Regulation of plasminogen activators and type-1 plasminogen activator inhibitor by cyclic AMP and phorbol ester in rat astrocytes. Two plasminogen activators (PAs): tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), as well as the type-1 plasminogen activator inhibitor (PAI-1) are synthesized and secreted by rat astrocytes. Preliminary studies suggest that PA activity plays a role in astrocyte development and differentiation. We have examined the regulation of the PA system by the cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in purified rat astrocyte cultures. PKA activity was increased by exposing cultured astrocytes to forskolin or dibutyryl cyclic AMP, whereas PKC activity was stimulated with phorbol-12-myristate 13-acetate (PMA). Activation of both second-messenger pathways produced a time- and dose-dependent increase in the total PA activity. However, based on SDS-PAGE/zymography we found that forskolin increased t-PA activity and reduced u-PA activity, whereas PMA treatment caused a significant increase in u-PA activity without altering t-PA activity. Reverse zymography analysis revealed that astrocyte PAI-1 activity is decreased by forskolin and increased by PMA. Together, these results demonstrate that the components of the PA system in rat astrocytes are independently and reciprocally regulated by PKA and PKC. Our findings raise the possibility that the plasminogen activator system could be involved in some of the actions of growth factors and/or neuromodulators that modulate PKC or PKA in astrocytes.


Protein kinase C isoforms in human glioblastoma cells. Protein kinase C (PKC), an enzyme involved in signal transduction, responds to diacyl glycerol and also to phorbol ester, a ligand analogous to diacyl glycerol. We have studied the expression of the major isoforms (alpha, beta I, beta II, and gamma) in eight human glioblastoma cell lines. In all eight lines, PKC-alpha mRNA and protein were expressed. In none of the eight did a probe for PKC-beta I and -beta II mRNA give positive results nor were Western blots for PKC-beta II positive. The half-life for PKC alpha mRNA was approximately 16 h and levels of the mRNA were increased slightly following addition of phorbol myristate acetate (PMA) or transforming growth factor-beta (TGF beta). PKC-gamma was present in most of the glioblastomas. In cell line A172, 82% of the PKC-alpha was present in the cytosol with the remainder evenly divided between plasma membrane and nucleus. Thirty minutes after addition of PMA, 33% of the total original protein was in the plasma membrane and 48% in the nuclear fraction. By 21 h, no PKC-alpha was recovered from any fraction. PKC-gamma was also down-regulated in the presence of PMA, but there was no evidence for translocation to the plasma membrane or nuclear fraction. In a more detailed study, translocation of PKC-alpha in the presence of PMA was complete by 10 min, and a major decrease in the PKC translocated to the plasma-membrane fraction occurred some time between 2 and 4 h after PMA addition, while a major decrease in the translocated nuclear fraction occurred some time after 6 h. cAMP alone had no effect on the PKC alpha protein level or distribution, nor did it alter the translocation and down-regulation due to PMA exposure. In these studies the level of PKC-alpha mRNA in tumors was similar to that in normal glial cells.


Zinc toxicity and induction of the 72 kD heat shock protein in primary astrocyte culture. Zinc is a potent inducer of the 72 kD heat shock protein (HSP72). In brain, pathological conditions such as ischemia and seizures increase extracellular zinc. The present study examines the effect of zinc on HSP72 expression in rat primary cortical astrocyte culture. Astrocytes were grown to confluence and exposed to zinc chloride in CO2-equilibrated Earle's buffered salt solution. Expression of HSP72 was examined using immunocytochemistry. HSP72 was induced with zinc concentrations of 5 to 100 microM after 4 h exposures, or 200 to 300 microM after 15 min exposures. At the lower concentrations expression occurred in small clusters of contiguous cells. At concentrations high enough to cause cell death, HSP72-positive astrocytes formed a continuous margin around patches of dead cells. These patterns of HSP72 expression are similar to the patterns seen after cerebral ischemia in vivo. Exposure to zinc at 100 microM for 4 h or 400 microM for 15 min caused greater than 90% cell death. Increases in extracellular zinc may contribute to HSP72 induction and astrocyte death under ischemia and other pathological conditions in brain.


