development of vanishing bile duct syndrome. Time-dependent analysis of cytomegalovirus infection after transplantation as a risk factor for vanishing bile duct syndrome, in a multivariate analysis with human leukocyte antigen match, showed no statistical significance. In our study, no association was found between cytomegalovirus infection alone or in relation to class I or II human leukocyte antigen match and the subsequent development of vanishing bile duct syndrome.


The mechanism of inhibition of hepatitis B virus replication by the carbocyclic analog of 2'-deoxyguanosine. The carbocyclic analog of deoxyguanosine inhibits hepatitis B virus replication by greater than 95% in the hepatitis B virus-producing cell line (2.2.15) as monitored by decreases of secreted hepatitis B virus DNA, hepatitis B virus polymerase activity and intracellular episomal hepatitis B virus DNA. Transcription of hepatitis B virus RNA from chromosomally integrated hepatitis B virus DNA was unaffected. Radioactive carbocyclic 2'-deoxyguanosine was directly phosphorylated within the 2.2.15 cells and was incorporated exclusively into DNA. In contrast, radioactive deoxyguanosine was presumably metabolized through the "salvage" pathway in which the guanine was primarily incorporated into cellular RNAs. The rate of incorporation of carbocyclic 2'-deoxyguanosine in 2.2.15 cells was similar to that in the parental cell line (HepG2), which does not contain hepatitis B virus sequences. Greater than 90% of the analog was present at internal sites within the DNA, indicating that the analog did not function as a DNA chain terminator. Kinetic analysis of the Km and Ki of dGTP and carbocyclic 2'-deoxyguanosine 5'-triphosphate, respectively, using both hepatitis B virus polymerase and DNA polymerase delta indicated that the analog is a competitive inhibitor for dGTP. Although both polymerases had similar Km's for dGTP, the Ki for carbocyclic 2'-deoxyguanosine 5'-triphosphate was about 6 times lower using the hepatitis B virus polymerase. This would indicate that, at low concentrations of intracellular carbocyclic 2'-deoxyguanosine 5'-triphosphate, the hepatitis B virus polymerase would be preferentially inhibited. We propose this to be the mechanism acting to inhibit preferentially hepatitis B virus replication in the tissue culture cells.


Desensitization of adenylate cyclase and increase of Gi alpha in cardiac hypertrophy due to acquired hypertension. The present study investigated whether reduced adenylate cyclase activity and an increase in inhibitory guanine nucleotide binding proteins (Gi alpha), which have been observed in the failing human heart, already occur in myocardial hypertrophy before the stage of heart failure. In membranes of hypertrophic hearts from rats with different forms of experimentally induced hypertension without heart failure (one-kidney, one clip rats, deoxycorticosterone-treated rats, and rats with reduced renal mass), basal as well as isoprenaline-, 5'-guanylylimidodiphosphate-, and forskolin-stimulated adenylate cyclase activity was reduced. The activity of the catalyst was depressed in deoxycorticosterone but unchanged in one-kidney, one clip and reduced renal mass compared with controls. The number of beta-adrenergic receptors was similar in all groups. Radioimmunological quantification of Gi alpha proteins revealed an increase by 73% in one-kidney, one clip, 67% in reduced renal mass, but only 20% in deoxycorticosterone compared with sham-operated, age-matched control rats. The increase of Gi alpha was accompanied by smaller changes of pertussis toxin-induced [32P]ADP-ribosylation of a 40-kd membrane protein. It is concluded that Gi alpha contributes to the reduced adenylate cyclase activity in cardiac hypertrophy in one-kidney, one clip and reduced renal mass and to a smaller extent in deoxycorticosterone. It is suggested that an enhanced expression of Gi alpha could occur not only in severe heart failure but also in cardiac hypertrophy and could, therefore, contribute to myocardial depression and progression of disease in heart failure. In addition, Gi alpha might represent an important regulatory mechanism for cardiac adenylate cyclase activity and thus, might play an important role in various cardiac diseases.


Effects of OPC-21268, an orally effective vasopressin V1 receptor antagonist in humans. An orally effective, nonpeptide vasopressin V1 receptor antagonist, OPC-21268 was produced for possible human use. We investigated the effects of OPC-21268 on the vascular effects of intra-arterially infused arginine vasopressin in human forearm vessels. The brachial artery was cannulated for drug infusions and direct measurement of arterial pressure. Forearm blood flow was measured by a strain gauge plethysmograph, and forearm vascular resistance was calculated. Arginine vasopressin was infused intra-arterially at doses of 0.02, 0.06, 0.09, 0.2, 0.6, and 1.2 ng/kg/min. The lower doses of arginine vasopressin increased, whereas the higher doses of arginine vasopressin decreased forearm vascular resistance (p less than 0.01). Intra-arterial infusion of phenylephrine at doses of 0.2, 0.4, and 2.4 micrograms/min increased forearm vascular resistance dose-dependently (p less than 0.01). OPC-21268 (50 mg for two, 100 mg for six, and 200 mg for two subjects) given orally did not alter resting arterial pressure, forearm vascular resistance, or heart rate. OPC-21268 decreased vasoconstrictor responses to arginine vasopressin at doses of 0.02 (p less than 0.02) and 0.09 (p less than 0.05) ng/kg/min and augmented vasodilator responses to arginine vasopressin at a dose of 1.2 ng/kg/min (p less than 0.01). However, the vasoconstrictor responses to phenylephrine were not altered by OPC-21268. These results demonstrated that OPC-21268 effectively and specifically antagonized the V1 receptor-mediated vasoconstriction in human forearm resistance vessels. These results suggest that OPC-21268 may be useful therapeutically to antagonize the vasoconstriction caused by arginine vasopressin in some pathological states.


Chronic kinin receptor blockade attenuates the antihypertensive effect of ramipril. The contribution of endogenous kinins to the chronic antihypertensive effect of angiotensin converting enzyme inhibitors was investigated in two-kidney, one clip hypertensive Wistar rats, using the new bradykinin B2-receptor antagonist HOE 140 (D-Arg, [Hyp3, Thi5, D-Tic7, Oic8]-bradykinin). In a first protocol, rats were pretreated orally with the angiotensin converting enzyme inhibitor ramipril (1 mg/kg per day), for 4 weeks. Acute blockade of bradykinin receptors by intravenous injections of HOE 140 at doses of 8.4 and 100 micrograms/kg, which inhibited the depressor responses to exogenous bradykinin, did not affect the antihypertensive effect of ramipril in these animals. Bradykinin receptors were then blocked chronically by subcutaneous infusion of HOE 140 (500 micrograms/kg per day) via osmotic minipumps for 6 weeks, while ramipril treatment was continued. HOE 140 partially reversed the antihypertensive effect of ramipril from 115.3 +/- 4.6 to 123.8 +/- 3.3 mm Hg (mean arterial blood pressure) after 3 weeks and to 121.3 +/- 2.9 mm Hg after 6 weeks. In contrast, in controls (ramipril plus subcutaneous vehicle infusion) mean arterial blood pressure decreased further from 112.0 +/- 6.0 to 110.3 +/- 4.9 mm Hg after 3 weeks and to 103.7 +/- 5.0 mm Hg after 6 weeks (p less than 0.05 and p less than 0.01, HOE 140 versus controls). Plasma catecholamines were not significantly different between the two groups at the end of the experiment, indicating that the partial reversal of the antihypertensive effect was not due to a bradykinin-like agonistic effect on catecholamine release.(ABSTRACT TRUNCATED AT 250 WORDS)


Treatment of cocaine dependence in methadone maintenance clients: a pilot study comparing the efficacy of desipramine and amantadine. We conducted a pilot study (N = 22) comparing the efficacy of desipramine and amantadine for treatment of cocaine dependence in methadone maintenance clients. The study which lasted 12 weeks, was double-blind, randomly assigned, and placebo-controlled. Subjects met DSM-III-R criteria for active cocaine dependence. All three groups' cocaine use, craving, and depressive symptoms declined significantly, but intergroup differences were not significant. Clients receiving desipramine were significantly more likely to remain in treatment and to be cocaine free at study completion. The results emphasize the importance of delivering comprehensive services to the cocaine user in methadone treatment. Further evaluations of these two medications as adjuncts in the treatment of cocaine dependence are needed.


Innervation of lymphoid organs and implications in development, aging, and autoimmunity. We now have substantial evidence demonstrating noradrenergic sympathetic and peptidergic innervation of both primary and secondary lymphoid organs. We have established criteria for norepinephrine, and some of the neuropeptides, as neurotransmitters, and have found changes in immune responsiveness following pharmacological manipulation of noradrenergic sympathetic or peptidergic nerves. Classic receptor binding studies have demonstrated a wide variety of target cells that possess beta-adrenoceptors and receptors for neuropeptides on cells of the immune system, including lymphocyte subsets, macrophages, accessory cells, or stromal elements. In this chapter we describe noradrenergic and peptidergic innervation of primary and secondary lymphoid organs in development, at maturation and during the normal aging process, and discuss possible functional implications of direct neural signals onto cells of the immune system at critical time points in the lifespan of an animal. Further, we examine for involvement of noradrenergic sympathetic and peptidergic innervation in the development and progression of several autoimmune disorders, including adjuvant-induced arthritis, New Zealand mice strains as a model for hemolytic anemia and lupus-like syndrome, and the experimental allergic encephalomyelitis model for multiple sclerosis.


