Long-term effects of a somatostatin analogue on renal haemodynamics and hypertrophy in diabetic rats. 1. To determine whether treatment with octreotide, a somatostatin analogue, may diminish or prevent long-term diabetic renal hypertrophy and nephropathy, uninephrectomized streptozotocin-diabetic rats maintained under moderate glycaemic control (approximately 300 mg/dl) were treated with either placebo (n = 10 rat/group) or octreotide for 14 weeks. Uninephrectomized non-diabetic rats given either placebo or octreotide served as controls. 2. Average body weight was diminished and kidney weight, daily urinary protein excretion, glomerular filtration rate and renal plasma flow were elevated in both diabetic groups relative to controls. 3. Administration of octreotide reduced average body weight and packed cell volume in non-diabetic and diabetic rats compared with their respective controls, but did not affect glomerular hyperfiltration or the increase in urinary protein excretion. 4. Histological examination at 14 weeks disclosed unequivocal glomerular hypertrophy and mild glomerular and tubulointerstitial lesions consistent with early diabetic renal alterations in all diabetic rats, but there was no independent effect of octreotide treatment. 5. Thus, long-term treatment with octreotide did not afford protection against the development of renal hypertrophy-hyperfiltration and the evolution of early diabetic nephropathy in rats.


Metabolic clearance rate of arginine vasopressin in severe chronic renal failure. 1. The metabolic clearance rate of arginine vasopressin was determined using a constant infusion technique in normal subjects and patients with chronic renal failure immediately before commencing dialysis. Endogenous arginine vasopressin was suppressed in all subjects before the infusion with a water load. 2. Plasma arginine vasopressin concentrations were determined using a sensitive and specific radioimmunoassay after Florisil extraction. The detection limit of the assay was 0.3 pmol/l, and intra- and inter-assay coefficients of variation at 2 pmol/l were 9.7% and 15.3%, respectively. 3. In normal subjects, the metabolic clearance rate was determined at two infusion rates producing steady-state concentrations of arginine vasopressin of 1.3 and 4.4 pmol/l. In the patients with renal failure, a single infusion rate was used, producing a steady-state concentration of 1.5 pmol/l. 4. At comparable plasma arginine vasopressin concentrations, metabolic clearance rate was significantly reduced in patients with renal failure (normal 1168 +/- 235 ml/min versus renal failure 584 +/- 169 ml/min; means +/- SD; P < 0.001). 5. Free water clearance was significantly reduced in normal subjects during the arginine vasopressin infusion from 8.19 +/- 2.61 to -1.41 +/- 0.51 ml/min (P < 0.001), but was unchanged in the patients with renal failure after attaining comparable plasma arginine vasopressin concentrations. 6. In normal subjects there was a small but significant fall in metabolic clearance rate at the higher steady-state arginine vasopressin concentration (1168 +/- 235 ml/min at 1.3 pmol/l versus 1059 +/- 269 ml/min at 4.4 pmol/l; P = 0.016).(ABSTRACT TRUNCATED AT 250 WORDS)


Weight loss normalizes the inhibitory effect of N6-(phenylisopropyl)adenosine on lipolysis in fat cells of massively obese human subjects. 1. Fat cells were isolated from massively obese patients at or before gastric bypass, from other patients after normalization of body weight after gastric bypass or gastroplasty (post-bypass patients) and from control subjects of a stable normal body weight. 2. The inhibition of isoprenaline-stimulated lipolysis by N6-(phenylisopropyl)adenosine in the presence of adenosine deaminase was much attenuated in cells from the massively obese patients as compared with those from normal-weight control subjects, but was normal in cells from post-bypass patients. 3. Isolated fat cells of the massively obese patients were larger (913 +/- 197 pl, mean +/- SEM) than those of the normal-weight group (437 +/- 95 pl). The volume of cells from the post-bypass patients was only 125 +/- 49 pl, although the body mass index of this group was almost exactly the same as that of the normal-weight control subjects. 4. Although epidemiological studies have suggested that genetic factors are important in the development and maintenance of obesity, these results demonstrate that the changes observed in the inhibitory regulation of lipolysis in obesity are secondary.


Serum sialic acid concentration in patients with hypertriglyceridaemia showing the Frederickson's IIB phenotype. 1. Serum total sialic acid concentration, recently shown to be a cardiovascular risk factor, and also serum lipid-associated sialic acid concentration were measured in 15 patients with hypertriglyceridaemia (fasting serum triacyglycerol concentration > 2.3 mmol/l) showing a Frederickson's type IIB phenotype, 15 patients with hypercholesterolaemia showing a IIA phenotype and 15 age- and sex-matched normal control subjects. 2. Total serum sialic acid concentration was significantly raised in the hypertriglyceridaemic group (84.9 +/- 21.5 versus 64.9 +/- 20.8 mg/dl, P < 0.03, Mann-Whitney U-test) compared with the normal control group, as was serum lipid-associated sialic acid concentration (23.0 +/- 4.3 versus 12.0 +/- 3.2 mg/dl, respectively, P < 0.001, Mann-Whitney U-test). 3. Serum total sialic acid concentration was also significantly elevated in the hypertriglyceridaemic group as compared with the IIA phenotype hypercholesterolaemic group (84.9 +/- 21.5 versus 58.4 +/- 11.7 mg/dl, P < 0.03, Mann-Whitney U-test), as was serum lipid-associated sialic acid concentration (23.0 +/- 4.3 versus 14.9 +/- 4.7 mg/dl, P < 0.001, Mann-Whitney U-test). 4. We suggest that serum concentrations of both total sialic acid and lipid-associated sialic acid may be useful markers of cardiovascular risk which could, in part, be related to hypertriglyceridaemia.


Twenty-four hour C-peptide and insulin secretion rates and diurnal profiles of glucose, lipids and intermediary metabolites in cirrhosis. 1. To examine the contributions of hypersecretion and decreased insulin clearance to the hyperinsulinaemia of cirrhosis, insulin secretion was calculated over the day from serum C-peptide concentrations and C-peptide metabolic clearance rate. The latter was measured during infusions of recombinant human C-peptide. In cirrhotic patients (n = 9) insulin secretion rate was twice that of normal control subjects (n = 10), both in the basal state [02.00-07.00 hours, 15.7 +/- 2.1 (mean +/- SEM) nmol/h (2.6 +/- 0.4 units/h) versus 7.0 +/- 0.9 nmol/h (1.2 +/- 0.2 units/h), P < 0.002] and over 24 h [787 +/- 93 nmol (132 +/- 16 units) versus 346 +/- 34 nmol (58 +/- 6 units), P < 0.001]. However, the area under the serum insulin concentration curve was approximately six times greater in the cirrhotic patients (24 h basal, 6.3 +/- 1.0 versus 1.1 +/- 0.3 nmol l-1 h, P < 0.001; 24 h total, 21.7 +/- 3.2 versus 3.7 +/- 0.7 nmol l-1 h, P < 0.001). Thus, despite impairment of insulin clearance there is continuing hypersecretion of insulin in cirrhosis. 2. The relationship of carbohydrate and lipid metabolism with insulin secretion was assessed. In cirrhotic patients, 24 h blood glucose profiles showed a worsening of glucose tolerance over breakfast, despite greater insulin secretion compared with other meals, suggesting that the insulin insensitivity of cirrhosis is worse at this time. 3. Cirrhotic patients showed impaired suppression of blood glycerol levels after meals but normal suppression of serum non-esterified fatty acid concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)


Post-operative changes in hepatic, intestinal, splenic and muscle fluxes of amino acids and ammonia in pigs. 1. After operation, changes in nitrogen metabolism occur. Although increased flux of amino acids from peripheral to splanchnic organs after operation has been described, substrate utilization by the individual organs in the splanchnic area is less well characterized. We were specifically interested in substrate flux across the spleen as it is an organ with important immunological functions. 2. Therefore, hindquarter, gut, spleen and liver fluxes of amino acids, ammonia, glucose, lactate and blood gases were measured for 4 days after a standard operation in pigs. In a separate control group, fluxes were measured 2-3 weeks after this operation and these values were assumed to represent the normal situation. 3. One day after operation, the hindquarter effluxes of glutamine, alanine and several essential amino acids were increased (P > 0.001), but these normalized at the end of the observation period. In the same period, liver glutamine uptake increased (P < 0.01), concomitantly with increased HCO3-, glucose and urea production, which also normalized. Portal drained viscera ammonia production decreased, concomitant with decreased glutamine uptake (P < 0.001). After operation, the splenic release of ammonia increased sevenfold (P < 0.05) and that of lactate increased from -158 +/- 544 to 3294 +/- 642 nmol min-1 kg-1 body weight (P < 0.001). Glucose uptake increased from -964 +/- 632 to -3933 +/- 1524 nmol min-1 kg-1 body weight and glutamine efflux (391 +/- 143) reversed to uptake (-752 +/- 169 nmol min-1 kg-1 body weight) (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Salbutamol, a beta 2-adrenoceptor agonist, increases skeletal muscle strength in young men. 1. beta 2-Adrenoceptor agonists have been shown to increase rapidly lean body mass and reverse muscle wasting in several animal models of human illness. However, no published information is available for humans. In the present study, we investigated the effects of a slow-release preparation of salbutamol or a placebo on skeletal muscle functional capacity in 12 healthy men. The strength of different muscle groups was measured on two occasions before and after 14 and 21 days of treatment. 2. No significant changes in muscle strength were observed with placebo during the trial. In contrast, the strength of both quadriceps muscles increased significantly (12 +/- 3%) after 14 days on salbutamol, and remained elevated at 21 days. Whereas the strength of the hamstring muscles of the dominant leg significantly increased after 21 days on salbutamol (22 +/- 6%), the strength of the non-dominant hamstring muscles returned to baseline values. 3. There was no significant change in the grip strength of either hand in these subjects during the trial. The maximal static inspiratory mouth pressure increased significantly (7 +/- 2%) after 14 days on salbutamol, and increased further after 21 days (15 +/- 4%); the expiratory mouth pressure remained constant. No significant changes in body weight, skinfold thickness, lean body mass or limb circumferences were measured in either group. 4. These data demonstrate that short-term administration of salbutamol increases voluntary muscle strength in man. However, the magnitude and duration of this effect vary between muscle groups. This study implies that the beta 2-adrenoceptor agonists may be of therapeutic potential in altering skeletal muscle function in humans.