The metabolic response to two very low energy diets (VLED) of differing amino acid composition during weight reduction. Nitrogen (N) sparing and even equilibrium have been achieved in obese subjects with all-protein weight-reducing very low energy diets (VLED) apparently independently of the content of essential amino acids. This study assessed whether the metabolic response observed at week 3 of an all-protein VLED with 46% of amino acids (aa) as essential was modified during week 4, when consuming a protein source that provided 16% of amino acids as essential. Six healthy obese subjects (BMI: 35.3 +/- 1.3 kg/m2, weight 90 +/- 9 kg) were given a 1.72 MJ (400 kcal) all protein (93 g) VLED and a multi-vitamin-mineral supplement daily for four weeks. During weeks 1 to 3, the protein was casein-soy (46% essential aa) and during week 4, tryptophan- and methionine-supplemented collagen hydrolysate (16% essential aa). At week 3, decreases in plasma glucose, insulin, cholesterol, blood pH and bicarbonate, and increases in plasma free fatty acids, serum urea, uric acid and blood and urine ketones occurred compared to baseline. These adaptations were unchanged at week 4. N balance returned toward equilibrium by day 23 remaining at values close to 0 despite the change in diet composition. Mean negative N balance did not differ between weeks 3 and 4 (-1.1 +/- 0.5 g vs. -0.6 +/- 0.5 g/day) and neither did mean urinary ammonium N excretion (0.71 +/- 0.08 vs. 0.73 +/- 0.07 g/day). Urinary urea N excretion tended to increase with the collagen-based diet reflecting the greater proportion of N in this protein source (18 vs. 15%).(ABSTRACT TRUNCATED AT 250 WORDS)


The Christmas factor in obesity therapy. The effect of Christmas time on body weight development was measured in 46 obese patients in maintenance therapy at the Obesity Unit of the Karolinska Hospital and in 76 hospital staff controls of similar sex and age distribution. In controls a statistically significant weight increase of 0.4 +/- 0.8 (s.d.) kg (P < 0.001) was found, whereas the mean increase in the obese patients of 0.6 +/- 2.4 kg was not statistically significant. Little variation in weight development over Christmas was found in controls, whereas the weight change in the obese group ranged from +6.1 to -8.8 kg over the 2-3 weeks. Seasonal variation in body weight has been attributed to circadian rhythms. Cultural food habits may also play a role.


Obesity and fat distribution in women from Saudi Arabia. Anthropometric measurements were carried out on 100 women selected randomly from the population of two cities in the eastern province of Saudi Arabia. The data obtained in this study were then compared to those reported earlier for European countries. This comparison indicates that the Saudi women are on average more obese than their European counterparts, with a preponderance of abdominal obesity. This is discussed in terms of socio-economic changes which this area has undergone in the last 50 years.


Human obesity: a defect in lipid oxidation or in thermogenesis? Numerous animal studies have shown that the development of obesity can depend on a decreased sympathetic nervous system activity but it has not been possible to convincingly demonstrate in humans that an excessive accumulation of adipose tissue might also be attributable to this factor. However, results from recent studies suggest that the contribution of the sympathetic nervous system to the development of human obesity may be greater than that generally believed by the scientific community. Indeed, experimental data show that the decrease in lipid oxidation induced by beta adrenergic blockade under free living conditions is substantially higher than the decrease in energy expenditure. Moreover, there are individual variations in the relative capacity to use lipid as an energy substrate under standardized conditions of diet composition. If the contribution of lipid to daily energy expenditure is reduced, there are two options to reach energy balance if physical activity habits are not modified. The first possibility is reduction in the relative lipid content of the diet to match the lipid content of the fuel mix oxidized. The second option is a gain in body weight and fat until the associated increase in the lipid content of the substrate mix is sufficient to re-equilibrate substrate and energy balance. Under high fat diet conditions, this gain in body weight and fat can be large enough to lead to an obese state. As described in this report, an integration of these observations suggests that further research pertaining to the role of sympathetic nervous system activity in human obesity should be focused as much on lipid oxidation as on thermogenesis.