Immune modification due to chemical interference with transmembrane signalling: application to polycyclic aromatic hydrocarbons. Triggering of the T-cell antigen receptor complex and some other surface molecules is coupled to the phosphodiesterase (phospholipase C)-mediated hydrolysis of membrane phosphoinositides, in particular, phosphatidylinositol-4,5-biphosphate (PiP2). PiP2 hydrolysis generates two products, inositol 1,4,5-triphosphate and diacylglycerol, which act in concert as second messengers to increase the free intracellular calcium concentration and activate protein kinase C, respectively, thereby stimulating subsequent events leading to cellular activation and proliferation. Transmembrane signalling in T-lymphocytes represents a potential target for designated drugs as well as immunotoxicants. Immunotoxic effects of polycyclic aromatic hydrocarbons are discussed in the view of interaction with transmembrane signalling in the T-lymphocyte.


Leukotriene B4 in the immune system.

Leukotriene B4 in the immune system. Leukotriene (LT) B4 is a biologically active molecule derived from arachidonic acid via the 5-lipoxygenase pathway. It mediates certain inflammatory and immunological reactions. The role of LTB4 in the immune system has been questioned since lymphocytes have been regarded to lack the enzymes involved in LTB4 formation. This review focuses on the recently described biosynthesis of LTB4 in B-lymphocytes and the effects of this compound on lymphocyte functions.


Targeting growth factor receptors with fusion toxins. Recombinant toxins which bind to growth factor receptors have been prepared and used to kill cells responsible for malignant or autoimmune disease. Our strategy has been to genetically fuse ligands to different forms of Pseudomonas exotoxin which due to mutations or deletions do not bind to normal cells. The resulting recombinant chimeric toxins, in concentrations often less than 1 ng/ml, selectively kill cells expressing the appropriate growth factor receptor. The ligand may be a growth factor, such as transforming growth factor alpha (TGF alpha), interleukin 6 (IL6) or interleukin 2 (IL2), or single chain antigen binding proteins, such as the variable heavy and light regions of the monoclonal antibody anti-Tac. These chimeric toxins kill not only established cell lines but also fresh tumor cells from patients and display anti-tumor activity toward human malignant tumors in nude mice. While clinical trials are beginning with some of these agents, work continues to improve the effectiveness of recombinant chimeric toxins, and to widen the scope of disorders which might be treated by this approach.


Development of dietary obesity in rats: influence of amount and composition of dietary fat. This study was conducted to examine long-term effects of amount and type of dietary fat on body weight and body composition. Adult male Wistar rats were fed high fat (HF; 60% of calories) or low fat (LF; 20% of calories) diets for 28 weeks. Half of the rats in each condition received diets with saturated fat (lard) (S) and the remainder received diets with polyunsaturated fat (corn oil) (U). From 28-39 weeks, HF rats were switched to LF diets (fat type remained constant). From 40-50 weeks, previously HF fed rats were weight-matched to rats in the LF fed groups. HF rats became fatter than LF rats during weeks 1-28 and remained heavier and fatter from weeks 28-39. During weeks 1-28, type of dietary fat had no effect on total body fat in either HF or LF rats, but during period 2 (weeks 28-39), U rats were heavier and fatter than S rats. There was some indication that U diets were associated with greater accumulation of fat in subcutaneous adipose tissue depots than S diets. From 40-50 weeks, rats previously fed the HF diet required less food to maintain their body weight than did LF diet rats. In summary, these results suggest that although both amount and type of dietary fat can affect body weight and body composition, the effects of the type of fat are less than those of amount of dietary fat.


Family practice residents' identification and management of obesity. This study, involving 25 family practice residents and 2746 patients in a family practice residency programme, addressed four hypotheses regarding the identification and management of obesity in the primary care setting: (i) the physician-identified prevalence of obesity is significantly lower than the actual prevalence in the population, (ii) obesity is more likely to be addressed with management actions when it is recorded on the medical record problem list than when it is not recorded, (iii) physician actions dealing with obesity are influenced by the patient's age, sex, level of motivation, and body mass index (BMI) value, and (iv) the type of physician management actions taken are affected by the patient's age, sex, level of motivation, and level of BMI value. Obesity was identified as a risk factor by physicians for 51.6% of all patients with a BMI greater than or equal to 30. Obesity was recorded on the medical record problem list for 70.6% of the physician-identified obese patients. When obesity was recorded on the problem list, management actions were taken for 92.9% of patients. However, when obesity was recorded on a risk factor evaluation form but not on the problem list, management actions were taken for only 56.6% of patients. Self-care strategies were selected as the management strategy more frequently than return visits. Demographic characteristics, BMI value and level of patient motivation did not influence the selection of follow-up management strategies. Given the potential for significant improvement in a patient's health status through early recognition and aggressive management of obesity, the barriers to physician identification and involvement in clinical management of obesity deserve further investigation.


Potentiating effects of cigarette smoking and moderate exercise on the thermic effect of a meal. The purposes of this study were: (i) to determine the relative thermic effects of smoking and exercise in fasted and postprandial states, and (ii) to determine whether there is a weight-controlling caloric advantage of moderate exercise in the postprandial state which might be used upon smoking cessation. The subjects were ten physically fit, young, male smokers. Twenty minutes of smoking (two cigarettes) while fasted resulted in a 6.0% (12.5 kcals/3h) increase in resting metabolic rate (RMR), and 20 minutes of walking (5.8 km/h) while fasted increased post-exercise RMR by 5.8% (11.7 kcals/3h). The thermic effect of a meal (TEM) (740 ml, 874 kcals) increased RMR 21.0% (42.3 kcals/3h), which was increased to 22.1% by smoking (45.0 kcals/3h). Exercise after a meal increased RMR 29.1% (58.1 kcals/3h). Post-prandial exercise potentiated the TEM, as the energy cost was 4.0 kcals/3h greater than the sum of the thermic effect of the meal plus the thermic effect of exercise in the fasted state. Postprandial exercise expended 112.2 kcals/20 min vs. 101.6 kcals/20 min for fasted exercise, a difference of 10.4%. The difference increased to 14.6% when the post-exercise thermic effect was included. It was concluded that the thermic effect of smoking and exercise were similar in the fasted state, but were substantially different postprandially. Moderate postprandial exercise appears to offer a substantial weight-controlling advantage when compared with fasted exercise.


Norepinephrine inhibits rat pre-adipocyte proliferation. Hormonal and neural status are major determinants for cellular growth. The purpose of this study was to assess the influence of the adrenergic hormones on pre-adipocyte growth in primary cell culture. Stromal-vascular cells were obtained from the inguinal pad of young rats and grown in culture for two weeks. Cells were exposed to norepinephrine (NE) during the proliferative phase of growth, labelled by [3H]-thymidine incorporation and then placed on a differentiation promoting medium. Adipocytes and stromal cells were separated using a density gradient, and [3H]-thymidine content was determined for both cell types. NE reduced [3H]-thymidine uptake indicating a reduction in pre-adipocyte proliferation. NE-induced inhibition of pre-adipocyte growth was blocked by the presence of propranolol, whereas phenoxybenzamine had no effect, thereby suggesting that NE-inhibition is through beta-adrenoceptors. Pre-adipocytes were treated with NE for varying lengths of time to investigate whether cells were desensitized to chronic beta-adrenergic stimulation. In addition, adenosine deaminase (ADA) was also applied to eliminate adenosine which may accumulate during NE stimulation. Neither the duration of NE exposure nor ADA treatment affected adrenergic control of adipocyte growth. These studies indicate that NE reduces pre-adipocyte proliferation and therefore may be an important negative regulatory component of adipocyte growth.


Eating behaviour in obese and normal weight 11-year-old children. The eating behaviour of 23 normal weight and 20 obese 11-year-old children was measured by the computerized eating monitor VIKTOR. The total intake of food, duration of consumption, rate of consumption and the relative rate of consumption were measured during two lunch meals. Subjective motivation to eat and food preferences were also measured. The obese children ate faster (P less than 0.05) and did not slow down their eating rate towards the end of the meal (P less than 0.05) as much as normal weight children. The obese children also described themselves as having less motivation to eat before lunch than normal weight children (P less than 0.05). A deficient satiety signal or an impared response to such signals in obese subjects could possibly explain these differences found.


Changes in hypothalamic neurotensin concentrations and food intake in rats fed a high fat diet. Neurotensin (NT), a peptide present both in the brain and in the gastrointestinal tract, has potent anorexigenic effects when centrally injected in rats and is secreted after fat ingestion. High fat diets are often associated with increased energy intake. The aim of this study was therefore to evaluate the role of neurotensin in the feeding behaviour of rats fed on a high fat (HF) diet. Adult Long-Evans rats were fed for two weeks either a control (C) well-balanced diet (n = 10) or a fat-rich diet containing about two-thirds of its energy as fat (margarine and peanut oil; n = 10). Neurotensin was measured by a specific radioimmunoassay in the plasma and in several microdissected brain nuclei involved in the regulation of feeding behaviour. Ingestion of the HF diet induced an increased body weight gain (47.6 +/- 7.7 g (HF) vs. 37.6 +/- 9.3 g (C); P less than 0.05) and an increased energy intake (+ 7.2%; P less than 0.05). Plasma fasting NT concentrations were not affected by the HF diet. In the hypothalamus, significant decreases in NT concentrations were measured in the HF rats in two nuclei important in the regulation of food intake, i.e. the paraventricular nucleus (1.72 +/- 0.16 (HF) vs. 2.27 +/- 0.15 (C) ng/mg protein; P less than 0.05) and the lateral hypothalamus (1.87 +/- 0.16 (HF) vs. 2.37 +/- 0.19 (C) ng/mg protein; P less than 0.05). On the other hand, no variations were measured in the ventral tegmental area, an important site for the metabolism and regulatory action of neurotensin and in other hypothalamic nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)


Prevalence of obesity in Bombay.