Sulphation of colonic and rectal mucin in inflammatory bowel disease: reduced sulphation of rectal mucus in ulcerative colitis. 1. Normal colonic mucin is heavily sulphated and this increases its resistance to degradation by bacterial enzymes. Any defect in mucus sulphation could therefore be important in the pathogenesis of ulcerative colitis. 2. Rectal biopsies taken at colonoscopy from patients with ulcerative colitis (n = 9), patients with Crohn's disease (n = 6) and control subjects (n = 16) were cultured for 24 h in the presence of N-[3H]acetylglucosamine and [35S]sulphate. Mucin was then extracted and purified, and the ratio of [35S]sulphate to N-[3H]acetylglucosamine incorporated into pure mucin was assessed. 3. The ratio of [35S]sulphate to N-[3H]acetylglucosamine incorporated into mucin was significantly reduced in rectal biopsies taken from patients with ulcerative colitis (0.463, 0.305-0.703, geometric mean and 95% confidence intervals) compared with control subjects (0.857, 0.959-1.111, P < 0.01). In patients with Crohn's disease the reduction in this ratio (0.559, 0.378-0.829) did not quite reach statistical significance (P = 0.06). There was no difference between the ratio of [35S]sulphate to N-[3H]acetylglucosamine incorporated into mucin in Crohn's disease and that in ulcerative colitis (P = 0.26). 4. In control subjects the ratio of [35S]sulphate to N-[3H]acetylglucosamine incorporated into mucin was higher in the rectal biopsies (0.882, 0.618-1.022) than in their paired proximal colonic biopsies (0.602, 0.421-0.861; P < 0.01), but this regional variation was not observed in either ulcerative colitis (rectum: 0.450, 0.262-0.773; right colon: 0.470, 0.321-0.690, P = 0.3) or Crohn's disease (rectum: 0.459, 0.260-0.815; right colon: 0.492, 0.260-0.929, P = 0.8).(ABSTRACT TRUNCATED AT 250 WORDS)


Colonic absorption of human calcitonin in man. 1. Human calcitonin was administered into the distal colon and by intravenous infusion in eight healthy subjects in an open, fixed sequence, cross-over bioavailability study. 2. Intravenously infused human calcitonin elicited a standard pharmacokinetic profile in eight healthy subjects with a biphasic elimination with half-lives of 10.2 +/- 0.7 min and 37.8 +/- 2.5 min. 3. Colonoscopically administered human calcitonin was absorbed across the distal colonic mucosa in low amounts with a bioavailability of 0.00-0.22%. 4. Absorption from the distal colon was impeded by the presence of faecal material in three of the eight subjects. 5. We conclude that human calcitonin crosses the gastrointestinal epithelium of man. This may demonstrate the feasibility of an oral form for clinical use.


Paradoxical effects of mild hypoxia and moderate altitude on human visual perception. 1. Prolonged (> 10 h) exposure to hypoxia and high altitude (> 5000 m) invariably have detrimental effects on cognitive performance. Paradoxically, mild improvements in cognitive function in patients with chronic obstructive pulmonary disease after cessation of oxygen therapy have been reported. 2. We studied in each of 10 healthy subjects the effect of an acute altitude challenge [rapid helicopter transport to the Jungfraujoch (3450 m), experiment 1] and of an acute exposure to mild hypoxia (fractional inspiratory oxygen concentration 14.5% experiment 2) on a simple test of cognitive performance (the time needed to read briefly displayed letters). 3. Under both hypoxic conditions the time needed to read briefly presented letters decreased, from 12.1 +/- SD 3.8 ms to 8.3 +/- 1.5 ms (P < 0.01) in experiment 1, and from 11.9 +/- 1.9 ms to 8.1 +/- 1.1 ms (P < 0.01) in experiment 2. 4. A rapid and mild hypoxic challenge seems to improve a simple measure of cognitive performance above normal values. The common notion that exposure to hypoxia and altitude invariably impairs cognitive performance may have to be re-evaluated.


Gastroduodenal polyps in patients with familial adenomatous polyposis. A review of the endoscopy reports and pathology results from esophagogastroduodenoscopy (EGD) of all patients with familial adenomatous polyposis (FAP) undergoing such an examination was performed. Two hundred forty-seven patients were identified, with an overall prevalence of duodenal adenomas of 66 percent and of fundic gland polyps of 61 percent. Analysis of our more recent experience (1986 to 1990) shows the prevalence to be 88 percent and 84 percent, respectively. A normal-appearing papilla was adenomatous in 50 percent of cases. No case of periampullary carcinoma developed in patients under surveillance. Routine EGD is indicated for patients with FAP. Duodenal adenomas and fundic gland polyps will occur in the majority of patients.


Agents capable of eliminating reactive oxygen species. Catalase, WR-2721, or Cu(II)2(3,5-DIPS)4 decrease experimental colitis. Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, hydroxyl radical, and hypochlorous acid have been implicated in the pathogenesis of inflammation and tissue injury in colitis. To determine whether or not anti-ROS agents can decrease the severity of colitis, we evaluated the effects of three known anti-ROS agents: catalase, WR-2721, and Cu(II)2(3,5-DIPS)4 on acetic acid-induced colonic inflammation in rats. Histologically, all three compounds significantly decreased the severity of colonic inflammation. The anti-ROS activity of these compounds was also tested using the luminol-enhanced chemiluminescence assay. Catalase, WR-2721, or Cu(II)2(3,5-DIPS)4 significantly inhibited luminol-enhanced chemiluminescence produced by inflamed colonic mucosa. These findings suggest that ROS, and in particular superoxide, hydrogen peroxide, and/or one of its secondarily derived species, may play an important role in acetic acid-induced colitis. Further studies are needed to determine the potential effectiveness of these compounds in human colitis.


Postoperative and long-term results of ileal pouch-anal anastomosis for ulcerative colitis and familial polyposis coli. The immediate postoperative and long-term functional results of 51 ulcerative colitis patients and 21 familial polyposis patients who underwent ileal J-pouch-anal anastomosis were compared in this study. The incidence of postoperative complications requiring reoperation was not statistically different in both groups. The mean daily stool frequency was significantly higher in colitis patients. Pouchitis occurred in 44% of colitis patients but not in polyposis patients (P < 0.005). Symptoms of pouchitis included bloody diarrhea, urgency, abdominal pain, weight loss, fever, and arthritis. Six colitis patients required pouch excision because of intractable pouchitis. The overall pouch excision rate was 22% in ulcerative colitis patients and 5% in familial polyposis patients. Patient satisfaction was good in 46% of ulcerative colitis patients and 76% of polyposis patients (P < 0.05). Our data demonstrate that the long-term outcome of ileal pouch-anal anastomosis is more favorable in polyposis patients than in colitis patients. Pouchitis is a major long-term complication occurring exclusively in colitis patients.


World Health Organization clinical case definition for AIDS in Africa: an analysis of evaluations. In 1985 at a World Health Organization (WHO) workshop on AIDS in Bangui, Central African Republic, a clinical case definition of Acquired Immune Deficiency Syndrome (AIDS) was developed for developing countries, such as sub-Saharan Africa, where sophisticated diagnostic equipment is not widely available. A particular cachectic syndrome, the "slim disease", which is highly suggestive of AIDS in Africa, constitutes the substratum for the clinical definition for AIDS. The WHO/Bangui definition in adults has a sensitivity of 60%, a specificity of 90%, and a high predictive value especially in endemic areas. The WHO/Bangui clinical case definition for paediatric AIDS is less easy to use in practice. Its low sensitivity (about 35%) is in relation to its incapacity to diagnose many of the frequently observed secondary infection for paediatric AIDS according to the CDC criteria. The WHO/Bangui clinical definition for AIDS seems to be convenient for epidemiological surveillance of the HIV epidemic in Africa. Nevertheless, the low sensitivity and the low specificity result in the failure to detect some cases of full blown AIDS.


Correlation of colposcopy and histology in cervical biopsies positive for CIN and/or HPV infection. The purpose of our study was the investigation of colposcopic findings in 108 patients with histologically proved CIN and/or HPV infection in colposcopically directed cervical punch biopsies. The chi square test with Yates' correction was used for the statistical analysis. In 82 cases there were histological changes suggestive of HPV infection, 91 cases showed CIN while in 65 cases CIN was superimposed on HPV infection. The sensitivity of colposcopy was 89% for the detection of CIN and 61% for HPV. In 45% of CIN cases colposcopy underdiagnosed the CIN level, especially in more severe lesions (level II or higher, P < 0.001). We have also observed that the HPV-related colposcopic findings significantly increased the probability of underdiagnosing CIN (P < 0.05). Our results suggest that biopsy is always necessary in order to establish the diagnosis and determine the severity of CIN, especially if the colposcopist suspects HPV infection. We have also shown that many cases of HPV infection are not detected by colposcopy, because they are masked by the coexisting CIN.


Distribution of human papillomavirus 16 in the blood of women with uterine cervix carcinoma. In the present study HPV 16 DNA was discovered in the cervical cancer tissue and in blood of women with cancer. In the blood of patients with advanced cancer the HPV appeared in granulocytes and was anchored to the lymphocytic membrane. It may be therefore concluded that the presence of HPV 16 DNA in the blood of women with cervical cancer reduced immunocompetence.


Atrial natriuretic peptide binds to ANP-R1 receptors in neuroblastoma cells or is degraded extracellularly at the Ser-Phe bond. ANP-R1 receptors for atrial natriuretic peptide (ANP) showed the following rank order of affinity in intact human neuroblastoma cells NB-OK-1: human ANP-(99-126) approximately human ANP-(102-126) approximately rat ANP-(99-126) (K1 17-32 pM) > human ANP-(103-126) > porcine brain natriuretic peptide (BNP). Analogues truncated at the C-terminal extremity or devoid of a disulphide bridge, such as rat ANP-(103-123), rat C-ANP-(102-121), rat ANP-(111-126), rat ANP-(99-109) and rat [desCys105,Cys121]ANP-(104-126) and chicken C-type natriuretic peptide, were not recognized. The occupancy of these high affinity ANP-R1 receptors led to marked cyclic GMP accumulation in the presence of 3-isobutyl 1-methylxanthine. An ectoenzymic activity, partly shed in the incubation medium, provoked the stepwise release of Phe-Arg-[125I]Tyr, Arg-[125I]Tyr and [125I]Tyr from rat [125I]ANP-(99-126), at an optimal pH of 7.0. Its inhibition by 1,10-phenanthroline, EDTA and bacitracin but not by thiorphan suggests the contribution of at least one neutral metalloendopeptidase, distinct from EC 3.4.24.11, for which ANP showed high affinity.


kappa-Opioid binding sites on a murine lymphoma cell line. As a first step in determining whether any subset of lymphocytes expresses opioid receptors, membranes prepared from mouse lymphoma cell lines were screened for [3H]naloxone binding sites. Membranes from the R1.1 cell line specifically bound [3H]naloxone. The Hill coefficient for [3H]naloxone binding was 0.93 +/- 0.18, and nonlinear regression analysis indicated that a one-site model was the best fit of the [3H[naloxone saturation binding data. Low concentrations of kappa-selective opioids, but neither mu nor delta opioids, inhibited [3H]naloxone binding. Saturation binding studies with the kappa-selective compound [3H]U69,593 revealed a single binding site with a KD value of 0.204 +/- 0.039 nM and a Bmax value of 31.7 +/- 3.1 fmol/mg of membrane protein. The Hill coefficient for [3H]U69,593 binding was 1.03 +/- 0.11, indicative of a single site. Time courses for the association and dissociation of [3H]U69,593 binding at 25 degrees C exhibited properties consistent with a single class of binding sites. Low concentrations of kappa-selective opioids, including dynorphin peptides, inhibited [3H]U69,593 binding, while high concentrations of mu opioids were needed to inhibit binding, and the delta-selective ligands were ineffective at concentrations up to 10 microM. Stereoselectivity of the binding site was demonstrated by the finding that the Ki value for (-)-pentazocine in inhibiting [3H]U69,593 binding was 25 times less than for the (+)-isomer. Based on its high affinity for U69,593, alpha-neo-endorphin, and dynorphin B, the kappa opioid binding site on R1.1 cell membranes belongs to the kappa 1b subtype. As observed with brain kappa opioid binding sites, sodium inhibited [3H]U69,593 binding to R1.1 cell membranes in a concentration-dependent manner. These data demonstrate that the murine lymphoma cell line R1.1 expresses kappa opioid binding sites that are very similar to brain kappa opioid binding sites.