Sympathoadrenal activity in human obesity: heterogeneity of findings since 1980. Alterations in sympathetic nervous system (SNS) activity are widely believed to contribute to the pathophysiology of the obese state. Disagreement, however, exists as to whether the predominant sympathetic abnormality is a decrease in neuronal activity (leading to diminished sympathetically-mediated energy expenditure and weight gain) or an increase (leading to hypertension). Findings summarized from over 40 separate studies support both hypotheses as well as the alternative thesis that SNS activity does not differ in obese humans compared to lean controls. Another abnormality being noted with increasing frequency in human obesity is reduced adrenaline (Ad) levels in plasma, both at rest or in response to a stimulus such as physical activity. Whether diminished adrenal medullary function is a cause or consequence of the obese state and whether the adrenal medulla plays any role in the regulation of energy metabolism on a daily basis are not known at the present time. Thus, while depressed SNS activity may be a sufficient explanation for the development of obesity, it is not a necessary condition. Suppressed adrenal medullary function may also contribute to this disorder.


What are the dietary energy needs of elderly adults? The energy requirements of healthy elderly men were investigated by measuring total energy expenditure (TEE), body composition and resting energy expenditure (REE), using 2H2(18)O techniques and indirect calorimetry during ten days when a weight maintaining diet was consumed. Values for TEE/REE (1.75 +/- 0.05, s.e.) were significantly higher than the recommended dietary allowances (RDAs) for energy in this age group, as were previously reported values for TEE/REE in a group of young men in the same experimental protocol. There was a significant negative association between TEE/REE (a physical activity indicator) and body fat mass. Combining data from the elderly men in this study and the young men studied previously, age and TEE/REE together accounted for 73% of the variation in body fat mass between individuals. These results lend additional weight to the suggestion that the current RDAs for energy may significantly under-estimate usual energy requirements. They also indicate that the low levels of energy expenditure suggested by the RDAs may favour unnecessarily high levels of body fat mass and that increased levels of energy expenditure are desirable.


Onset of obesity and puberty in genetically obese SHHF/Mcc-cp rats. SHHF/Mcc-cp rats, as a model of obesity and diabetes, were followed through breeding and throughout development to determine timing of obesity and sexual development. The obesity or corpulency gene (cp) follows recessive transmission characteristics with no segregation between sexes. Although the frequency of litter sizes was different, the mean litter size of heterozygous mating (8.9 +/- 0.3 pups/litter) was not different from homozygous lean matings (7.9 +/- 0.3 pups/litter). Body weights of the population of female obese rats statistically deviated from lean females at day 35 and obese males deviated from lean males at day 37. Vaginal opening of obese and lean females did not differ in time of occurrence (day 34.6 +/- 0.2 for lean and 33.6 +/- 0.4 for obese). To further evaluate development and examine onset of diabetes, animals were killed at six, eight and ten weeks of age and development of reproductive organs and plasma levels of insulin, glucose, and testosterone or oestradiol determined. Testes development was slightly retarded in the obese male with smaller testes at six weeks of age, however testes size increased at eight and ten weeks of age and was not significantly less than lean males. In contrast, testes function was impaired with smaller seminal vesicles and lower testosterone levels in the obese male rats. Both ovarian and uterine weights were significantly less in obese females. However, oestradiol levels were not significantly different at any of the time points examined. Development of elevated insulin levels were first noted in the obese female at six weeks of age, however marked hyperinsulinemia developed only in the obese males at ten weeks of age.(ABSTRACT TRUNCATED AT 250 WORDS)