Prevalence of obesity in Bombay. Obesity has been reported in developed as well as developing countries. However, data on a large sample of the Indian population are lacking. This study analysed the prevalence of obesity among 1,784 adults in Bombay from various sections of society. Since the prevalence of obesity depends upon the criteria used, prevalence was judged by three criteria viz. percentage excess of body weight, body mass index, and body fat content. The data were classified and analysed according to occupation, age group, income, diet type, and also with respect to family history of obesity. The three methods gave a different prevalence of obesity. In general, the criterion of body mass index under-estimated, and body fat content over-estimated the prevalence as compared to that obtained by percentage excess body weight. An extremely high prevalence of obesity was found in all sub-groups of the sample. As judged by a body mass index of 25 and above, male students had the lowest (10.7%) and male medical doctors had the highest (53.1%) prevalence of obesity. Prevalence was highest for the age group 31-50 years for males and females, and declined on either side of this age range. Prevalence was directly proportional to financial income, and subjects with a family history of obesity had a greater prevalence of obesity compared to those without. This study indicates the gravity of the problem of obesity in Bombay, and provides directions for nutritional planning in the future.


Corticosterone and tri-iodothyronine transport into brain of obese (ob/ob) mice. Unidirectional influx of [3H]-corticosterone and [125I] 3,5,3'-tri-iodothyronine (T3) into brain, and of [3H]-corticosterone into livers, of 8-10 week old lean and obese (ob/ob) mice was measured with a bolus injection, tissue-sampling technique. Uptake of corticosterone by brain and liver of ob/ob mice was comparable to that observed in lean mice, and unaffected by corticosterone concentration in the bolus (17-32 nM or 25 microM) or by addition of lean or ob/ob mouse blood serum to the bolus injectate. Uptake of T3 by ob/ob mouse brain was lower than observed in lean mice when the bolus contained physiological concentrations of T3 (2.6-19 nM). An increase in T3 concentration in the injectate to 5 microM depressed T3 uptake by brain of lean mice, but not of ob/ob mice. Addition of blood serum from ob/ob mice to the bolus further lowered influx of T3 into the brain of lean and ob/ob mice. Even though unidirectional influx of T3 into the brain of ob/ob mice was impaired, steady-state T3 concentrations in ob/ob mice brain were similar to lean values. The enhanced sensitivity of ob/ob mice to the obesity-producing effects of glucocorticoids is not caused by an increased influx of corticosterone into brain or liver.


Analysis of uncoupling protein and its mRNA in adipose tissue deposits of adult humans. Brown adipose tissue (BAT) is a specialized adipose tissue whose specific marker is the uncoupling protein (UCP). UCP and its mRNA were previously detected in the perirenal fat of several adult subjects undergoing surgery for pheochromocytoma. We have investigated the possible association of the presence of UCP and its mRNA with pathological conditions other than pheochromocytoma. We obtained adipose tissue from both the periadrenal and the perirenal regions of 36 subjects: group A: human infants (n = 6); group B: adult subjects (n = 11) undergoing surgery for pheochromocytoma; group C: adult subjects (n = 9) undergoing surgery for other endocrine pathologies; group D: adult patients (n = 10) operated for non-endocrine pathologies. In all subjects of group A UCP was detectable by Western analysis. Interestingly, in two newborns, we also found a positive signal for UCP in the peristernal and the retroperitoneal adipose tissues as well as in the perirenal fat. We also identified UCP in eight cases in group B, in five cases in group C and six cases in group D. The human H-UCP-0.5 genomic probe detected a typical BAT mRNA in the periadrenal adipose tissue of all subjects of groups B, C and D showing a positive Western blot. Our results confirm the presence of well-developed BAT in human infants, as well as in adults with pheochromocytoma. They also suggest that human BAT UCP and UCP mRNA are present in adult subjects in pathological conditions other than pheochromocytoma. It might be argued that certain hormones distinct from catecholamine could activate BAT development in human adults.(ABSTRACT TRUNCATED AT 250 WORDS)


An assessment of the thermogenic effects of fluoxetine in obese subjects. Fluoxetine is an antidepressant drug with weight reducing properties. To assess whether fluoxetine has an ability to promote diet induced thermogenesis (DIT), 30 obese subjects (BMI 30-45 kg/m2) underwent a double blind, randomized, cross-over trial of 60 mg fluoxetine versus placebo. A two week single blind, run-in period on placebo was incorporated into the study to allow for placebo responders. The first stage of the study lasted for 14 days followed by a six week cross-over wash-out phase and concluded with the second 14 day stage of the study. An estimate of resting metabolic rate (RMR) was measured by continuous indirect calorimetry using the ventilated hood technique. Metabolic measurements were performed on six occasions, immediately before the first tablet was taken in the first stage, 24 hours after the first tablet was consumed and on the last day of the first stage; these three recordings were repeated on the second stage of the study. On each occasion a control reading of RMR was taken for 30 minutes then DIT was measured for 90 minutes following a lemon and glucose drink (1 g/kg body weight). Whilst a significant weight reduction (1.16 kg, P less than 0.05) occurred in the active stage, no such effect was achieved in the placebo phase. No differences were found between the two stages with regard to RMR, total DIT, peak DIT and time taken to reach peak DIT. We conclude that fluoxetine does not stimulate metabolism and that the weight reduction after 14 days therapy is due to other mechanisms.


Regulatory functions of soluble auxin-binding proteins. Since the effects of auxin on plant tissues are complex, the mode of action of auxin at the molecular level may not depend on a single mechanism. There may be a mechanism by which the interaction of auxin with receptors localized in the cytoplasmic membranes activates certain enzymes which are necessary to generate the putative second messengers. On the other hand, soluble auxin-binding proteins have been isolated from a variety of plant tissues. Some of these proteins have a high affinity for auxins and the binding is auxin specific, reversible, and saturable, characteristics which suggest that these proteins may be auxin receptors. Although these criteria are often used to distinguish real receptors from nonfunctional binding proteins, it is necessary to clarify the biological function of the binding proteins to classify them as putative receptors. The reported results on the function of soluble auxin-binding proteins demonstrate that, in the transcription system composed of isolated nuclei, auxin interacts with soluble auxin-binding proteins and stimulates the expression of specific genes. Thus, one of the mechanisms of action of auxin may involve a direct interaction with a soluble receptor protein, such that the resultant auxin-receptor complex, possibly together with other protein factors, can subsequently recognize the promoter region of specific gene(s) and interact with RNA polymerase II to cause a transcription of the gene(s).


Nile red staining of lysosomal phospholipid inclusions. We have employed the fluorescent dye nile red to distinguish between normal cells and cells containing lysosomal accumulations of phospholipids. When fibroblasts from an individual with a genetic deficiency in lysosomal sphingomyelinase activity (Niemann-Pick disease) were stained with nile red and visualized by fluorescence microscopy, orange-colored inclusions were observed throughout the cytoplasm. The orange fluorescent bodies could be distinguished from the neutral lipid droplets that fluoresce a brilliant yellow-gold in the presence of nile red. These inclusions were also observed in alveolar macrophages obtained from rats treated with amiodarone, an antiarrhythmic agent known to produce lysosomal phospholipidosis. Flow cytofluorometric analysis revealed that staining of these phospholipid-rich macrophages with nile red can distinguish them from control alveolar macrophages. These results demonstrate that nile red can be employed for the rapid staining of cellular phospholipid inclusions.


Determination of diborane by adsorption sampling using modified silica gel and the chromotropic acid-HPLC method. A method for determining atmospheric diborane in concentrations higher than 1/10 of TLV, i.e., 0.01 ppm, has been developed using the adsorption sampling method. Silica gel impregnated with potassium permanganate, synthetic resin activated carbon impregnated with or without mercury(II) chloride and activated carbon impregnated with chromate salt showed adsorption capacities larger than 18 l of 3 ppm diborane test gas when the test gas was drawn at 300 ml/min. Complete desorption of the adsorbed diborane was possible only from silica gel impregnated with potassium permanganate into a hydroxylamine hydrochloride solution. As methods for determining the desorbed boron, both the chromotropic acid-HPLC method and ICP-AES were applied. The former was more sensitive, but the latter was less influenced by coexistent substances. The most sensitive and reproducible procedure for diborane determination was as follows: diborane is collected with silica gel impregnated with potassium permanganate (0.05% (w/w)) and desorbed into hydroxylamine hydrochloride solution (400 micrograms/ml) followed by the determination of boron by the chromotropic acid-HPLC method. When diborane in 3 l of 0.1 ppm test gas was collected, the desorption efficiency was 105.3% with an RSD of 13.5%. The limit of quantitation of this method was 0.0026 ppm in 3 l air. Much lower concentrations can be determined by sampling larger amounts of air.


Response of xenografts of human malignant gliomas and squamous cell carcinomas to fractionated irradiation. The response of xenografts of five human malignant glioma cell lines and two human squamous cell carcinomas to fractionated irradiation was studied. For this, the tumors were transplanted into nude mice which had been further immunosuppressed by 6 Gy whole-body irradiation. Radiation was given as 30 fractions applied under normal blood flow conditions in two sessions per day over 15 days. Absolute and specific tumor growth delay after 48 Gy, and tumor control dose 50% (TCD50) were evaluated. Using local tumor control as experimental endpoint, four out of five malignant gliomas were more resistant to fractionated radiation therapy than the two squamous cell carcinomas. The TCD50s of these four gliomas ranged from 73 Gy to more than 120 Gy, whereas the TCD50s of the squamous cell carcinomas were 51 and 60 Gy. Absolute tumor growth delay correlated well with TCD50, but no correlation was obtained between specific growth delay and TCD50. The response of the human tumor xenografts in vivo did not correlate with the surviving fractions at 2 Gy of the same cell lines in vitro which have been previously obtained in our laboratory. The results suggest that the unique radioresistance observed in malignant gliomas in patients is at least in part reflected in human tumor xenografts. The lack of correlation between the surviving fraction at 2 Gy in vitro and the tumor response in vivo could be a consequence of an immune response by the host, a difference in cell radiation sensitivity between cell lines and xenografted tumors, or of differences of parameters such as hypoxic fraction, rate of repopulation, and cell cycle effects between the different tumor lines studied. It illustrates the difficulties which might be involved in the prediction of the response of individual tumors to radiation therapy based solely on the intrinsic radiosensitivity of the tumor cells as assayed by in vitro assays of colony formation.