Characterization of the angiotensin II receptor expressed by the human hepatoma cell line, PLC-PRF-5. Radioligand binding studies were undertaken to establish the expression of angiotensin II (AII) receptors on the human hepatoma cell line, PLC-PRF-5. Cell membranes were shown to express a large number of AII receptors with high and low affinity binding sites having Bmax values of 1269 +/- 365 and 4190 +/- 1055 fmol/mg protein and affinities (Kd) of 2.0 +/- 0.3 nM and 8.7 +/- 0.4 nM, respectively. In intact cells a single class of AII binding site was seen with an affinity (Kd) of 6.7 +/- 1 nM and a Bmax value of 315 +/- 32 fmol/mg. In both membranes and intact cells AII, AIII and the selective angiotensin AT1 receptor antagonist, DuP 753, all had a high affinity for the receptor (Ki values in the nanomolar range), but the selective angiotensin AT2 ligands, PD 123177 and p-aminophenylalanine6 AII, had low affinity (Ki values in the micromolar range). These results indicate that the PLC-PRF-5 cells express the angiotensin AT1 receptor subtype. This was further supported by the demonstration of the sensitivity of the receptor to dithiothreitol (DTT). Pretreatment of membranes with DTT reduced [3H]AII binding in a concentration-dependent manner with an IC50 of 4.2 +/- 0.9 mM. The coupling of the AT1 receptor to signal transduction pathways was investigated. In intact cells AII (100 nM) evoked an increase in intracellular calcium ([Ca2+]i). This increase in [Ca2+]i was unaffected by PD 123177 (100 microM) but was abolished by DuP 753 (100 microM). Furthermore, AII (100 nM) did not inhibit forskolin (0.1-10 microM) stimulated cyclic AMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)


Affinity cross-linked delta-opioid receptor in NG108-15 cells is low molecular weight (25 kDa) and coupled to GTP-binding proteins. The affinity cross-linking of the delta-opioid receptor in neuroblastoma x glioma NG108-15 cells was undertaken using (3-[125I]iodotyrosyl27)human-beta-endorphin ([125I]beta-endorphin) and disuccinimidyl suberate (DSS) or bis(sulfosuccinimidyl) suberate (BS3) in order to estimate molecular size. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, two radioactive bands were observed. Labeling of a major band of 29 kDa diminished in the presence of unlabeled selective delta-opioid agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), in a concentration-dependent manner, while labeling of a minor band of 58 kDa was hardly affected. The labeling intensity of the 29 kDa band decreased by addition of guanosine 5'-(3-o-thio)triphosphate (GTP gamma S) or by pretreatment of cells with pertussis toxin. These results, taking the molecular weight of covalently bound beta-endorphin (3.6 kDa) into consideration, suggest that the delta-opioid receptor in NG108-15 cell membrane is a 25 kDa protein which is coupled to pertussis toxin-sensitive guanosine triphosphate-binding proteins (G-proteins).


Characterization of bradykinin B2 receptors on human IMR-90 lung fibroblasts: stimulation of 45Ca2+ efflux by D-Phe substituted bradykinin analogues. [3H]Bradykinin binds to intact human IMR-90 fetal lung fibroblasts in a time and dose-dependent manner. Binding equilibrium was attained by 120 minutes at 4 degrees C. [3H]Bradykinin binding was saturable; Scatchard analysis of saturation binding data demonstrated a single binding site having a KD = 1.8 +/- 0.2 nM and a receptor concentration of 17.4 +/- 4.0 fmol/10(5) cells. The calculated value for KD(k-1/k1) from the association (k1 = 4.71 x 10(6) mol-1 min-1) and dissociation (k-1 = 1.13 x 10(-2) min-1) rate constants was 2.4 nM. The rank order of potency observed for bradykinin peptide agonists, bradykinin > Lys-bradykinin > Met,Lys-bradykinin > Ile,Ser-bradykinin >> des-Arg9-bradykinin, is consistent with that of a bradykinin B2 receptor. Bradykinin stimulated efflux of 45Ca2+ from IMR-90 cells dose dependently with an EC50 = 331 +/- 50 pM. 45Ca2+ efflux was also demonstrated with Lys-bradykinin and Met-Lys-bradykinin but not by des-Arg10-kallidin (100 nM) or NKA (1 microM). Hoe-140 inhibited bradykinin-induced 45Ca2+ efflux (IC50 = 3 +/- 2 nM). D-Phe7-substituted bradykinin analogues stimulated 45Ca2+ efflux dose dependently and this stimulation of 45Ca2+ efflux was inhibited by Hoe-140. These results suggest that D-Phe7 substituted bradykinin analogues are agonists at the bradykinin B2 receptor in IMR-90 cells.


Interaction of opioid drugs with human platelet alpha 2-adrenoceptors. Morphine, naltrexone and naloxone inhibited the binding of [3H]clonidine to the alpha 2-adrenoceptors in human platelet membranes, provided that Mg2+ was present in the medium. In the presence of 5'-guanylyl imidophosphate (Gpp(NH)p) or in the absence of Mg2+ morphine did not modify the binding of [3H]clonidine. Neither [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAGO), nor [D-Pen2,D-Pen5]enkephalin (DPDPE), nor dynorphin-(1-17) affected the [3H]clonidine binding. The presence of 1 mM naloxone did not alter the affinity of either [3H]clonidine or [3H]yohimbine, but reduced the number of binding sites of [3H]clonidine, having no effect on [3H]yohimbine. Naloxone inhibited the binding of adrenaline to high- but not low-affinity sites. It is concluded that morphine and semisynthetic antagonist derivatives interact with alpha 2-adrenoceptors only in the high-affinity state.


Activation of protein kinase C and elevation of cAMP interact synergistically to raise c-Fos and AP-1 activity in Jurkat cells. We have earlier found that in Jurkat cells activation of protein kinase C (PKC) enhances the cyclic adenosine monophosphate (cAMP) accumulation induced by adenosine receptor stimulation or activation of Gs. Here we have therefore examined the effect of the phorbol ester PMA (phorbol 12-myristate 13-acetate) which stimulates PKC and a combination of the adenosine receptor agonist NECA (5'-(N-ethyl)-carboxamido adenosine) and forskolin to raise cAMP, on the levels of c-Fos and Jun and on the binding and transcriptional activity of the transcription factor, activator protein-1 (AP-1). PMA treatment caused a concentration- and time-dependent increase in both c-Fos and Jun immunoreactivity in contrast to cAMP elevation that had only a slight effect. Both PMA and the combination of NECA and forskolin acted together either to increase (c-Fos) or decrease (Jun) protein levels as well as increasing AP-1 binding, as judged by gel-shift assay, and AP-1 transcriptional activity. Furthermore there was a clear-cut synergy between the PKC stimulator and the cAMP elevating agents. The results demonstrate that the simultaneous activation of PKC and elevation of cAMP leads to an enhanced AP-1 transcriptional activity in a T-leukemia cell line, suggesting that the previously observed interaction between the parallel signal transduction pathways may have functional consequences at the level of gene transcription.


Protective effects of E3330, a novel quinone derivative, on galactosamine/tumor necrosis factor-alpha-induced hepatitis in mice. Oral pretreatment with E3330, a novel quinone derivative, attenuated liver injury induced with tumor necrosis factor-alpha in galactosamine-sensitized mice. Tumor necrosis factor-alpha is known to induce inflammatory mediators such as leukotrienes and prostanoids. An in vitro study showed that E3330 inhibited the generation of leukotriene B4 and thromboxane B2, but enhanced prostaglandin E2 generation from rat peritoneal exudate cells stimulated with the Ca(2+)-ionophore, A23187. These findings suggest that the protective effect of E3330 on galactosamine/tumor necrosis factor-alpha hepatitis is due at least in part to its inhibition of the generation of leukotrienes. The inhibition of thromboxane B2 generation or the enhancement of prostaglandin E2 generation by E3330 may also contribute to its hepatoprotective effect.


Ultradian variations in sensitivity of rat aorta rings to noradrenaline. The in vitro sensitivity of the rat aorta to the vasoconstrictor effect of noradrenaline was found to vary according to the time the animals were killed, with a minimum at 10:00 h and a maximum at 16:00 h. This ultradian rhythm was not influenced by the presence of the endothelium but was modified by some, as yet unidentified, circulating factor(s). The results also strongly suggested an ultradian rhythm of in vitro sensitivity to the beta-adrenoceptor agonists, salbutamol and isoprenaline. This rhythm appears to be opposite to that for the sensitivity of aortic alpha-adrenoceptors to stimulation. In conclusion, our results demonstrate that the regulation of aortic tone follows an ultradian rhythm.


TNF-specific deactivation of granulocytes in vivo: a possible mechanism of self-protection. Granulocytes (PMN) have recently been shown to be specifically deactivated towards tumor necrosis factor (TNF) by preexposure to TNF itself and to other stimuli such as endotoxin and complement C5a in vitro. When TNF (200 micrograms/m2) was infused to a male volunteer, we found ex vivo an almost complete TNF-specific deactivation of PMN adhesion (57 +/- 3* vs 6 +/- 2%**), of exocytosis of secondary granules (37 +/- 1* vs 7 +/- 1%**) and of the respiratory burst as measured by hexose monophosphate shunt activation (74 +/- 1* vs 12 +/- 3** nM/10(7) PMN/45 min). Furthermore, up- and down-regulation, respectively, of the PMN surface adhesion molecules Mac-1 and LAM-1 became TNF-resistant. Similar to the results obtained in vitro, deactivation correlated with a decrease in the number of cellular TNF receptors. In PMN of 4 patients exposed to activated complement during hemodialysis, a state of TNF-specific cross-deactivation could also be demonstrated.


Expression and function of beta-adrenergic receptors in human hematopoietic cell lines. We investigated the expression and functional characteristics of beta-adrenoceptors in a panel of 10 phenotypically different human hematopoietic cell lines. A binding assay with [125I]iodocyanopindolol as the ligand revealed that cell lines of myelomonocytic or histiocytic derivation (HL-60, ML-2, RC-2A, U-937) expressed high numbers of beta-adrenoceptors. An intermediate density of receptors was found in a non-T, non-B cell leukemia line (Nall-1), whereas T-cell (JM, CCRF-CEM), B-cell (Raji) or erythroleukemic cell lines (K-562, HEL) displayed minimal or undetectable binding of the radioligand. Isoprenaline-stimulated cAMP production by the cells correlated to their extent of beta-adrenoceptor expression. Southern blot hybridization analysis of genomic DNA from the cell lines with a 32P-labelled beta 2-adrenoceptor cDNA probe revealed no evidence for major rearrangement or amplification of the receptor gene. Incubation with isoprenaline in vitro suppressed the proliferation of the receptor-rich RC-2A cells but did not affect the growth rate of the receptor-deficient K-562 cells. Treatment with propranolol slightly enhanced the proliferation of the RC-2A cells but did not markedly alter the growth rate of two other cell lines, regardless of their beta-adrenoceptor status. These findings indicate a regulatory influence by the sympathoadrenergic system on selected cells of the myelomonocytic lineage.