Increased gluconeogenesis in hepatocytes from GTG-obese mice is insensitive to inhibition by insulin. The effect of a supraphysiological concentration of insulin on gluconeogenesis from L-[U14C] lactate was studied in hepatocytes isolated from control mice and mice made obese by a single injection of gold-thioglucose (GTG). At the time of experimentation (10-12 weeks post GTG injection) the obese mice weighted significantly more than controls (41.7 +/- 0.5 vs. 29.6 +/- 0.8 g respectively; P < 0.001), and exhibited fasting hyperinsulinaemia (35.9 +/- 4.6 vs. 21.3 +/- 4.2 microU/ml; P < 0.05) and hyperglycaemia (16.4 +/- 1.2 vs. 9.2 +/- 1.1 mmol/l; P < 0.001). The amount of lactate converted to glucose by hepatocytes isolated from GTG-obese mice was significantly greater than from lean controls (322 +/- 44 vs. 209 +/- 20 nmol/30 min/10(6) cells; P < 0.05). The addition of 10(-6)M insulin to the incubations significantly reduced lactate conversion to glucose by hepatocytes isolated from control mice (209 +/- 20 vs. 123 +/- 22 nmol/30 min/10(6) cells; P < 0.02), but there was no effect of insulin on glucose production from lactate by hepatocytes isolated from GTG-obese mice (322 +/- 44 vs. 294 +/- 47 nmol/30 min/10(6) cells). Glycogen production and triacylglycerol glycerol production from L-[U14C] lactate were also significantly increased in hepatocytes from GTG-obese mice compared with controls. There was no effect of 10(-6)M insulin on glycogen or triacylglycerol glycerol production from lactate by hepatocytes from GTG-obese mice but the addition of 10(-6)M insulin to the incubations of control hepatocytes significantly reduced the amount of lactate converted to glycogen and triacylglycerol glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)


The effects of testosterone treatment on body composition and metabolism in middle-aged obese men. Twenty-three middle-aged abdominally obese men were treated for eight months with testosterone or with placebo. Testosterone treatment was followed by a decrease of visceral fat mass, measured by computerized tomography, without a change in body mass, subcutaneous fat mass or lean body mass. Insulin resistance, measured by the euglycemic/hyperinsulinemic glucose clamp method, improved and blood glucose, diastolic blood pressure and serum cholesterol decreased with testosterone treatment. A small increase in prostate volume was noted, but serum prostate specific antigen concentrations were unchanged and no adverse functional side-effects were found. Insulin sensitivity improved more in men with relatively low testosterone values at the outset. The mechanisms involved in these changes might act either via effects on visceral fat accumulation, followed by metabolic improvements, and/or via direct effects on muscle insulin sensitivity, as suggested by results of other recent studies. It is concluded that testosterone treatment of middle-aged abdominally obese men gives beneficial effects on well-being and the cardiovascular and diabetes risk profile, results similar to those observed after hormonal replacement therapy in postmenopausal women.


Body weight and psychological distress in NHANES I. This study examined the relationship between body mass index, smoking status, and depressive symptoms reported on the Center for Epidemiologic Studies Depression (CES-D) scale in the First National Health and Nutrition Examination Survey (NHANES I). Among women, but not men, greater body mass index was weakly associated with elevated reports of depressive symptoms. This relationship remained significant after controlling for age, years of education, and smoking status. A history of smoking (current and ex-smoking) and body weight in the highest weight quintile (> or = 28.96 kg/m2) was marginally related to increased risk of depression (CES-D score > or = 16) among women only. These results indicate that relative body weight is weakly related to psychological distress among women but not men, and that cigarette smoking does not significantly modify this relationship.


Bradykinin and angiotensin II analogs containing a conformationally constrained proline analog. Three analogs of bradykinin and one of angiotensin II have been prepared in which the naturally occurring proline residues have been replaced by the bicyclic amino acid, 2,4-methanoproline (2,4-MePro). The relative binding affinities for these analogs were determined to be significantly reduced in the cases of the three bradykinin analogs; [2,4-MePro3]-BK retains 1.3%, [2,4-MePro7]-BK retains 0.3% and [2,4-MePro2]-BK retains 0.021% of the binding affinity of bradykinin. Results from other modification at positions three and seven indicate preference for the trans-amide bond preceding these residues implying that other factors, either steric or conformational, are responsible for the decreased affinity for the receptor seen with 2,4-MePro substitution. The retention of significant binding affinity (26%) in the case of [Ile5,2,4-MePro7]-angiotensin II gives direct evidence that the trans-conformation of the proline amide bond is the one recognized by the AII receptor. Only significant retention of activity can be interpreted unambiguously with the use of this proline analog because of its known conformational differences from Pro as well as its increased steric requirements at the receptor.