Radioprotection by vitamin E: injectable vitamin E administered alone or with WR-3689 enhances survival of irradiated mice. Radioprotection by injectable vitamin E (alpha-tocopherol) was investigated in mice exposed to 60Co radiation (0.2 Gy/min). Vitamin E injected subcutaneously either 1 hr before or within 15 min after irradiation significantly increased 30-day postirradiation survival in CD2F1 male mice. A dose reduction factor (DRF) of 1.11 (95% confidence interval [1.08, 1.14]) was observed for vitamin E at a dose of 100 IU/kg body weight administered within 15 min after irradiation. Combination studies with the phosphorothioate WR-3689 (S-2([3-methylaminopropyl]amino)ethylphosphorothioic acid) were undertaken to determine whether radioprotection by WR-3689 could be enhanced by vitamin E. Mice were given WR-3689 (150-225 mg/kg, intraperitoneally) 30 min before irradiation and were given vitamin E (100 IU/kg) either 1 hr before or within 15 min after irradiation. Survival was significantly increased in mice given vitamin E and WR-3689 before irradiation as compared to mice given WR-3689 alone: the DRF for WR-3689 (150 mg) was 1.35 [1.32, 1.38]; for WR-3689 combined with vitamin E (100 IU), the DRF was 1.49 [1.45, 1.53].


Thermoradiotherapy of recurrent malignant brain tumors. In an attempt to improve local control and survival over those achieved with brain implant alone, a Phase I/II study of interstitial thermoradiotherapy was undertaken for recurrent malignant gliomas and recurrent solitary brain metastases. Between June 1987 and September 1990, 49 tumors in 48 patients were treated with thermoradiotherapy, including 26 glioblastoma multiforme (GM), 16 anaplastic astrocytomas (AA), 4 adenocarcinomas, and 3 melanomas. Patient age ranged from 18 to 71 years and Karnofsky Performance Status from 40 to 90. Stereotactically implanted catheters were used for both hyperthermia and brachytherapy. Hyperthermia was administered immediately before and after brachytherapy, heating as much of the tumor as possible to 42.5 degrees C for 30 min using helical coil microwave antennas. High-activity iodine-125 sources delivered tumor doses of 32.6 to 63.3 Gy. Complications included reversible neurologic changes in 13 patients, 9 seizures, 4 infections, 1 deep venous thrombosis with pulmonary embolus, and 1 scalp burn. Eighteen patients underwent reoperation for tumor and/or necrosis. Follow-up ranged from 9 to 166+ weeks. The median follow-up for living patients with GM and AA was 37 weeks and 92 weeks, respectively. Actuarial median survival was 47 weeks for patients with GM. For patients with AA, actuarial survival was 65% at 18 months and median survival has not yet been reached. Multivariate analysis showed a strong correlation between freedom from local tumor progression and "T90" temperature or minimum tumor temperature. Interstitial brain thermoradiotherapy is now being evaluated in a randomized Phase II trial for previously untreated GM.


Natural alpha-interferon in combination with hyperfractionated radiotherapy in the treatment of non-small cell lung cancer. Our previous study in patients with small-cell lung cancer indicated that natural alpha interferon might be a radiosensitiser. In this study we considered 20 patients with inoperable non-small cell lung cancer, who were randomly assigned to receive either hyperfractionation radiotherapy alone, 1.25 Gy twice a day (6 hr interval), 60 Gy/48F/32d; or the same radiotherapy concurrently with alpha interferon. Patients in the radiotherapy+alpha interferon arm received 3 x 10(6) IU natural alpha interferon intramuscularly and 1.5 x 10(6) IU inhaled via a dosimeter-equipped jet nebulizer 30 min before each radiotherapy session. Tumor response and radiation-induced lung injury were assessed by serial chest radiographs, computerized tomography scans and lung function studies, during a 1 year follow-up period. No patient in either arm achieved complete response. On the other hand, five patients in the radiotherapy arm and six in the radiotherapy+interferon arm experienced partial response, and the corresponding figures for stable disease were three and one. Combined treatment with radiotherapy and inhaled and intramuscular interferon proved feasible but laborious, for both patients and staff. Pneumonitis and/or oesophagitis in the radiotherapy+interferon arm were moderate to severe, and only two patients tolerated the treatment without any modifications. No treatment modifications were necessary in the radiotherapy arm. The early deaths in the radiotherapy+interferon arm may have been treatment-related. The optimal way to combine interferon and radiotherapy to further evaluate its role as a radiosensitiser needs further studies in larger series.


32P-postlabeling analysis of DNA adducts in rats during estrogen-induced hepatocarcinogenesis and effect of tamoxifen on DNA adduct level. DNA adduct formation in the liver, pancreas, kidneys and uterus in ethynylestradiol (EE)-induced carcinogenesis and the effect of tamoxifen (TAM) on DNA adduct formation were evaluated in female Wistar JCL rats using the 32P-postlabeling method. Hyperplastic nodules were noted in the liver of all rats 4 months after the first oral administration of 0.075 mg of EE, and hepatocellular carcinoma was detected in 8.1% of rats treated with EE for 12 months. DNA adducts increased in the liver for 4 months, reaching a level of 7.3 adducts/10(7) nucleotides and decreasing thereafter. Formation of DNA adducts was also noted in the pancreas and kidney, but the adduct levels were lower than those in the liver. TAM inhibited estrogen receptors (ER) in liver tissues and completely suppressed the development of hyperplastic nodules or hepatocellular carcinoma but did not affect DNA adduct formation in the liver. In this model, therefore, EE is considered to cause mutations of hepatocytes due to DNA adduct formation without mediation by ER and to induce initiated cells to develop into hepatocellular carcinoma in the presence of ER-mediated hormonal activities.


Impaired long-term T cell immunity to Epstein-Barr virus in patients with nasopharyngeal carcinoma. The long-term T cell immunity to Epstein-Barr virus (EBV) is considered to play an important role in suppressing proliferation of EBV-infected B cells and outgrowth of EBV-associated tumors. It can be manifested and quantified by the EBV-induced focus regression assay. In the present study, we examined the strength of T cell immunity to EBV in patients with nasopharyngeal carcinoma (NPC) and other cancers originating from the head and neck region. In contrast to patients with other types of cancers, including EBV-negative NPC, patients with EBV-positive NPC were found to have a profound impairment in the long-term T cell immunity to EBV.


Human T-cell leukemia virus-1-positive cell line established from a patient with small cell lung cancer. A stable cell line, KHM-3S, was established from a patient with small cell lung cancer (SCLC), who had a high serum level of soluble interleukin 2 receptors (sIL2-R) and was seropositive for human T cell leukemia virus (HTLV)-1. KHM-3S cells were positive for IL2-R (Tac) and NKH-1, but negative for other lymphocytic markers such as OKT 11, OKT 4, OKT 8, T cell receptor (WT 31), B 1, and B 4. Moreover, the KHM-3S cells were negative for leukocyte common antigen and strongly positive for neuron-specific enolase (NSE). Secretion of sIL2-R and NSE by the KHM-3S line was detected by an enzyme-linked immunosorbent assay. Rearrangement of the T cell receptor gene and monoclonal HTLV-1 integration were found by Southern blot analysis of KHM-3S DNA. However, Northern blot analysis showed no T cell receptor mRNA. KHM-3S may be useful for studies on the role of HTLV-1 in carcinogenesis and IL2-R expression in SCLC.


Phenotypic and genotypic lineage switch of a lymphoma with shared chromosome translocation and T-cell receptor gamma gene rearrangement. A case of non-Hodgkin's lymphoma showed a phenotypic and genotypic cell lineage switch twice during nine years of his clinical history; first, T-cell type, pleomorphic small cell lymphoma developed, followed by B-cell type, diffuse centroblastic/centrocytic lymphoma, and finally T-zone lymphoma without follicles again developed, from which AST-1 cultured cell line was established. Karyotype analysis demonstrated a shared abnormal chromosome, der(1)t(1;?)(p36;?), among the first relapsed B-cell tumor, the second relapsed T-cell tumor and AST-1 cell line. Furthermore, T-cell receptor (TCR) gamma gene rearrangement bands of the same size were observed in the first relapsed B-cell tumor and the second relapsed T-cell tumor as well as AST-1 cell line. These results suggested that both relapsed tumors of different cell lineages are derived from a common malignant clone, presumably a committed lymphoid stem cell. A unique translocation, t(2;14)(q37;q11.2), which may involve TCR delta/alpha gene complex, was observed in the second relapsed tumor and AST-1 cells. To attempt to isolate the breakpoint of this translocation, the configuration of TCR delta/alpha gene complex was studied. The result showed that two rearrangements of TCR alpha gene detected with J alpha probes were the products of the normal TCR rearrangement process, and were not involved in the translocation at this region. This patient, together with the AST-1 cell line, provided us a unique opportunity to study the development and clonal evolution of malignant lymphoma.