Surface potential changes of mitoplasts in the presence of pyridoxal phosphate modified cytochromes c. 1. The addition of native cytochrome c to mitoplasts leads to a decrease of surface potential of the mitoplast membrane. However the surface potential is slightly decreased (approximately 3 mV) when PLP(Lys 86)-cytochrome c and PLP(Lys 79)-cytochrome c were added. 2. The native and PLP-modified cytochromes c do not influence the order parameters S and isotropic constant a when both spin probe I and probe II were used. It is shown that cytochrome c binding to the membrane does not affect the hydrophobic intermembrane area as well as the lipid arrangements of the mitoplast membrane. 3. At low ionic strength there was observed a significant difference in the membrane potential when PLP-cytochromes c were added to the mitoplasts. 4. At high ionic strength the addition of native or PLP-modified cytochromes c does not change the membrane potential.


[Mapping of plasmid genes responsible for microcin R51 synthesis and immunity to it]. Microcin R51 is plasmid-determined low-molecular-weight peptide antibiotic produced by Escherichia coli. The spectrum of its action includes many different species of gram negative and some gram positive bacteria. Microcinogenic strains are immune to the action of the microcin they synthesize. As shown earlier, genes responsible for MccR51 production and immunity are located in a continuous 11.1 kb DNA fragment. These genes were cloned in pUC19 and pACYC184 plasmid vectors. Deletion derivatives and Tn5 insertion mutant plasmids which determined no microcin synthesis and immunity were obtained. Analysis of clones' phenotypes and physical mapping of mutant plasmids demonstrated that the 5 kb DNA fragment was indispensable for microcin production. The region of about 4.6 kb confers complete immunity of the producing strains, while partial immunity is provided by 1.8-1.9 kb DNA fragment.


The biological reductive capacity of tissues is decreased following exposure to oxidative stress: a cyclic voltammetry study of irradiated rats. The reductive capacity of rat tissue homogenates and body fluids was determined by cyclic voltammetric measurements. The reductive capacity of rat lung, liver and kidney homogenates was significantly reduced four days after total body gamma-ray irradiation with 5.5 Gy as compared to controls. In parallel, reduced ability of the irradiated organ homogenates to scavenge hydroxyl radicals and to destroy hydrogen peroxide was recorded. However, no difference in their superoxide dismutase activity was found. The possible use of cyclic voltammetry as a method for qualitative evaluation of the ability of biological tissues to cope with oxidative stress is discussed.


In vitro study of the phagocytic processes in splenic granulocytes of the tench (Tinca tinca, L.). The different stages of the phagocytic process by splenic granulocytes of Tinca tinca were studied. Adherence capacity to both endothelium and tissue substrate, mobility rate, the phagocytosis capacity for both cells (Candida albicans) and inert particles (latex beads), candidicide power, and capacity of digestion measured by nitroblue tetrazolium (NBT) reduction were evaluated in splenic granulocytes of healthy adult tench. The capacity of adherence to nylon fiber was possessed by 51% of the granulocytes. The percentage capable of adherence to smooth plastic surfaces rose with incubation time. Casein, an effective chemoattractant, increased the random mobility of the granulocytes. Phagocytosis was greater for opsonized C. albicans than for nonopsonized. However, the number of phagocytosed yeast cells destroyed by the granulocytes did not depend on whether or not the C. albicans had been previously opsonized. The phagocytosis indices and the percent phagocytosis of latex beads were greater than those obtained for the phagocytosis of C. albicans in the absence of serum. Finally, the metabolic activity in these cells following the digestion of ingested material showed a 148 +/- 31% stimulation. The results show that splenic cells of tench have the capacity to make a phagocytic response against both cells (C. albicans) and inert particles (latex beads).


Correlation between destruction of malarial parasites by polymorphonuclear leucocytes and oxidative stress. The role of reactive oxygen species (ROS) generated by polymorphonuclear leucocytes (PMNs) in the host response against malaria was investigated. Non-activated human PMNs were added to cultures of P. falciparum in microtitre cells. Parasite viability was evaluated by the incorporation of radioactive hypoxanthine. Using PMN/RBC = 1/150 (starting parasitemia was 1%) the incorporation on the second day in culture was only 61% of the control cultures. An effect could be observed already after two hours of incubation (30% reduction at a 1/50 PMN/RBC ratio). A direct contact between the effector and target cells was obligatory for the expression of the damage. Parasites within G6PD-deficient erythrocytes were more sensitive to the PMNs than normal parasitized erythrocytes. This difference could be attributed to the production of reactive oxygen intermediates in the experimental system, since G6PD-deficient erythrocytes are generally more sensitive to oxidant stress. Salicylic acid was used as a scavenger and reporter molecule for hydroxyl radical fluxes. It is converted to the corresponding dihydroxybenzoic acid derivatives, which could be detected by HPLC. Uninfected NRBC or parasitized erythrocytes containing young ring forms could trigger the PMNs to produce much less ROS than the mature forms of the parasites. Other factors associated with PMNs may inactivate the parasites, such as phagocytosis, lysosomal enzymes or degradation toxic products of the PMNs. However our results indicate that increased oxidative stress induced by PMNs interfere with the growth of P. falciparum and could play a role in human evolution of abnormal erythrocytes.


Toxic effects and detection of oxygen free radicals on cultured articular chondrocytes generated by menadione. The aim of this work was to study the proliferation pathological perturbations of cultured chondrocytes in response to menadione, an oxygen free radicals producing drug. Rabbit articular chondrocytes in monolayer culture were treated with 10(-5) M, 1.5.10(-5) M and 2.10(-5) M of menadione during three days. A dose dependent decrease of the proliferative capacity was observed. Flow cytometry analysis revealed a perturbation of the cell cycle progression consisting in an accumulation of cells in the S and G2 + M phases. This growth perturbation was due to oxygen radicals production since a treatment with catalase suppressed these toxic effects. Furthermore, to identify oxygen derived radicals in the cellular suspension of cultures treated with menadione, we used a technique of spin-trapping coupled with electron spin resonance (ESR). The ESR signal corresponding to the DMPO hydroxyl radical adduct (DMPO-OH) has been detected. The spectra observation indicated the actual production of hydroxyl radical. However, superoxide anions have not been identified; this fact can be explained by the low reactivity of these anions with DMPO and by the decomposition of signal DMPO-OOH to DMPO-OH.


Prognostic significance of polymerase chain reaction detected human papillomavirus of tumors and lymph nodes in surgically treated stage IB cervical cancer. This study describes the prognostic role of polymerase chain reaction detected human papillomavirus (HPV) in Stage IB cervical cancer patients treated with radical hysterectomy and pelvic and paraaortic node dissection. All tumors were confined to the cervix and all margins and nodes were disease free. Twenty-one patients were analyzed: 6 patients recurred within 20 months of initial therapy, while 15 had no evidence of disease with a minimum follow-up of 36 months. Polymerase chain reaction (PCR) was performed on paraffin-block tissue of the hysterectomy specimen cervical tumor and lymph nodes. Oligonucleotide probes for HPV types 6, 11, 16, 18, 31, 33, and 35 were used with consensus primers for uncharacterized HPV types created from an L1 constant region. Control tissues were run with each tumor sample to assure against contamination. HPV type confirmation was performed using diagnostic restriction sites. HPV was detected in all cervical tumors. Recurring tumors were infected with multiple types of HPV in all 6 tumors versus only 5 of 15 nonrecurring tumors being multiply infected (P = 0.023). No tumor had HPV 6 or 11, and the incidence of HPV 16, 31, 33, and 35 was not significantly different for recurrent versus nonrecurrent groups. HPV 18 was found in 5 of 6 recurring cancers versus 1 of 15 nonrecurring tumors (P = 0.0029). PCR typing of the histologically negative nodes that had been obtained at radical hysterectomy was done in all 6 recurring patients and in 6 nonrecurring patients. The recurrent patients had a significantly higher incidence of lymph nodes positive for HPV DNA (71%) than the nonrecurring patients (35%) (P = 0.0047). These observations suggest that HPV 18 cervical cancer patients, those with infections of multiple types, and those with HPV DNA in histologically negative lymph nodes may be at increased risk for recurrence.


Intraabdominal hemorrhage with pulmonary large cell carcinoma metastatic to the ovary. Secondary tumors comprise nearly 10% of ovarian malignancies; however, metastatic cancers arising from the lung are uncommon, with fewer than 15 cases reported. A patient with pulmonary large cell carcinoma and ovarian metastases resulting in recurrent refractory intraabdominal hemorrhage is presented. Metastatic involvement of the ovary from pulmonary malignancy is reviewed.


[Sutureless intestinal anastomosis with a biofragmentable anastomotic ring]. There have been efforts to develop an ideal method of intestinal anastomosis throughout medical history. In ancient China, Egypt and India living ants were used to approximate the transected edges of bowel. Many other devices and techniques have since been introduced, using organic or synthetic, nonabsorable or absorbable materials. Lately mechanical stapling devices have become exceedingly popular. The recently introduced biofragmentable anastomotic ring (BAR) is composed of an absorbable polyglycolic acid in 12.5% barium sulfate suspension to permit x-ray visualization. Prospective randomized studies have shown it to be both effective and safe as compared to conventional handsutured or stapled anastomosis. Its main advantages are simplicity and ease of handing, short operating time and the absence of foreign bodies at the anastomotic site that might interfere with imaging methods, such as CT or MRI. We present our preliminary, satisfactory experience with BAR in 14 patients with a mean age of 67 years. 8 underwent right colectomy, 2 left colectomy, 1 sigmoidectomy, 2 resection of upper rectum, 1 subtotal colectomy, 1 excision of a giant lymphoma of the cecum, and 1 had had recurrent bouts of volvulus of the sigmoid.


Development of preneoplastic lesions in the liver and nasal epithelium of rats initiated with N-nitrosodimethylamine or N-nitrosopyrrolidine and promoted with polybrominated biphenyls. In rats, N-nitrosodimethylamine (NDMA) and N-nitrosopyrrolidine (NPYR) induce liver tumours and, to a lesser extent, nasal tumours. Polybrominated biphenyls (PBBs) are liver tumour promoters and are highly persistent in tissues of rats. To characterize the development of preneoplastic lesions in the liver and nasal cavity, female Sprague-Dawley rats were initiated with NDMA or NPYR and promoted with Firemaster (FM), a commercial mixture of PBBs. Rats were killed after 30, 120 or 180 days of promotion. Liver and nasal tissues were stained with haematoxylin and eosin and were tested immunohistochemically for glutathione S-transferase placental form (GST-P). Significantly more altered hepatocellular foci (AHF) were evident in rats initiated with NDMA or NPYR and promoted with FM compared with non-promoted groups or rats given only FM. Appreciable numbers of AHF were seen at 120 and 180 days in livers of rats in all other treatment groups, whereas the untreated control rats had no AHF. The percentage volume of the liver occupied by AHF was significantly higher in promoted rats given NDMA than in rats given only NDMA or FM. These results indicate that a single oral dose of PBB can significantly enhance development of AHF in rats initiated with NDMA or NPYR. Preneoplastic lesions in nasal tissues were not detected by staining with GST-P.