Development of endocochlear potential and compound action potential in the rat. The present study was designed to investigate the developmental changes of the endocochlear potential and compound action potential simultaneously from rat pups of various ages. Animals were anesthetized with ketamine/xylazine, and the endocochlear potential was measured with a glass microelectrode. At the same time, a wire electrode was placed on the round window to record the click-evoked compound action potential. The endocochlear potential was found to be very low during the first few days of postnatal life. A rapid increase in the value of the endocochlear potential was noted between eleven and thirteen days of age, and adult-like values were recorded by seventeen days of age. Compound action potential responses were recorded at thirteen days of age to high intensity clicks, followed by a progressive improvement of thresholds and reduction of latencies. The development of the endocochlear potential and compound action potential was found to be reciprocally related - as the magnitude of the endocochlear potential increased, the compound action potential threshold declined with increasing age. The development of the endocochlear potential was found to closely approximate the development of enzymatic activity of sodium, potassium-ATPase in the stria vascularis reported by Kuijpers (1974).


Mechanism of synergistic cytotoxic effect between tumor necrosis factor and hyperthermia. We previously reported that recombinant human tumor necrosis factor (rhTNF) and hyperthermia had a synergistic effect against tumors, in vitro and in vivo. We have now investigated the mechanism of this synergy by measuring the lysosomal enzyme activity and hydroxyl radical production of L-M cells treated with rhTNF and/or hyperthermia. A synergistic activation of lysosomal enzyme and the induction of hydroxyl radical production in L-M cells treated with both rhTNF and hyperthermia was observed. A synergistic cytotoxic effect was observed when rhTNF and hyperthermia were combined, and was inhibited by the addition of a reactive oxygen scavenger, dimethyl sulfoxide or bipyridine. The results show that the augmenting effect of hyperthermia on lysosomal enzyme activation and induction of hydroxyl radical production by rhTNF plays an important role in the synergistic cytotoxic effect.


Cholinergic regulation of the phosphoinositide second messenger system in the guinea pig organ of Corti. The effect of cholinergic agents on the phosphoinositide second messenger system was investigated in the cochlea of the adult guinea pig in vivo and in vitro. In vivo, phospholipids were labeled with [32P]-orthophosphate by perilymphatic perfusion and their hydrolysis assayed in 'chase' experiments with non-radioactive orthophosphate. Carbachol (1 mM) reduced the content of 32P-labeled phosphatidylinositol 4,5-bisphosphate in the organ of Corti from 31% to 21% of total 32P-lipids, indicating stimulated hydrolysis. The pharmacology of this effect was studied in detail in vitro via the release of inositol phosphates from phosphoinositides pre-labeled with 3H-inositol. Release was increased 2-fold by 1 mM carbachol, 1.6-fold by 1 mM muscarine, but was unaffected by dimethylphenylpiperazinium; the stimulation was blocked by 1 microM atropine but not mecamylamine. These responses indicate the coupling of phosphoinositides to a muscarinic receptor. Furthermore, stimulated inositol phosphate release was higher in the base of the organ of Corti than in the apex which correlates with the increased cholinergic efferent innervation of outer hair cells in the basal region. These results suggest that muscarinic-stimulated inositol phosphate release occurs at the level of the outer hair cell and thus may have an important modulatory role in auditory transduction.


Increase in potential activities of protein phosphatases PP1 and PP2A in lymphoid tissues of autoimmune MRL/MpJ-lpr/lpr mice. The differential assay conditions for protein phosphatases PP1, PP2A, and PP2C were extensively studied by using crude extracts from mouse lymphoid tissues as enzyme sources. Under these conditions, the protein phosphatase activities were measured in MRL/MpJ-lpr/lpr mice (MRL/lpr mice), autoimmune-prone mice, and MRL/MpJ(-)+/+ mice (MRL/+/+ mice) and C3H/HeJ mice as the controls. In MRL/lpr mice, significant alterations in protein phosphatase activities from those in the control mice were demonstrated. In spleen and liver from MRL/lpr mice, potential activities of PP1 and PP2A were distinctly elevated over those of the control mice. These elevations appeared to be due to accumulation of the abnormal lymphocytes that emerged in MRL/lpr mice. Although the PP1 activity in MRL/lpr lymph nodes was lower than those of normal spleen and thymus, it was greatly increased by Co(2+)-trypsin treatment so that the PP1 activity of MRL/lpr lymph nodes was the highest among those of all the tissues examined. In contrast, PP2C activity showed no remarkable alteration in the autoimmune disease model mice as compared with that in the control mice. These results demonstrated a specific elevation in potency of protein dephosphorylation in the tissues of MRL/lpr mice, suggesting a new explanation for the defect in signal transduction in this disease.


Minimal model analyzing response of glycine receptors expressed in Xenopus oocyte: inhibition by a lipid hydroperoxide. Glycine receptor (GlyR) was expressed in Xenopus oocytes by injecting rat brain mRNA. Glycine (Gly)-elicited responses in the oocyte were measured by the voltage-clamping method. The following measurements were made to establish the relationship between Gly concentration and the current: 1) Gly-induced membrane current before desensitization, 2) Gly-induced membrane current after desensitization equilibrium, 3) fraction of the active form of the receptor after desensitization equilibrium, 4) rate of recovery of the desensitized receptors upon removal of Gly. These results were analyzed on the basis of the minimal model proposed for nicotinic acetylcholine and gamma-aminobutyric acid A receptor. The equilibrium and rate constants of the model were evaluated for GlyR. The effects of procaine and 13-L-hydroperoxylinoleic acid (LOOH) on GlyR were examined electrophysiologically. LOOH noncompetitively inhibited the receptor with the inhibition constant of 27 microM, while 1 mM procaine, a local anesthetic, did not inhibit GlyR at all.


1H-NMR assignment and secondary structure of human insulin-like growth factor-I (IGF-I) in solution. Human insulin-like growth factor-I (IGF-I) was studied by two-dimensional 1H-NMR spectroscopy. Resonance assignments were obtained for all the backbone protons and almost all of the sidechain protons of the total 70 amino acid residues, using sequence-specific assignment procedures. The secondary structure elements of human IGF-I were identified by investigation of the sequential and medium range NOEs as a preliminary step in determining the three-dimensional structure of this protein by means of distance geometry calculations. The typical NOEs of d alpha beta(i,i + 3) and d alpha N(i,i + 3), as well as the successive strong NOEs of dNN connectivities and slowly exchanging amide protons confirmed the presence of three helical segments corresponding to the sequence regions, Ala8-Cys18, Gly42-Cys48, and Leu54-Cys61, and the existence of a beta-turn in the Gly19-Gly22 region. Our results definitely indicate that the secondary structure of human IGF-I in solution is consistent with that of insulin in the crystalline state.


Partial purification and reconstitution of inositol 1,4,5-trisphosphate receptor/Ca2+ channel of bovine liver microsomes. The binding of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] to bovine liver microsomes was characterized. The Ins(1,4,5)P3 receptor of the microsomes was solubilized by 1% Triton X-100 and purified by sucrose density gradient, Heparin-Sepharose, DEAE-Toyopearl, ATP-Agarose, and Ins(1,4,5)P3-Sepharose column chromatographies. More than 1,000-fold enrichment of the Ins(1,4,5)P3-binding activity was achieved. Kd values of the binding activity were 2.8 nM in microsomes and 3.0 nM in the partially purified receptor, respectively, and the binding activity was optimal in the medium containing 100 mM KCl and at pH between 7.5 and 8.5. The presence of Ca2+ failed to inhibit the binding. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PtdIns), and phosphatidylinositol-4-monophosphate [PtdIns(4)P] showed no effect on the Ins(1,4,5)P3 binding. However, soybean phospholipids asolectin and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] strongly inhibited the binding activity. PtdIns(4,5)P2 inhibited the activity competitively with a half-maximal inhibitory concentration of 30 micrograms/ml. The partially purified Ins(1,4,5)P3 receptor was reconstituted into proteoliposomes. Fluorescence measurements using Quin 2 indicated that Ins(1,4,5)P3 stimulated Ca2+ influx into the proteoliposomes. The EC50 of Ins(1,4,5)P3 on Ca2+ influx was 50 nM. This result strongly suggest that Ins(1,4,5)P3 binding protein of liver microsomes acts as a physiological Ins(1,4,5)P3 receptor/Ca2+ channel.


Identification of an N-terminal domain of eukaryotic DNA topoisomerase I dispensable for catalytic activity but essential for in vivo function. We have found that deletion of a 70-amino acid domain, spanning from position 141 to 210 in the N-terminal part of human topoisomerase I, has no effect on the catalytic activity of the enzyme in vitro but suppresses the lethal consequence of overexpressing human topoisomerase I in a rad52 top1 Saccharomyces cerevisiae strain. By immunostaining, the 70-amino acid domain is shown to be necessary for nuclear location of topoisomerase I. We demonstrate that the nuclear localization signal from the SV40 large T antigen can substitute for the 70-amino acid domain, restoring both the lethal effect of overexpression and the correct subcellular localization of topoisomerase I. Thus, we have identified a domain in the N-terminal part of human topoisomerase I, nonessential for catalytic activity in vitro but serving an in vivo function by directing the enzyme to the nucleus. Based on sequence comparisons, we suggest that this domain is a conserved element in the apparently non-homologous N-terminal parts of yeast and human topoisomerase I.


Calcium and muscarinic agonist stimulation of type I adenylylcyclase in whole cells. The type I adenylylcyclase which was originally purified and cloned from bovine brain is stimulated by Ca2+ and calmodulin in vitro. Although it has been proposed that this enzyme may couple elevations in intracellular Ca2+ to increases in cAMP in whole cells, this has not been demonstrated in vivo. In this study, the type I adenylylcyclase was expressed in human 293 cells, and the influence of extracellular Ca2+ and Ca2+ ionophore on intracellular cAMP levels was examined. The cAMP levels of control cells were unaffected by Ca2+ and A23187. In contrast, intracellular cAMP in 293 cells expressing type I adenylylcyclase was markedly elevated by addition of A23187 and extracellular Ca2+. In the presence of forskolin, the muscarinic agonist carbachol also increased cAMP in 293 cells expressing the type I adenylylcyclase. These data indicate that the type I adenylylcyclase can be stimulated by Ca2+ in vivo, and that muscarinic agonists may indirectly stimulate the enzyme by increasing intracellular free Ca2+.


Coupling of two endothelin receptor subtypes to differing signal transduction in transfected Chinese hamster ovary cells. We examined the intracellular signal transduction of two endothelin receptor subtypes (ETA and ETB) by transfection and stable expression of individual receptor cDNAs in Chinese hamster ovary cells. Both receptors showed a rapid and marked stimulation of phosphatidylinositol hydrolysis and arachidonic acid release in response to agonist interaction. The two receptors, however, exhibited different responses in the cyclic AMP transduction cascades. ETA mediated the accumulation of cyclic AMP formation, whereas ETB displayed an inhibitory action on the forskolin-stimulated cyclic AMP accumulation. In both receptors, the responses of phosphatidylinositol hydrolysis, arachidonic acid release, and cyclic AMP formation were induced in complete agreement with the endothelin-binding selectivity of each receptor subtype. Endothelin, added together with GTP, activated the adenylate cyclase activity in membrane preparations of ETA-expressing cells, indicating the direct linkage of ETA to the adenylate cyclase system. Pertussis toxin treatment of ETA-expressing cells resulted in partial inhibition of the endothelin-induced cyclic AMP accumulation, whereas the same treatment of ETB-expressing cells completely abolished the endothelin-induced inhibition of cyclic AMP formation. Thus, the two endothelin receptor subtypes are coupled to multiple but distinct signal transduction cascades through different G proteins.


Chemical modification of the neutral amino acid transport system L of Chinese hamster ovary cells with p-chloromercuribenzene sulfonate. Branched-chain and aromatic neutral amino acids enter mammalian cells predominantly through a Na(+)-independent transport agency called System L. The sulfhydryl specific reagent p-chloromercuribenzene sulfonate (pCMBS) has been shown to be a potent inactivator of System L transport activity in Chinese hamster ovary cells, however, inactivation by pCMBS can be prevented by the presence of System L-specific substrate amino acids during the inactivation reaction. In addition, the presence of amino acids that are not substrates for System L have no effect on pCMBS inactivation of System L. Inactivation of System L activity by pCMBS was sensitive to pH and reversible by incubation with dithiothreitol. These findings suggest that there is a sulfhydryl group in, or very near, the amino acid-binding site of the System L transporter of CHO cells. Substrate protection, however, could be explained by conformational changes in the transporter associated with substrate binding. The presence of a substrate protectable sulfhydryl group on the System L transporter would aid in the attempt to identify this transporter using the technique of differential labeling.


Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells. Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.


Thapsigargin inhibits contraction and Ca2+ transient in cardiac cells by specific inhibition of the sarcoplasmic reticulum Ca2+ pump. Regulation of the level of ionized calcium, [Ca2+]i, is critical for its use as an important intracellular signal. In cardiac and skeletal muscle the control of fluctuations of [Ca2+]i depend on sarcolemmal and sarcoplasmic reticulum ion channels and transporters. We have investigated the sesquiterpine lactone, thapsigargin (TG), because of its reported action to alter cellular calcium regulation in diverse cell types, including striated muscle cells. We have combined biochemical and physiological methods at the cellular level to determine the site of action of this agent, its specificity, and its cellular effects. Using a patch-clamp method in whole cell configuration while measuring [Ca2+]i with Indo-1 salt, we find that TG (100 nM) largely blocks the contraction and the [Ca2+]i transient in rat ventricular myocytes. Analysis of these data indicate that no sarcolemmal current or transport system is directly altered by TG, although indirect [Ca2+]i-dependent processes are affected. In permeabilized myocytes, TG blocked oxalate-stimulated calcium uptake (half-maximal effect at 10 nM) into the SR. However, TG (100 microM) had no effect on Ca(2+)-induced Ca(2+)-release in purified muscle (ryanodine-receptor enriched) vesicles while clearly blocking Ca(2+)-ATPase activity in purified (longitudinal SR) vesicles. We conclude that in striated muscle TG markedly alters calcium metabolism and thus alters contractile function only by its direct action on the Ca(2+)-ATPase.


Evidence for the functional involvement of protein kinase C in the action of 1,25-dihydroxyvitamin D3 in bone. In the present study the involvement of protein kinase C in the action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on osteoblast-like cells and in the stimulation of in vitro bone resorption by 1,25(OH)2D3 was examined. Incubation for 24 h with 1,25(OH)2D3 potently stimulated osteocalcin synthesis by ROS 17/2.8 cells. This stimulation was inhibited (30-70% inhibition) by 25 microM of the protein kinase C (PKC) inhibitors 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG) and sphingosine without affecting basal osteocalcin synthesis. 1,25(OH)2D3-stimulated osteocalcin secretion by nontransformed isolated fetal rat osteoblasts was also inhibited (30-55%) by AMG. Also, AMG inhibited 10(-9) M 1,25(OH)2D3-induced up-regulation of vitamin D receptor in ROS 17/2.8 cells. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) did not cause an increase in osteocalcin secretion, while only a small increase in cellular content of osteocalcin in ROS 17/2.8 cells was observed. Addition of PMA together with 1,25(OH)2D3 did not change the response to 1,25(OH)2D3. The PKC inhibitors were not toxic for the cells. 1,25(OH)2D3 did not stimulate diacylglycerol production in ROS 17/2.8 cells up to 5 min after administration. However, 4- and 24-h incubation with 10 nM 1,25(OH)2D3 increased phorbol ester binding in ROS 17/2.8 cells. 1,25(OH)2D3 potently stimulated bone resorption after 3 and 6 days of culture in fetal mouse long bones and calvaria. Both the PKC inhibitors AMG (25 microM) and staurosporine (50 nM) strongly inhibited (60-86% inhibition) 1,25(OH)2D3-stimulated bone resorption without affecting basal 45Ca release. These effects were not due to a cytotoxic effect of both PKC inhibitors. Nor is it likely that the effects of AMG and staurosporine are due to inhibition of cell proliferation as hydroxyurea did not affect 1,25(OH)2D3-stimulated bone resorption. The inhibition of 1,25(OH)2D3-stimulated bone resorption by PKC inhibitors suggests that besides osteocalcin synthesis PKC is also involved in other responses of 1,25(OH)2D3 in bone. 1,25(OH)2D3 does not directly activate PKC via an increase in diacylglycerol production but more likely via an increase in PKC. Together, the present study demonstrates a functional involvement of PKC in the action of 1,25(OH)2D3 in bone and bone cells which may have consequences for the development of 1,25(OH)2D3 analogs, e.g. with less hypercalcemic and relatively more antiproliferative activity.


Characterization of beta-125I-endorphin cross-linked proteins in NG108-15 cell membranes. A 25-kilodalton protein with properties of delta-opioid-binding site. Cross-linking of beta-125I-endorphin to NG108-15 cell membranes labeled bands with molecular masses of 55, 35, and 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We applied several criteria to evaluate the relevance of these cross-linked bands to delta-opioid receptors, including selectivity, stereospecificity, affinity, G-protein coupling, down-regulation, and correlation with opioid receptor level in different well-characterized cell lines. Only the 25 kDa protein adequately fulfilled all these criteria. Thus, cross-linking to the 25-kDa band was selectively inhibited by ligands with delta-opioid affinity, but not by mu-opioid, kappa-opioid, or optically inactive opioid ligands or by non-opioid ligands. Based on inhibition of cross-linking, we calculated an affinity of [D-Ala2,D-Leu5]enkephalin binding to the 25-kDa and (Kd = 6 nM) that is similar to that reported for [D-Ala2,D-Leu5]enkephalin binding to NG108-15 membranes; this affinity decreased approximately 10-fold in the presence of Na+/guanyl-5'-yl imidodiphosphate. Chronic agonist treatment of NG108-15 cells reduced cross-linking to the 25-kDa band, but not to others, in a manner parallel to down-regulation of opioid receptors. Finally, the amount of the 25-kDa band was roughly proportional to the level of opioid receptors present in N18TG2, NS20Y, ST7-3, and ST8-4 cells. The 25-kDa band was absent in PC12h, NIH3T3, and C6BU1 cells as well as in liver, all of which had no detectable opioid binding.


Interaction of the -170 cyclic AMP response element with the adjacent CCAAT box in the human fibronectin gene promoter. In the fibronectin gene promoter the cAMP response element (CRE) and the CCAAT box are separated by only 20 base pairs (bp), i.e. two turns of double helix. Binding of nuclear proteins to these elements, assessed by DNase I footprinting, differs in the different cell types. While in a variety of cells tested (HeLa, granulosa, brain, and adenocarcinoma) only CRE binding activity is observed, liver extracts show both CRE and CCAAT binding activities. Competitions with CRE oligonucleotides were able to prevent the binding of both liver factors, while competitions with CCAAT oligonucleotides only abolished the binding to the CCAAT box. Consistently, the occupation of the CCAAT box was reduced when the distance between the CRE and CCAAT elements was increased in a series of spacing mutants in which DNA fragments of 20, 28, or 44 bp were inserted, and in a construct where the CRE sequence was deleted. Furthermore, the mutants are less efficient than the wild type as templates for in vitro transcription elicited by liver nuclear extracts. Transcriptional activity decreases with the 20- and 28-bp insertions but is partially recovered with the 44-bp insertion. Partial purification of liver CRE- and CCAAT-binding proteins by high performance liquid chromatography on a Mono Q column and recombination of column fractions showed that a novel 73-kDa CRE-binding protein facilitates the association of the CCAAT-binding protein to the CCAAT site of the fibronectin gene.