Cytosolic Ca2+ overload and macromolecule permeability of endothelial monolayers. It was investigated how cytosolic Ca2+ overload affects the cytoskeletal structure and macromolecule permeability (for albumin) of monolayers of endothelial cells (from porcine aorta). States of cytosolic Ca2+ overload were produced either 1. by metabolic inhibition (5 mM KCN plus 5 mM 2-deoxyglucose) or 2. by increasing membrane permeability with the use of a Ca2+ ionophore (10 microM A 23187). The effects of cytosolic Ca2+ overload on the structure of F-actin filaments and monolayer permeability were monitored. ATP stores were rapidly degraded (> 90% in 15 minutes) in the presence of metabolic inhibitors, but only partially reduced in the presence of A 23187 (30%) in two hours). Concomitantly with ATP loss, cytosolic Ca2+ levels were increased in metabolically inhibited cells. Two-hour exposure to the Ca2+ ionophore A 23187 mimicked the effect of two-hour metabolic inhibition on F-actin filaments and monolayer permeability, in spite of the divergence in energy metabolism. Disintegration of F-actin filaments in presence of metabolic blockers or ionophore was accompanied by appearance of F-actin clumps in the cells, but total contents of F-actin remained unaltered. Within three hours after removal of these agents, a normal F-actin structure and normal macromolecule permeability were re-established in the monolayers. The results show that cytosolic Ca2+ overload causes disintegration of F-actin filaments and a subsequent increase in macromolecule permeability. These changes are readily reversible as long as the dis-integration is based on fragmentation and not depolymerization of F-actin filaments.


Inhibition of vascular smooth muscle cell proliferation by endothelium-dependent vasodilators. This review considers the hypothesis that the endothelium-derived vasodilator agents, prostacyclin and nitric oxide, also function physiologically to inhibit vascular smooth muscle cell (VSMC) proliferation. The underlying biochemical mechanisms are also discussed. Prostacyclin and other agents that increase intracellular cAMP concentration are potent and effective inhibitors of the proliferation of isolated VSMC in culture. Such agents inhibit the initiation of proliferation in quiescent cells and the proliferation of logarithmically growing cells from a variety of sources, including man. The data implicate prostacyclin as an important regulator of VSMC proliferation, although there is little direct in vivo evidence. Nitric oxide-releasing drugs (and atriopeptins which increase intracellular cGMP concentration by a different mechanism) also inhibit proliferation of cultured VSMC. The effects are, however, partial and obtained at higher concentrations than those required for vasodilatation. Even allowing for the instability of the agents under tissue culture conditions, cGMP-elevating agents appear to be poorer at inhibiting proliferation than cAMP-elevating agents, despite similar or greater vasodilator potency. These data imply that nitric oxide is less likely than prostacyclin to be a physiological regulator of VSMC proliferation, although definitive experiments in vivo are again lacking. It also follows that nitrovasodilators are less attractive as therapy for VSMC proliferation than prostacyclin analogues or other cAMP-elevating agents, such as phosphodiesterase inhibitors. By analogy with the mechanisms of vasodilatation, inhibition of calcium mobilization and the subsequent activation of protein kinase C are considered as possible mechanisms underlying inhibition of proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)


Recruitment of dorsal column fibers in spinal cord stimulation: influence of collateral branching. An electrical network model of myelinated dorsal column nerve fibers is presented. The effect of electrical stimulation was investigated using both a homogeneous volume conductor and a more realistic model of the spinal cord. An important feature of dorsal column nerve fibers is the presence of myelinated collaterals perpendicular to the rostro-caudal fibers. It was found that transmembrane potentials, due to external monopolar stimulation, at the node at which a collateral is attached, is significantly influenced by the presence of the collateral. It is concluded that both excitation threshold and blocking threshold of dorsal column fibers are decreased up to 50% compared to unbranched fibers.


Paget's disease of the breast: a study of 43 cases. Paget's Disease of the breast is caused by spread of duct carcinoma cells along the mammary ducts to the epidermis of the nipple and areola. This is a study of 43 cases of Paget's Disease of the breast. Though only few patients presented with a lump; a carcinoma, either DCIS or IDC or both were found in all cases. The presence of an underlying breast carcinoma in Paget's Disease of the breast suggests that radical mastectomy is the treatment of choice in this condition.


Beta-adrenoreceptor blockade attenuates heat-induced tachycardia,but not the tolerance to the stress. Ten healthy males (age 34 +/- 3 yr 9 SE) underwent 40 min of heat exposure (WD 39.7.C) after 2 hours of ingesting 120 mg of Propranolol (Inderal; ICI), or a placebo, in a random manner, the exposures being about a week apart. That there was no placebo effect was ensured by giving a control run (no medication). In the placebo trials, the end-experiment heart rate had increased by 52%, while after propranolol the increase was only 43%. Regression analysis showed that with the placebo, the HR increased by 22 beats/min/o rise in core (aural) temperature, while with propranolol, the rise (14 beats/min) was significantly lower (P < 0.02). The various heat strain indices viz the Craig's Index, the Body heat storage (Kilocals/m2/hr), and the effective heat storage were also similar for both the treatments. We conclude that beta-adrenoreceptor activity plays a significant role in producing tachycardia of heat exposure in humans, but blocking this activity with propranolol does not affect tolerance to heat stress.


Influence of two guar preparations on glycosylated hemoglobin, total cholesterol and triglycerides in patients with diabetes mellitus. Guar smoothens postprandial glucose peaks and reduces serum cholesterol. Both properties are advantageous for diabetic patients and can be realized, in part, by adding fiber to the diet. Depending on the blood glucose concentration, a small portion is incorporated into hemoglobin (HbA1), building a largely irreversible complex, making it a long-term indicator of blood glucose status. HbA1 may increase from a normal value of about 5% to as much as 20% of total hemoglobin in diabetics. This study tested the efficacy of two guar preparations in diabetic patients over 3 months by measuring HbA1, cholesterol and triglycerides. Forty diabetic patients with HbA1 > 10% ingested either 3 x 4 g daily of a new guar preparation (GU-052, Steigerwald, Darmstadt, Germany) or 3 x 5 g daily of Glucotard (Boehringer, Mannheim, Germany) for 12 weeks. Before and after 30, 60 and 90 days of treatment, HbA1, cholesterol and triglycerides were determined. After 90 days of treatment, GU-052 caused a reduction in HbA1 from 12.6 +/- 2.6% to 10.5 +/- 1.5% (-15.6 +/- 9.0%) in the GU-052 group and from 12.0 +/- 2.6% to 10.9 +/- 1.82% (-8.6 +/- 7.2%) in the Glucotard group. Cholesterol was reduced from 269 +/- 28 mg/100 ml on day 0 to 227 +/- 18 mg/100 ml on day 90 in the GU-052 group and from 261 +/- 40 to 235 +/- 26 mg/100 ml in the Glucotard group. Both GU-052 and Glucotard had little effect on plasma triglycerides. Fewer unpleasant side effects were reported for the GU-052 preparation than for Glucotard.(ABSTRACT TRUNCATED AT 250 WORDS)


Metabolic aspects of acute tissue hypoxia during extracorporeal circulation and their modification induced by L-carnitine treatment. In this study the authors examine the effects of acute hypoxia due to extracorporeal circulation (ECC) and the role played by L-carnitine treatment on some plasmatic metabolites linked to glycolytic cellular metabolism. To obtain biochemical data, 120 patients in extracorporeal circulation during aortopulmonary bypass surgery were evaluated. The patients received either sodium bicarbonate (40 patients), or L-carnitine during ECC (40 patients) or before and during ECC (40 patients), and plasma samples were collected before ECC, during ECC and after ECC. The levels of lactate and pyruvate showed significant alterations in sodium bicarbonate-treated patients, and there was also a considerable imbalance in the succinate/fumarate ratio. This means that tissue hypoxia due to ECC leads to cellular oxidative damage and to a considerable decrease in the intracellular energy pools. The use of L-carnitine antagonizes the oxidative stress, as is well documented by the levels of plasmatic metabolites which remain confined to normal amounts.


Interactions of vitamin D with sodium zeolite A in rats from low and adequate vitamin D colonies. Weanling rats (21 days old) from either a low vitamin D colony or an adequate vitamin D colony fed either a vitamin D adequate or a vitamin D deprived AIN-76A purified diet were used as a model to investigate interactions of vitamin D status with dietary sodium zeolite A (100 mg/kg body wt/day). Rats with adequate or replete vitamin D status had greater overall body weight gain, dry tibia weights and femur densities and ash weights than rats fed a vitamin D deprived diet. Addition of sodium zeolite A to the diets did not have an effect on plasma calcium, body weight gain or on femur density, ash and percent ash. Dietary sodium zeolite A increased total tibia fat in rats fed the vitamin D adequate diet and decreased total tibia fat in rats fed the vitamin D deprived diet. This effect of sodium zeolite A appeared to be beneficial to bone status in the groups fed adequate vitamin D, since these treatment groups had higher, although not significant, dry tibia weights with and without fat.


[Detection of varicella zoster virus infections using polymerase chain reaction]. The polymerase chain reaction (PCR) was used to detect varicella zoster virus (VZV) DNA in vesicle samples from patients with varicella and zoster. Primers and the oligonucleotide probe were chosen from the region of the immediate early gene 63. Procedures for preparing the DNA from the specimens were omitted, and the amplified DNA was directly detected in ethidium bromide-stained polyacrylamide or agarose gels, thus providing a rapid and less laborious assay. A total of 66 vesicle specimens including 3 crusts (collected on days 1-14 after the onset of exanthem) were tested by the simplified VZV-PCR, and 64 (97%) were positive. When the direct visualization of the amplified DNA was confirmed by DNA hybridization, a non-radioactive hybridization assay involving a digoxigenin-labelled oligonucleotide probe and detection by chemiluminescence proved as adequate as a radioactive hybridization assay. Thus, the VZV PCR described appears to be a useful diagnostic test for detecting and identifying varicella zoster virus.


[Peptidergic innervation of human salivary glands (parotid gland and submandibular gland)]. Immunocytochemistry and a radioimmunoassay were used to investigate the existence and distributions of various regulatory peptide immunoreactivities (ir) in human submandibular and parotid glands. Numerous nerve fibers containing vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM), or neuropeptide tyrosine (NPY) and C-flanking peptide of NPY (CPON)-ir were found in close proximity to acini, ducts and blood vessels. Only a few calcitonin gene-related peptide (CGRP)- and substance P (SP)-ir nerve fibers could be demonstrated and were mainly localized around blood vessels and ducts. Galanin and the recently discovered peptides helospectin and pituitary adenylate cyclase activating peptide were unable to be detected in the salivary glands studied. Preliminary quantitative investigations of four human submandibular glands using radioimmunoassay showed that VIP-ir had the highest concentration, followed by NPY-ir and CGRP-ir; SP-concentrations were below the detection limit. The possible physiological significance of these peptides for salivary secretion is discussed.


[Scintigraphic imaging of head and neck cancers with 99m technetium (v) dimercaptosuccinic acid. A prospective clinical study]. In patients with cervical metastases conventional examination by ultrasound, CT or MRI imaging often fails to identify an unknown primary tumor. Also the retrieval of a recurrent malignancy may be difficult. Scintigraphy, utilizing technetium-99m (v) dimercaptosuccinic acid was chosen for a prospective study in 17 patients to evaluate its properties for imaging metastasizing squamous cell carcinoma of the head and neck. Scintigraphic findings were correlated with the results of clinical examination and conventional imaging techniques. In all cases the primary tumor revealed good uptake of 99mTc(v)DMSA. Manifest cervical metastases could only be imaged in some cases. In future, therefore, 99mTc(v)DMSA scanning may be used for the detection of unknown primary tumors. However, it does not appear helpful in the evaluation of cervical nodes.


[Peripheral facial paralysis as the first symptom of unknown metastatic primary tumor]. The spread of metastases to the brain and the leptomeninges is usually a terminal event in patients with known malignancies. We report on two patients, in whom the diagnosis of a diffusely metastasizing carcinoma was considerably delayed by the initial symptom of a peripheral facial nerve paralysis. The clinical, neuroradiologic and cerebrospinal fluid findings are discussed. A malignant neoplasm with diffuse or focal metastasis to the leptomeninges is a rare cause of peripheral facial paralysis of unclear genesis. The necessity of a lumbar puncture plus magnetic resonance imaging of the brain to enable an early diagnosis is emphasized.


[Pleomorphic adenoma of the epiglottis. Case report and review of the literature]. A rare case of a pleomorphic adenoma of the epiglottis in an 82-year-old patient is presented. Resection of the tumor was accomplished by using the CO2-laser and required a partial epiglottectomy. Postoperatively there were no functional impairments. This is the third case of a pleomorphic adenoma of the epiglottis, published in literature, but the first case in which epiglottectomy was performed by endoscopic laser resection.


Cyclooctatin, a new inhibitor of lysophospholipase, produced by Streptomyces melanosporofaciens MI614-43F2. Taxonomy, production, isolation, physico-chemical properties and biological activities. Cyclooctatin has been isolated from Streptomyces melanosporofaciens MI614-43F2 as part of a program designed to find microorganism-produced inhibitors of lysophospholipase. It was purified by chromatography on silica gel, Capcell Pak C18 (HPLC) and Sephadex LH-20 followed by solvent extraction and then isolated as a colorless powder. Cyclooctatin has the molecular formula of C20H34O3. It is competitive with the substrate, and the inhibition constant (Ki) was 4.8 x 10(-6) M.


Nutritive quality and palatability of switchgrass hays for sheep: effects of cultivar, nitrogen fertilization, and time of adaptation. Feeding and palatability trials were conducted with four cultivars (cv) of switchgrass (Panicum virgatum L.), fertilized at three levels of N (0, 75, or 150 kg of N/ha) in 2 yr. Wether sheep had ad libitum access to chopped hays, and intake, apparent digestibility, particle passage rates, and concentrations of blood metabolites were determined. Palatability was measured with mature sheep in cafeteria trials. Nitrogen fertilization did not affect (P > .05) DM digestibility (DMD) or DMI, but there was a year x N interaction (P < .01) for NDF digestibility. Dry matter digestibility values for combined years and N levels were 56.9, 54.4, 56.6, and 57.9% (P < .01) for Pathfinder, New Jersey 50, Kentucky 1625, and Trailblazer cv, respectively; mean DMI values were 60.4, 60.8, 57.7, and 64.0 g/kg BW.75 (year x cv, P < .01). An apparently greater quality of Trailblazer was masked by weed invasion of N-fertilized stands of this cv in yr 2, with changes in hay composition. Lambs adapted to hay diets with time; mean DMI for cv and N levels combined increased (P < .001) from 50.5 to 71.4 g/kg BW.75 between wk 2 and 3 and wk 10 and 11, with no change in DMD. Intakes of NDF increased from 37.6 to 55.6 g/kg BW.75, an increase of 48%. Marker (Yb) measurements indicated little change in particle passage rates with treatment. There were no major differences in blood composition, apart from increases in blood urea N, as a result of N fertilization. Cafeteria trials showed preference by sheep for Trailblazer and KY 1625 compared with NJ 50 and Pathfinder, with a N x cv interaction (P < .01). Trailblazer was preferred to KY 1625 in a two-choice situation (P < .01). Results show relatively small effects of cv and N fertilization on quality of switchgrass and indicate the need for a lengthy period of adaptation by sheep to the feeding of a warm-season grass.


Effects of supplemental protein source and alkaline hydrogen peroxide treatment of wheat straw on site of nutrient digestion and flow of nitrogenous compounds to the duodenum of steers. The objective of this study was to determine the effects of soybean meal (SBM) or spray-dried blood meal (BM) supplementation of diets based on untreated (UNT-WS) or alkaline hydrogen peroxide-treated wheat straw (AHP-WS). A 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments was used. Variables included nutrient digestion and flow to the duodenum. Four Simmental steers (average weight 477 kg) fitted with ruminal and duodenal cannulas were fed 65% UNT-WS or AHP-WS based diets in 12 equal portions daily. Diets were formulated to contain 10% CP. Chromic oxide was used as the digesta flow marker and purines were used as the microbial marker. There were no straw type x protein source interactions. Total tract and ruminal OM digestibility were approximately 25% greater (P < .04) when AHP-WS was fed than when UNT-WS was fed. Source of protein did not affect (P > .10) OM or fiber digestion in the rumen or total tract. Ruminal digestion of NDF and ADF was increased (P < .01) by 51 and 40%, respectively, when AHP-WS was fed than when UNT-WS was fed. Main effect means (P > .10) for N flow to the duodenum as a percentage of N intake were 128.2, 142.5, 133.4, and 137.6 for UNT-WS, AHP-WS, SBM, and BM treatments, respectively. Despite increased (P < .01) ruminal OM digestion for AHP-WS, microbial N flow to the duodenum was greater (P < .01) when UNT-WS was fed than when APH-WS was fed.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of energy level and protein source on nitrogen kinetics in steers fed wheat straw-based diets. A 4 x 4 Latin square metabolism trial with a 2 x 2 factorial arrangement of treatments was conducted to determine N kinetics in steers. Steers were fed either untreated (UNT-WS) or alkaline hydrogen peroxide-treated wheat straw (AHP-WS) based diets supplemented with soybean meal (SBM) or blood meal (BM). Single doses of (15NH4)2SO4 were infused into ruminal pools to determine N kinetics. Ruminal NH3N concentrations (main effects) were 3.81, 1.65, 3.18, and 2.28 mg/dL in steers when fed diets that contained UNT-WS, AHP-WS, SBM, and BM, respectively. Ruminal N pool size was greater (P < .05) for UNT-WS than for AHP-WS diets and also was greater (P < .10) for SBM than for BM diets. Nitrogen flux rate into the rumen was not affected (P > .10) by diet. However, production rate of N from the ruminal pool was greater (P < .05) for UNT-WS than for AHP-WS diets and greater (P < .10) for SBM than for BM diets. Nitrogen recycled into the rumen was 33% greater (P < .05) for AHP-WS than for UNT-WS diets and 26% greater (P < .05) for BM than for SBM diets. Nitrogen recycling (percentage of N intake) was 33, 56, 36, and 49% for UNT-WS, AHP-WS, SBM, and BM diets, respectively. The blood urea N (BUN) concentrations were 10.23, 4.58, 7.15, and 7.65 mg/dL for UNT-WS, AHP-WS, SBM, and BM diets, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Reactive oxygen in skeletal muscle. II. Extracellular release of free radicals. We have tested the hypothesis that diaphragm muscle fibers release superoxide anion radicals (O2-.) into the extracellular space. Fiber bundles were isolated from rat diaphragm and incubated in Krebs-Ringer solution containing cytochrome c (10(-5) M), a standard assay for O2-.. Bundles were either passive or active, i.e., directly stimulated to contract rhythmically. After 1 h, absorbance of reduced cytochrome c in the incubation medium was measured at 550 nm. Absorbance was greater in medium exposed to passive muscle than in medium without muscle (P < 0.01), indicating O2-. release by passive muscle. Absorbance was greater in medium exposed to active muscle than in that exposed to passive muscle (P < 0.01), an increase inhibited by superoxide dismutase (10(3) U/ml). Active bundles fatigued; bundles developing the lowest final stresses produced the greatest absorbance increases (P < 0.001), suggesting that the magnitude of fatigue was inversely related to O2-. release. We conclude that O2-. is released by diaphragm myocytes into the interstitium and surrounding medium, a process accelerated by fatiguing muscular contractions.


Mechanisms of increased pulmonary microvascular permeability induced by FMLP in isolated rabbit lungs. We observed that the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L- phenylalanine (FMLP) induced pulmonary edema when polymorphonuclear leukocytes (PMNs) were added to isolated constant-flow buffer-perfused rabbit lungs. This study was designed to test the hypothesis that PMNs activated by FMLP induced lung injury by the modulation of reactive oxygen species (ROS), cyclooxygenase products, or cysteinyl leukotrienes (LTs). Addition of FMLP alone did not increase microvascular permeability (Kf). When PMNs were added to the isolated lung, FMLP caused an 80% increase in Kf. Wet-to-dry weight ratio was also significantly increased with PMNs + FMLP compared with FMLP only. There was a significant positive correlation between total myeloperoxidase activity in lung tissue and Kf values after FMLP (30 min). Pretreatment with two dissimilar cyclooxygenase inhibitors, meclofenamate or ibuprofen, had no effect on the PMN + FMLP-induced increase in Kf. However, the ROS inhibitor catalase and the nonantioxidant LT synthesis blocker MK 886 inhibited the PMN + FMLP increase in Kf. Perfusate levels of LTs (LTC4, -D4, and -E4) were significantly increased from baseline values 30 min after FMLP. Both MK 886 and catalase suppressed the elevation of LTs after PMN + FMLP. These results indicate that FMLP increased a pulmonary microvascular permeability in isolated buffer-perfused rabbit lungs that is PMN dependent and mediated by LT produced possibly by a result of ROS production.


Vascular beta-adrenoceptor-mediated responses in hypertension and ageing in rats. 1. In normotensive Wistar (W) and spontaneously hypertensive (SHR) rats between 5 and 20 weeks of age, there was an age-related increase in blood pressure (r2 = 0.770 and r2 = 0.801 respectively). Except for adrenaline (r2 = 0.979) in SHRs, plasma catecholamines and age were unrelated. 2. In ring segments of thoracic aorta from 5, 10, 15 and 20 week old rats, the respective EC80s (-log M) for phenylephrine (PE)-induced contractions were 8.20 +/- 0.37, 7.96 +/- 0.10, 7.15 +/- 0.12 and 7.12 +/- 0.21 in W and 7.73 +/- 0.13, 7.72 +/- 0.16, 7.37 +/- 0.08 and 7.40 +/- 0.03 in SHR tissues (means +/- SEM; n = 5-7). 3. In PE-preconstricted rings, the respective EC50s (-log M) for isoprenaline (IPNA)-induced relaxation were 7.97 +/- 0.11, 7.74 +/- 0.10, 6.96 +/- 0.19 and 6.57 +/- 0.26 in W and 8.03 +/- 0.24, 7.62 +/- 0.08, 6.88 +/- 0.13 and 6.73 +/- 0.14 in SHR tissues (n = 5-7). 4. In PE-preconstricted rings from 5 and 20 week old rats, a single concentration of IPNA (approximating the respective IPNA EC50s) induced relaxation which was sustained over 2 h in W but not SHR tissues. The SHR:W ratios of the net relaxant responses, at 5 and 20 weeks, were 0.6461 and 0.6167 respectively. 5. Thus, W rats exhibit an age-related loss in both vascular alpha- and beta-adrenoceptor responsiveness which appears unrelated to plasma catecholamines. SHRs also exhibit an age-related loss in vasodilator beta-adrenoceptor responsiveness, which may involve adrenaline-induced desensitization, but appear to maintain vasoconstrictor alpha-adrenoceptor responsiveness. It is proposed that an age-related decline in beta-adrenoceptor responsiveness coupled to maintenance of alpha-adrenoceptor responsiveness may lead to chronic blood pressure elevation, as observed in SHRs, while a parallel decline in both alpha- and beta-adrenoceptor responsiveness, as observed in W rats, may preclude hypertension development.