A Pro/Ser substitution in nucleoside diphosphate kinase of Drosophila melanogaster (mutation killer of prune) affects stability but not catalytic efficiency of the enzyme. Nucleoside diphosphate kinase of Drosophila, recently identified as the product of the awd gene, is essential for larval development. The conditional lethal mutation Killer of prune maps to the same gene. We purified the nucleoside diphosphate kinases from wild-type and mutant larvae by a simple procedure involving affinity chromatography on blue Sepharose. Both proteins are purified as hexamers in their native state. The mutant protein, which carries a serine instead of proline at position 97, has structural properties and catalytic efficiency that are very similar to the wild-type protein. However, the mutant protein has a much lower stability to denaturation by heat and urea. Following dilution of urea with buffer the urea-denaturated mutant nucleoside diphosphate kinase accumulates as folded monomers and cannot recover its quaternary structure and enzymatic activity. In contrast, the wild-type enzyme recovers hexameric structure and activity. This suggests that the mutation affects the folding/assembly pathway without affecting the function of the mature protein once folded and assembled into the mature hexameric structure.


Dissection of the beta subunit in the Escherichia coli RNA polymerase into domains by proteolytic cleavage. The 1342 amino acid long beta subunit of Escherichia coli RNA polymerase includes a dispensable region (residues 940-1040) that is absent in homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria (Borukhov, S., Severinov, K., Kashlev, M., Lebedev, A., Bass, I., Rowland, G. C., Lim, P.-P., Glass, R. E., Nikiforov, V., and Goldfarb, A. (1991) J. Biol. Chem. 266, 23921-23926). Genetic disruption of this region by in-frame deletion or insertion sensitizes the beta subunit in assembled RNA polymerase molecules to attack by trypsin. We demonstrate that RNA polymerase with the beta polypeptide cleaved in the dispensable region retains normal in vitro activity. Moreover, the RNA polymerase activity is completely restored after denaturation and reconstitution of the enzyme carrying cleaved beta subunit indicating that its carboxyl- and amino-terminal parts fold and assemble into RNA polymerase as separate entities.


Tumor necrosis factor-alpha activates nuclear factor kappa B and induces manganous superoxide dismutase and phosphodiesterase mRNA in human papillary thyroid carcinoma cells. Human papillary thyroid carcinoma (PTC) has a relatively benign prognosis despite a high frequency of lymphatic metastasis. This suggests that local anticancer factors, generated in lymph nodes, control PTC progression. The cytokine, tumor necrosis factor-alpha (TNF-alpha), may be one such factor. We have previously shown that a human PTC cell line (NP-PTC) has high affinity TNF-alpha receptors. We now report on the action of TNF-alpha in these cells. TNF-alpha decreased [3H]thymidine incorporation as well as cellular DNA content and cell number in a dose-dependent manner. The abundance of phosphodiesterase and manganous superoxide dismutase mRNA species was increased in a time- and dose-dependent manner in the NP-PTC cells after TNF-alpha treatment. TNF-alpha activated NF-kappa B, a nuclear factor thought to mediate multiple actions of TNF-alpha, in these cells with a maximum effect observed after 30 min of treatment. Thus, TNF-alpha has an antiproliferative action on NP-PTC cells, despite its ability to induce the accumulation of mRNA that encodes an enzyme (manganous superoxide dismutase), thought to be cytoprotective. The net antiproliferative effect must therefore be explained by a balance of protective and tumoricidal or static effects that ultimately result in control of tumor spread. These antiproliferative effects may be in part mediated by NF-kappa B and PDE.


Beta 1- and beta 3-subunits can associate with presynthesized alpha-subunits of Xenopus oocyte Na,K-ATPase. Oligomerization of newly synthesized alpha- and beta-subunits is a prerequisite for the structural and functional maturation of Na,K-ATPase. In this study, we have tested the competence of presynthesized alpha- and beta-subunits to assemble into functional enzyme complexes. Antisense oligonucleotides complementary to alpha-mRNA were used to inhibit alpha-subunit synthesis in Xenopus oocytes leaving a presynthesized trypsin-sensitive alpha-subunit pool. beta-Subunits expressed in these oocytes from injected cRNA assembled with the preexisting alpha-subunits, rendered them trypsin-resistant, and permitted the expression of more ouabain binding sites at the plasma membrane. Similarly, presynthesized beta 1- or beta 3-subunits produced in Xenopus oocytes by injection of beta-cRNA and later of specific antisense oligonucleotides were stabilized and transported out of the endoplasmic reticulum when alpha-cRNA was injected into oocytes. These data indicate that alpha- and beta-subunits can insert into endoplasmic reticulum membranes independent of each other in an assembly-competent form and retain their ability for oligomerization after synthesis.


Irradiation of the bovine mitochondrial F1-ATPase previously inactivated with 5'-p-fluorosulfonylbenzoyl-8-azido-[3H]adenosine cross-links His-beta 427 to Tyr-beta 345 within the same beta subunit. The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p'-fluorosulfonylbenzoyl-8-azidoadenosine (8-N3-FSBA) with an apparent Kd of 0.47 mM at pH 8.0 and 23 degrees C in the absence of light. Irradiation of dark-inactivated enzyme with long-wavelength UV light produced cross-linked dimers and, to a lesser extent, trimers made up of alpha and beta subunits. Two major radioactive peptides were resolved by high-performance liquid chromatography from tryptic digests of MF1 which had been inactivated with 8-N3-FSB[3H]A at pH 8.0 in the dark. Sequence analysis revealed that one contained Tyr-beta 368 and the other contained His-beta 427 which were labeled in the ratio of 18:15. Sequence analysis of radioactive tryptic peptides isolated from digests of irradiated MF1 derivatized with 8-N3-FSB[3H]A showed that photolysis induced cross-linking of His-427 to Tyr-345 within the same beta subunit in high yield. When MF1 derivatized with 8-N3-FSB[3H]A was irradiated in the presence of beta-mercaptoethanol, alpha-beta cross-links were eliminated, whereas those between His-beta 427 and Tyr-beta 345 were unaffected. Analysis of radioactive peptides in tryptic digests of MF1 derivatized with 8-N3-FSB[3H]A and then irradiated in the presence or absence of beta-mercaptoethanol showed that the nitrene generated from reagent attached to Tyr-beta 368 participates in formation of alpha-beta cross-links in the absence of beta-mercaptoethanol. Therefore, the nitrene generated from reagent tethered to His-beta 427 is shielded from solvent and reacts with the side chain of Tyr-beta 345. In contrast, the nitrene generated from reagent attached to Tyr-beta 368 is exposed to solvent, but in the absence of scavengers reacts with side chains present in the alpha subunit. Irradiation of MF1, partially inactivated with 8-N3-FSBA, led to loss of residual ATPase activity without affecting residual ITPase activity. The amount of photoinactivation was greater when partial dark inactivation was performed at pH 6.9, where modification of His-beta 427 predominates, than when performed at pH 8.0, where modification of Tyr-beta 368 predominates. This suggests that cross-linking of His-beta 427 to Tyr-beta 345, and not cross-linking of alpha and beta subunits, is responsible for the augmented inactivation induced by irradiation.


Protein phosphatase inhibitors induce the sustained expression of the Egr-1 gene and the hyperphosphorylation of its gene product. The immediate-early gene Egr-1 is strongly and rapidly induced in human and mouse Balb/c fibroblasts by okadaic acid and calyculin A, both specific inhibitors of protein serine/threonine phosphatases 1 and 2A. In contrast to the transient induction of the Egr-1 gene by serum or phorbol 12-myristate 13-acetate, these phosphatase inhibitors stimulated a sustained induction of the Egr-1 gene. The induction is shown to occur transcriptionally and is sustained post-transcriptionally. Okadaic acid-induced Egr-1 mRNA is significantly more stable than serum-induced Egr-1 mRNA. The half-life of serum-induced Egr-1 mRNA is estimated to be 12 min, compared with a half life of 2 h for okadaic acid-induced Egr-1 mRNA. Okadaic acid also induced the expression of the related immediate-early genes Egr-2 and Egr-3 albeit to a lesser extent than Egr-1. Treatment of cells with okadaic acid and calyculin A also induced the synthesis of Egr-1 protein. The Egr-1 protein is weakly or not phosphorylated in quiescent cells, but multiple species of the phosphorylated forms of the Egr-1 protein are detected in cells treated with either of the phosphatase inhibitors. Simultaneous treatment of cells with TPA and okadaic acid synergistically induced Egr-1 gene expression, and H7 strongly inhibits this induction. Taken together, the results indicate that the induction of Egr-1 gene transcription and the phosphorylation of the induced Egr-1 protein are under the control of protein kinase(s) and protein phosphatase(s). The phosphorylation and dephosphorylation of Egr-1 protein may play a role in controlling cell growth.


Activation of mitogen-activated protein (MAP) kinase by a MAP kinase-kinase. Previously it has been shown that acute 12-O-tetradecanoylphorbol-13-acetate treatment of intact U937 cells results in activation of mitogen-activated protein (MAP) kinase and a MAP kinase activator. MAP kinase activator induces phosphorylation of MAP kinase on tyrosine and threonine residues, thereby activating MAP kinase. Here, experiments with the irreversible kinase inhibitor, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), show that MAP kinase activator is in fact a MAP kinase-kinase. Treatment of MAP kinase activator with FSBA results in complete inactivation. This inactivation is prevented by a 10-fold excess of ATP. Inactivation of MAP kinase by FSBA does not affect the extent of threonine/tyrosine phosphorylation induced by MAP kinase-kinase.