Detection of infectious bursal disease virus infection using the polymerase chain reaction. The method of reverse transcription (RT) followed by the polymerase chain reaction (PCR) was used to amplify two different fragments of the infectious bursal disease virus (IBDV) genome. Two sets of primer framed two different regions within the genes coding for proteins VP2 and VP3, respectively. Both sequences were detected in five strains of IBDV, whereas, none were obtained from uninfected control cells. The sensitivity of RT-PCR was carried out on nucleic acids from the IBDV infected cell cultures. The detection limit was 10(0) to 10(-1) TCID50 in ethidium bromide stained gels and could be enhanced further to 10(-1) to 10(-3) TCID50 by hybridization after southern transfer. In addition, detection of IBDV infection in 12 out of 14 bursal specimens examined by this technique was shown to be entirely consistent with the clinical history and an alternative diagnostic method. The speed, sensitivity, and specificity of this method is relevant for the diagnosis of infection with IBDV.


Polymerase chain reaction for differentiation between pathogenic and non-pathogenic serotype 1 Marek's disease viruses (MDV) and vaccine viruses of MDV-serotypes 2 and 3. A polymerase chain reaction (PCR) test based on primers flanking the 132 bp tandem repeat in pathogenic MDV-1 DNA was developed. These primers amplify a dimer or a trimer 132 bp repeat in pathogenic MDV-1 DNA from blood and organs of commercial chickens with Marek's disease (MD) symptoms. Using the same primers in a radioactive PCR test, it was possible to distinguish between vvMDV-1 and the non-pathogenic MDV-1 CVI-988 vaccine in which the 132 bp repeats in the DNA were increased up to 9 repeats. The MDV-1 specific primers did not amplify MDV-2 (SB1) and MDV-3 (HVT) DNA. Primers prepared according to the nucleotide sequence of MDV-1 antigen A gene amplified MDV-1 DNA only. Specific primers prepared according to the nucleotide sequence of MDV-3 (HVT) antigen A gene amplified MDV-3 DNA but not MDV-1 nor MDV-2 DNA. The results of the present study show that the PCR tests can be used for the early identification of vvMDV-1 DNA in pathological samples from diseased commercial chickens and to distinguish between the vvMDV-1 and the three types of virus vaccines used to immunize chickens. The tests are accurate and can be performed in the presence of vaccine virus DNA in the sample.


In vitro reactivation of latent herpes simplex virus by the demethylating compound hexamethylene-bis-acetamide. We have characterized previously a model of herpes simplex virus (HSV) infection of rat dorsal root ganglia (DRG) following cutaneous infection. During acute infection HSV can be isolated from co-cultivated rat ganglia in (mean +/- S.E.M.) 4.8 +/- 0.33 days (d) and from latently infected ganglia in 7.8 +/- 0.53 (d) (P < or = 0.0001). We treated co-cultivated rat ganglia from acute and latent infected rats with the demethylating compound hexamethylene-bis-acetamide (HEX) to see what effect, if any, it would have on HSV infection. HEX-treated ganglia from rats with acute infection did not differ significantly from controls in the proportion of rats, skin or ganglia positive for HSV. The mean time to detect virus was not different between treated (3.6 +/- 0.38 d) and control (3.1 +/- 0.46 d) (P > 0.05) groups. In latent infected rats there was no difference between treated and controlled groups in the proportions of rats, skin and ganglia positive for HSV. There was a significant difference in the mean time to CPE between the HEX and control groups respectively (4.5 +/- 0.72 d vs 8.92 +/- 1.42 d, P < 0.01). We conclude that HEX converted latent HSV infection to a productive one.


Glucose metabolism and hypoglycaemia in SIDS. Once a child is born its survival depends on the maturation of the blood glucose homeostatic control mechanisms. When this fails or where there is an inborn error of metabolism the infant is susceptible to potentially fatal hypoglycaemic episodes. A variety of environmental stresses, either singly or in combination, such as inappropriate or low caloric intake, acute infections of childhood, endotoxaemia, fever, xenobiotic exposure, oxidative stress or anaphylaxis, can greatly exacerbate the deficiency of the normal homeostatic compensatory mechanism and result in the onset of hypoglycaemia. Various inborn errors have been found in infants who died of SIDS. Our approach to this problem has been to use the six microsomal glucose-6-phosphatase proteins as a model system to study defects in carbohydrate metabolism in cases of SIDS. Initial studies determined the ontogeny of the glucose-6-phosphatase proteins and showed that intact microsomes isolated from unfrozen liver samples can be used to study glucose-6-phosphatase in cases of SIDS that were presumably due to the low concentrations of liver lipid peroxidation. More recently we have used a combination of techniques to demonstrate the abnormalities of glucose-6-phosphatase in cases of SIDS. Classic gross pathology and histology have now clearly defined the various subgroups of sudden and unexpected deaths of infancy. This now enables us to develop new molecular approaches to predict and prevent hypoglycaemia in infants who are at risk of SIDS.


Diazepam effects on the P3 event-related potential. Cerebral event-related potentials arise from synchronous neural activity associated with cognitive processing. The P3 is a late positive component that is related to task complexity and is directly proportional to stimulus evaluation time. P3 latency and amplitude were examined after oral administration of diazepam, a commonly prescribed medication with known cognitive side effects. Latency of the P3 was significantly increased after a single dose of both 5 and 10 mg of diazepam but not after placebo. P3 amplitude decreased slightly, but not significantly after 10 mg of diazepam. Changes in P3 occurred in the absence of significant changes in the preceding evoked waves (N1, P2). These findings should be considered when the P3 is obtained for clinical or research purposes from medicated patients.


High-fiber diet and serum tricyclic antidepressant levels. The role of dietary fiber as a cause of antidepressant malabsorption has received little attention. Three patients are described who had previously been successfully treated with tricyclic antidepressants and subsequently became refractory to treatment after commencing a high-fiber diet. Serum antidepressant levels were decreased while the patients ingested the high-fiber diets and rose when the fiber content of the diet was reduced. Clinical improvement of their depression followed the concomitant rise in serum tricyclic antidepressant levels.


Sodium bicarbonate and yeast culture effects on ruminal fermentation, growth, and intake in dairy calves. Sodium bicarbonate and yeast culture effects on ruminal fermentation, intake, and growth were evaluated in young calves. In trail 1, nine ruminally cannulated Holstein calves averaging 12 wk of age were fed control starter (17% CP) or starters containing 3% sodium bicarbonate or .2% yeast (Saccharomyces cerevisiae) culture in a 3 x 3 Latin square. Calves were fed for ad libitum consumption for 10 d and then at 85% of ad libitum intake to d 14. Ruminal fluid taken at 0 h postfeeding tended to have higher pH and a greater proportion of acetate when calves were fed sodium bicarbonate, but other ruminal and blood parameters did not differ among treatments. By 4 h after feeding, ruminal VFA had increased to 120.7 mM, molar proportions of individual acids were altered, and blood ketones and VFA increased in treated calves. In trial 2, 42 Jersey calves were fed experimental starters for ad libitum consumption during a 12-wk study. Calves began the study at 3 to 5 d of age. There were no significant effects of yeast culture or sodium bicarbonate on DMI or intake of starter, rates of gain, or feed efficiency. Plasma urea N was reduced when sodium bicarbonate was fed. Both sodium bicarbonate and yeast culture affected blood and ruminal metabolites when calves were limit-fed but did not influence intake or daily gain when calves were fed for ad libitum consumption.


New heteromyeloma cell lines for the production of human monoclonal antibodies. The aim of this study was to establish hybridomas capable of long-term production of human monoclonal antibodies (mAbs). Heterohybridization was performed between the mouse myeloma cell line P3X63Ag8.653 and activated human peripheral blood lymphocytes (PBL). In order to achieve better retention of human chromosomes, as well as to improve the stability of the heterohybrids, one HAT-sensitive immunoglobulin (Ig)-non-secreting human x mouse (h x m) heteromyeloma was fused for a second time with activated human PBL. In this way, a panel of HAT-sensitive Ig-non-secreting h x h x m heteromyelomas was obtained and tested for its ability to generate stable human Ig-secreting heterohybrids with activated human PBL. Six lines were selected on the basis of their enhanced characteristics of fusion efficiency and genetic stability. When fused with in vitro immunized human PBL, they generated several h x h x h x m hybridomas stably secreting high yields (10-23 micrograms/ml/24 h) of human mAbs reactive with recombinant HBV core antigen (rHBcAg). Moreover, a continuous production of human Ig was observed when two h x h x m heteromyelomas, previously made ouabaine-resistant, were hybridized with EBV-transformed lymphoblastoid cell lines. These h x h x m heteromyelomas are ideal fusion partners for the production of human mAbs.


Preparative separation of foreign antigens for highly efficient presentation to T cells in vitro. A method is described for the separation and purification of proteins from complex mixtures of foreign antigens in a form suitable for stimulating T cells in vitro. The technique involves electrophoretic separation of proteins followed by elution, concentration and adsorption of the polypeptide subunits to latex microspheres. Alternatively, where a specific antibody is available, proteins may be affinity-purified from a heterogeneous mixture of antigens, using antibody-coated latex microspheres. Nanogram quantities of protein coupled to latex were shown to be highly efficient stimulators of antigen-specific T cells as tested by in vitro proliferation and cytokine release assays. The utility of this technique was demonstrated using poliovirus capsid proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and coupled to latex microspheres for specificity analysis of T cell clones. Antigen reactivity of the T cell clones was confirmed using recombinant baculoviruses expressing individual poliovirus proteins. Furthermore, recombinant proteins coupled to latex microspheres were used for efficient stimulation and in vitro propagation of T cell clones specific for the simian immunodeficiency virus (SIV) envelope (env) protein. Although the technique is illustrated in this report using viral antigens, it has also proved to be an efficient method for the separation of bacterial antigens in studies of polyclonal T cell responses to Bordetella pertussis antigens.


Detection of hypodense eosinophils by Percoll multilayer density gradient centrifugation in subjects with normal or slightly elevated eosinophilia. Poor reproducibility and eosinophils of density < 1.077 g/ml. In patients with marked hypereosinophilia 'hypodense' and 'normodense' eosinophils have been found after density gradient centrifugation. Subsequently this terminology has also been used in studies of patients with milder eosinophilia. However, in these cases the differentiation between normo- and hypodense eosinophils was less clear. This might be due to the high imprecision of the test of density gradient centrifugation, as demonstrated in the first part of this study: the mean within-assay variance of the number of eosinophils in the different density layers was 35%. It was calculated that the test must be performed eight times to obtain an estimate of the true mean for the individual patient. In the second part of the study, the absolute number of 'hypodense eosinophils' in groups of patients with asthma (adults and children) and rheumatoid arthritis (adults) were compared to normal controls. Although a difference in the absolute number of hypodense eosinophils between groups of patients and controls could be demonstrated, the high imprecision of the test of density gradient centrifugation suggested that the technique used was not useful in an individual with normal or slightly elevated eosinophils in the peripheral blood.