Tethered ligand agonist peptides. Structural requirements for thrombin receptor activation reveal mechanism of proteolytic unmasking of agonist function. The human platelet thrombin receptor is activated when thrombin cleaves its receptor's amino-terminal extension to reveal a new amino terminus that functions as a tethered peptide ligand. Exactly how this "agonist peptide domain" remains cryptic within the uncleaved receptor and becomes functional after receptor cleavage is unknown. In this report we define the structural features of the thrombin receptor's agonist peptide domain important for receptor activation. Studies with mutant thrombin receptors have suggested that agonist peptide domain residues 2-6 contained determinants critical for receptor activation, and the synthetic peptide SFLLR-NH2 representing the 1st 5 amino-terminal residues of the agonist peptide domain was sufficient to specify agonist activity. Acetylating or removing the agonist peptide's amino-terminal ammonium group greatly attenuated agonist activity. Agonist peptide residue Phe2 was vital for agonist function; residues Leu4 and Arg5 individually played less important roles. These structure-function relationships held for both platelet activation and activation of the cloned receptor expressed in transfected mammalian cells. Our studies suggest that structures at the extreme amino terminus of the thrombin receptor's agonist peptide domain, in particular the free ammonium group of Ser1 and the phenyl ring of Phe2, are critical for receptor activation and that the agonist function of this domain is expressed when receptor proteolysis unmasks such determinants. In addition to revealing details of the thrombin receptor's proteolytic triggering mechanism, these studies open avenues to the development of drugs targeting the thrombin receptor and to further definition for the role of the thrombin receptor in cellular regulation.


Cytotoxicity of quinolones toward eukaryotic cells. Identification of topoisomerase II as the primary cellular target for the quinolone CP-115,953 in yeast. The quinolone CP-115,953 (6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4- quinolone-3-carboxylic acid) represents a novel mechanistic class of drugs with potent activity against eukaryotic topoisomerase II in vitro (Robinson, M. J., Martin, B. A., Gootz, T. D., McGuirk, P. R., Moynihan, M., Sutcliffe, J. A., and Osheroff, N. (1991) J. Biol. Chem. 266, 14585-14592). Although the quinolone is highly toxic to mammalian cells in culture, its mechanism of cytotoxic action is not known. Therefore, yeast was used as a model system to determine whether topoisomerase II is the primary target responsible for the in vivo effects of CP-115,953. The quinolone was equipotent to etoposide at enhancing DNA breakage mediated by the Saccharomyces cerevisiae type II enzyme. Moreover, at concentrations as low as 5 microM, CP-115,953 was cytotoxic to yeast cells that carried wild type topoisomerase II (TOP2+). By utilizing a yeast strain that expressed the top2-1 temperature-sensitive mutant, the effect of topoisomerase II activity on quinolone cytotoxicity was determined. At the permissive temperature of 25 degrees C, cells were highly sensitive to CP-115,953. However, at the semipermissive temperature of 30 degrees C (where in vivo enzyme activity is present but is greatly diminished), cells displayed only marginal sensitivity to the quinolone at concentrations as high as 50 microM. These results strongly suggest that topoisomerase II is the primary physiological target responsible for quinolone cytotoxicity and that CP-115,953 kills cells by converting the type II enzyme into a cellular poison.


Transient alterations in the lateral mobility of erythrocyte membrane components during Sendai virus-mediated fusion. Destabilization of the target membrane structure by fusion-promoting viral glycoproteins is assumed to be an essential part of the fusion mechanism. To explore this possibility, we employed fluorescence photobleaching recovery to investigate changes in the lateral mobility of native membrane constituents in human red blood cells (RBCs) during the course of Sendai virus-mediated fusion. The mobile fraction of RBC membrane proteins labeled with 5-(4,6-dichloro-5-triazin-2-yl)aminofluorescein increased significantly in the course of fusion, relaxing back to the original values upon completion of the fusion process. A different effect was observed on the lateral mobility of a fluorescent lipid probe, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, incorporated initially into the external monolayer. In this case, the lateral diffusion coefficient (rather than the mobile fraction) increased during fusion; this increase was permanent in the absence of Mg-ATP and transient in its presence. An active viral fusion protein was required to mediate the effects on both protein and lipid mobility. These effects, which take place on the same time scale as that of the fusion process, suggest that the organization of the RBC membrane is perturbed during fusion and that the observed changes may be related to the fusion mechanism.


Characterization of the zinc binding site of bacterial phosphotriesterase. The bacterial phosphotriesterase has been found to require a divalent cation for enzymatic activity. This enzyme catalyzes the detoxification of organophosphorus insecticides and nerve agents. In an Escherichia coli expression system significantly higher concentrations of active enzyme could be produced when 1.0 mM concentrations of Mn2+, Co2+, Ni2+, and Cd2+ were included in the growth medium. The isolated enzymes contained up to 2 equivalents of these metal ions as determined by atomic absorption spectroscopy. The catalytic activity of the various metal enzyme derivatives was lost upon incubation with EDTA, 1,10-phenanthroline, and 8-hydroxyquinoline-5-sulfonic acid. Protection against inactivation by metal chelation was afforded by the binding of competitive inhibitors, suggesting that at least one metal is at or near the active site. Apoenzyme was prepared by incubation of the phosphotriesterase with beta-mercaptoethanol and EDTA for 2 days. Full recovery of enzymatic activity could be obtained by incubation of the apoenzyme with 2 equivalents of Zn2+, Co2+, Ni2+, Cd2+, or Mn2+. The 113Cd NMR spectrum of enzyme containing 2 equivalents of 113Cd2+ showed two resonances at 120 and 215 ppm downfield from Cd(ClO4)2. The NMR data are consistent with nitrogen (histidine) and oxygen ligands to the metal centers.


Mechanisms by which lipoprotein lipase alters cellular metabolism of lipoprotein(a), low density lipoprotein, and nascent lipoproteins. Roles for low density lipoprotein receptors and heparan sulfate proteoglycans. We sought to investigate effects of lipoprotein lipase (LpL) on cellular catabolism of lipoproteins rich in apolipoprotein B-100. LpL increased cellular degradation of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) by 277% +/- 3.8% and 32.5% +/- 4.1%, respectively, and cell association by 509% +/- 8.7% and 83.9% +/- 4.0%. The enhanced degradation was entirely lysosomal. Enhanced degradation of Lp(a) had at least two components, one LDL receptor-dependent and unaffected by heparitinase digestion of the cells, and the other LDL receptor-independent and heparitinase-sensitive. The effect of LpL on LDL degradation was entirely LDL receptor-independent, heparitinase-sensitive, and essentially absent from mutant Chinese hamster ovary cells that lack cell surface heparan sulfate proteoglycans. Enhanced cell association of Lp(a) and LDL was largely LDL receptor-independent and heparitinase-sensitive. The ability of LpL to reduce net secretion of apolipoprotein B-100 by HepG2 cells by enhancing cellular reuptake of nascent lipoproteins was also LDL receptor-independent and heparitinase-sensitive. None of these effects on Lp(a), LDL, or nascent lipoproteins required LpL enzymatic activity. We conclude that LpL promotes binding of apolipoprotein B-100-rich lipoproteins to cell surface heparan sulfate proteoglycans. LpL also enhanced the otherwise weak binding of Lp(a) to LDL receptors. The heparan sulfate proteoglycan pathway represents a novel catabolic mechanism that may allow substantial cellular and interstitial accumulation of cholesteryl ester-rich lipoproteins, independent of feedback inhibition by cellular sterol content.


Characterization of a rat liver protein carboxyl methyltransferase involved in the maturation of proteins with the -CXXX C-terminal sequence motif. We have utilized S-farnesyl-Leu-Ala-Arg-Tyr-Lys-Cys as a methyl-accepting substrate to characterize a membrane-bound C-terminal protein methyltransferase from rat liver. We have localized the activity to the microsomal fraction and show that the bulk of the enzyme fractionates by density gradient centrifugation with glucose-6-phosphatase, a marker of the endoplasmic reticulum, and not with 5'-nucleotidase, a marker of the plasma membrane, or galactosyl:N-acetylglucosamine transferase, a marker of the Golgi apparatus. This methyltransferase appears to form an integral part of the membrane structure. Its activity is markedly affected by a variety of detergents used to solubilize membrane proteins in their native form. All activity is lost when membranes are treated with seven different detergents at a concentration of 1% (w/v). The activity is inhibited by N-ethylmaleimide, although it can be protected against inactivation with its substrate S-adenosyl-L-methionine, or its product S-adenosyl-L-homocysteine. Finally, we find that 5'-methylthioadenosine, a substrate analogue reported to be an inhibitor of this activity in other studies, is not an effective inhibitor in vitro.


Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca2+ signal transduction. A cDNA clone for a new metabotropic glutamate receptor, mGluR5, was isolated through polymerase chain reaction-mediated DNA amplification by using primer sequences conserved among the metabotropic glutamate receptor (mGluR) family and by the subsequent screening of a rat brain cDNA library. The cloned receptor consists of 1171 amino acid residues and exhibits a structural architecture common to the mGluR family, possessing a large extracellular domain preceding the seven putative membrane-spanning segments. mGluR5 shows the highest sequence similarity to mGluR1 among the mGluR members and is coupled to the stimulation of phosphatidylinositol hydrolysis/Ca2+ signal transduction in Chinese hamster ovary cells transfected with the cloned cDNA. This receptor also resembles mGluR1 in its agonist selectivity and antagonist responses; the potency rank order of agonists for mGluR5 was determined to be quisqualate greater than L-glutamate greater than or equal to ibotenate greater than trans-1-aminocyclopentane-1,3-dicarboxylate. Blot and in situ hybridization analyses indicated that mGluR5 mRNA is widely distributed in neuronal cells of the central nervous system and is expressed differently from mGluR1 mRNA in many brain regions. This investigation thus demonstrates that there is an additional mGluR subtype which closely resembles mGluR1 in its signal transduction and pharmacological properties and is expressed in specialized neuronal cells in the central nervous system.