Regulation of adrenal steroidogenesis by adrenaline: expression of cytochrome P450 genes. The effect of adrenaline on the secretion of cortisol and cyclic AMP (cAMP) and on the accumulation of four different mRNAs encoding cholesterol side-chain cleavage cytochrome P450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450(17 alpha)), 21-hydroxylase cytochrome P450 (P450c21) and 11 beta-hydroxylase cytochrome P450 (P450(11 beta)) was studied in bovine adrenocortical cells in primary culture and compared with the effects of ACTH. Treatment of cultured cells with adrenaline (1-100 mumol/l) showed a biphasic response in cortisol release over 1-24 h. Concentration of cAMP in the culture media increased from a basal level of < 0.06 pmol/dish to a maximal level of 40.14 +/- 8.9 pmol/dish with a half-maximal release of 20.07 pmol cAMP/dish in the medium reached 1.2 h after treatment with 10 mumol adrenaline/l. This stimulation resulted in an uniform increase in the levels of all four P450 mRNAs as revealed by Northern blot analysis. Increasing doses of adrenaline produced a maximal mRNA accumulation at a concentration of 10 mumol adrenaline/l. Incubation of the cells with 10 mumol adrenaline/l for 1-24 h produced a biphasic time-course with a half-maximal stimulation after about 5-6 h. Maximal stimulation with ACTH (100 nmol/l) caused different accumulations of the four mRNAs: P450sec mRNA increased twice as much and P450(17 alpha) mRNA six times as much as the accumulation of P450c21 mRNA and P450(11 beta) mRNA, which was about ten-fold over basal values. Propranolol totally blocked the stimulatory effect of adrenaline but not the effect of ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)


A role for di-acetyl alpha-melanocyte-stimulating hormone in the control of cortisol release in the teleost Oreochromis mossambicus. In stressed tilapia, Oreochromis mossambicus, total alpha-melanocyte-stimulating hormone (alpha-MSH) levels and di-acetyl alpha-MSH/mono-acetyl alpha-MSH (di:mono) ratios are elevated. We therefore investigated the role of alpha-MSH in the regulation of the pituitary-interrenal axis. The corticotrophic activities of des-acetyl alpha-MSH, mono-acetyl alpha-MSH and di-acetyl alpha-MSH were compared. These forms of alpha-MSH were isolated from neurointermediate lobes and tested in a superfusion experiment with homologous interrenal tissue. The corticotrophic activity of di-acetyl alpha-MSH was the highest, followed by that of des-acetyl alpha-MSH and mono-acetyl alpha-MSH. Apparently, acetylation of alpha-MSH is of functional significance for corticotrophic action. Di-acetyl alpha-MSH proved to be about 100 times less potent than ACTH(1-39): the half-maximal stimulating concentrations for ACTH and di-acetyl alpha-MSH were 0.89 nmol/l and 110 nmol/l respectively. Surprisingly, a superfusate from neurointermediate lobes proved to be only about three times less active than a superfusate from the pituitary pars distalis, in which the corticotrophic activity is attributable to its ACTH content. When selectively stripped of all forms of alpha-MSH by passage through a Sepharose column coated with an antiserum against alpha-MSH, the neuro-intermediate lobe superfusate was devoid of corticotrophic activity. Thus alpha-MSH appears to be the corticotrophic factor in the superfusate of the neurointermediate lobe. After the same treatment, the corticotrophic activity of the pars distalis superfusate was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)


Intracellular pH and growth hormone-releasing factor-stimulated adenosine 3'5'-monophosphate, intracellular calcium and growth hormone release from rat anterior pituitary cells. In this study, we examined the effect of changes in intracellular pH (pHi) on basal and GH-releasing factor (GRF)-stimulated cyclic AMP (cAMP), intracellular Ca2+ and GH release using a static monolayer culture prepared from dispersed rat anterior pituitary cells. To modulate pHi, two approaches were used: variation of extracellular pH (pHo) and addition of sodium propionate and ammonium chloride which alter pHi directly. Direct pHi measurement with 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein showed that for pHo values between 6.9 and 7.6, a change in pHo of 0.1 units resulted in a change in pHi of 0.045 units. Sodium propionate (30 mmol/l) reduced pHi by 0.06 units whereas ammonium chloride (30 mmol/l) increased pHi by 0.1 units. Increasing pHo from 6.6 to 7.8 enhanced the maximal GRF-stimulated cAMP and GH responses by 80% and 300% respectively, indicating that the GRF-stimulated cAMP and GH release were both pH-dependent. Acute elevation of pHo from 6.6 to 7.8 also increased basal GH release by sixfold. Reduction of pHi by sodium propionate, however, had no significant effect on GRF-stimulated cAMP levels while the corresponding GRF-stimulated GH release was reduced by up to 40%. In comparison, elevation of pHi by ammonium chloride enhanced the GRF-stimulated cAMP release by up to 75% and the corresponding increase in GH was less than 20%. When the relationship between pHi and intracellular Ca2+ was determined with the fluorescent Ca2+ indicator, Fura-2, it was found that increasing pHo and treatment with ammonium chloride increased intracellular Ca2+, while sodium propionate and reducing pHi had no effect on intracellular Ca2+. These results indicate that activation of adenylate cyclase and mobilization of intracellular Ca2+, two intracellular signalling pathways of importance to GH secretion, are both sensitive to changes in pHi.


Inhibition of bovine adrenocortical steroidogenesis by benzodiazepines: a direct effect on microsomal hydroxylation or an inhibition of calcium uptake? We have previously reported that benzodiazepines inhibit microsomal steroid hydroxylases. We have now studied their effects at much lower drug concentrations and have also addressed the suggestion that benzodiazepines alter cellular calcium metabolism. We investigated the in-vitro effects of midazolam on microsomal steroid hydroxylation by measuring basal and ACTH-stimulated cortisol and 17 alpha-hydroxyprogesterone (17-OHP) synthesis. Threshold inhibition of basal cortisol production was achieved by 3.4 mumol midazolam/l while ACTH-stimulated production required 13.6 mumol/l. This was accompanied by a biphasic response of 17-OHP production, rising to a maximum at 13.6 mumol midazolam/l for basal and 6.8 mumol midazolam/l for ACTH-stimulated synthesis suggesting a preferential inhibitory effect on 21-hydroxylase activity at < 6.8 mumol/l and additional effects on 17 alpha-hydroxylation at higher drug concentrations. This explains the inhibition of ACTH-stimulated cortisol synthesis by midazolam (50% inhibitor dose (IC50) 22 mumol/l). Using 21-deoxycortisol as substrate, we have demonstrated that midazolam is a competitive inhibitor of 21-hydroxylase (inhibitory constant (KI) 35 mumol/l). Both midazolam and diazepam inhibited K(+)-stimulated aldosterone synthesis, with IC50 values of 1.2 mumol/l and 0.8 mumol/l respectively, which are far lower than those observed for ACTH-stimulated cortisol synthesis. With 11 beta-hydroxyprogesterone as substrate, the KI for the inhibition of aldosterone synthesis by midazolam was 54 mumol/l. Potassium stimulates aldosterone biosynthesis at least partly by changing intracellular free calcium levels. To investigate possible antagonistic effects of benzodiazepines on calcium metabolism, we measured 45Ca uptake in the presence of midazolam.(ABSTRACT TRUNCATED AT 250 WORDS)


Intracellular pH regulation in human thyrocytes: evidence of both Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange. The present study was undertaken to investigate the regulation of intracellular pH in human thyroid epithelial cells and to characterize the kinetics of the acid-extruding processes operating in the absence and presence of HCO3-, Na+ and Cl-. A dynamic technique of dual excitation microfluorimetry and the pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein was employed. The intracellular pH was 7.01 +/- 0.27 (n = 29) and 6.94 +/- 0.25 (n = 54) in the absence and presence of HCO3- respectively. Both in the absence and presence of HCO3-, the recovery from intracellular acid loads was not only due to an Na+/H+ exchange, but also to an Na(+)-dependent HCO3-/Cl- exchange. In alkaline conditions caused by NH4Cl pulsing, an HCO3-/Cl- exchange was also found. The cells in HCO3- responded with a wide range of maximal hydrogen efflux rates in experiments where cells were either pretreated with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid or incubated with amiloride. The heterogeneity might be due to subpopulations of thyrocytes in different metabolic states or at different points in the cell cycle. It is concluded that recovery from intracellular acidification in human thyroid cells is due to both Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange even in nominally HCO3(-)-free conditions, and that recovery from intracellular alkalinization is due to a Cl-/HCO3- exchange which needs to be characterized further.


George N. Papanicolaou, M.D. Father of modern cytology. A 30-year commemorative. Thirty years have passed since the death of the renowned Dr. George N. Papanicolaou, a man of conviction and discoverer of the Pap smear. Despite difficulties and opposition he remained persistent and developed a method for early cancer detection. Subsequently, use of the Pap smear has resulted in a 70% decrease in cervical and uterine cancer deaths in the last 40 years. It is considered a reliable technique and is still expanding by being applied to other organs and systems and in the use of fine needle aspiration.


The nss mutation or lanthanum inhibits light-induced Ca2+ influx into fly photoreceptors. Ion-selective calcium microelectrodes were inserted into the compound eyes of the wild-type sheep blowfly Lucilia or into the retina of the no steady state (nss) mutant of Lucilia. These electrodes monitored light-induced changes in the extracellular concentration of calcium (delta[Ca2+]o) together with the extracellularly recorded receptor potential. Prolonged dim lights induced a steady reduction in [Ca2+]o during light in the retina of normal Lucilia, while relatively little change in [Ca2+]o was observed in the retina of the nss mutant. Prolonged intense light induced a multiphasic change in [Ca2+]o: the [Ca2+]o signal became transient, reaching a minimum within 6 s after light onset, and then rose to a nearly steady-state phase below the dark concentration. When lights were turned off, a rapid increase in [Ca2+]o was observed, reaching a peak above the dark level and then declining again to the dark level within 1 min. In analogy to similar studies conduced in the honeybee drone, we suggest that the reduction in [Ca2+]o reflects light-induced Ca2+ influx into the photoreceptors, while the subsequent increase in [Ca2+]o reflects the activation of the Na-Ca exchange which extrudes Ca2+ from the cells. In the nss mutant in response to intense prolonged light, the receptor potential declines to baseline during light while the Ca2+ signal is almost abolished, revealing only a short transient reduction in [Ca2+]o. Application of lanthanum (La3+), but not nickel (Ni2+), into the retinal extracellular space of normal Lucilia mimicked the effect of the nss mutation on the receptor potential, while complete elimination of the Ca2+ signal in a reversible manner was observed. The results suggest that La3+ and the nss mutation inhibit light-induced Ca2+ influex into the photoreceptor in a manner similar to the action of the trp mutation in Drosophila, which has been shown to block specifically a light-activated Ca2+ channel necessary to maintain light excitation.