Constrained phenylalanine analogues. Preferred conformation of the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue. Three Tic-containing (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) model peptides were synthesized to assess the tendency of this constrained Phe analogue to fold into a beta-bend and a helical structure, and to adopt a preferred side-chain disposition. The results of the solution conformational analysis, performed by using Fourier transform infrared absorption and 1H nuclear magnetic resonance, indicate that in chloroform the -Aib-D-Tic-Aib-, -(Aib)2-D-Tic-(Aib)2-, and -L-Pro-D-Tic- sequences fold into intramolecularly H-bonded forms to a great extent. An X-ray diffraction analysis on p-BrBz-(Aib)2-DL-Tic-(Aib)2-OMe monohydrate and p-BrBz-L-Pro-D-Tic-NHMe allows us to conclude that, while the pentapeptide methylester forms an incipient (distorted) 3(10)-helix, the dipeptide methylamide adopts a type-II beta-bend conformation. In both cases, the D-Tic side-chain conformation is D, gauche(-). The implications for the use of the Tic residue in designing conformationally restricted analogues of bioactive peptides are briefly discussed.


Efficient solid phase peptide synthesis. Use of methanesulfonic acid alpha-amino deprotecting procedure and new coupling reagent, 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexafluorophosphate (BOI). An efficient method for solid phase peptide synthesis was developed, which consists of N alpha-selective deprotection by dilute methanesulfonic acid, in situ neutralization and rapid coupling reaction using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or 2-(benzotriazol-1-yl)oxy-1,3- dimethylimidazolidinium hexafluorophosphate (BOI) reagent. Selective removal of the N alpha-Boc group by dilute methanesulfonic acid was of more advantage than removal by TFA in terms of stability of semipermanent protecting groups and suppression of undesired side reactions. The use of in situ neutralization and rapid coupling method reduced intramolecular aminolytic cyclization by shortening exposure of the deprotected nucleophilic amino group. A successful synthesis of porcine brain natriuretic peptide (pBNP) has been achieved using this efficient solid phase peptide synthesis scheme.


Management and survival of patients affected with obstructive colorectal cancer. Between 1978 and 1984 a consecutive series of 571 patients with colorectal cancer were admitted to the First Department of Surgery of the University of Rome. Patients were divided into a group of 82 patients affected with obstructive cancer and a control group of 489 patients with non-obstructive tumors. In the obstructed group there was a significantly higher incidence of lesions localized in the left colon. Depending on the advancement of lesions a significantly higher incidence of Dukes D tumor, nodal involvement, hepatic metastases and peritoneal dissemination and a significantly lower incidence of Dukes A tumors, were found in the obstructed patients. No significant differences were found in the two groups according to age distribution, duration of symptoms and degree of differentiation of neoplasms. The mortality and morbidity rate were 9.7% and 12.2% respectively in the obstructed patients, and 3.5% and 8.3% respectively in the non-obstructed patients. The rate of complications was greater in the two groups when serum albumin values were under 3 g/l, being 40% vs. 3.3 and 20% vs. 5.2% in obstructed and nonobstructed groups respectively. When Hb levels were under 10 g/l the incidence of complications was 16.7% and 14.4% for the two groups, while when it was higher than 10 mg% the morbidity rate was 8.7% and 6.3% in obstructed and non-obstructed patients respectively. The execution of surgical treatment within 24 hours was related to a morbidity and mortality rate of 50% and 22.2% in obstructed patients, and 40% and 20% in the non-obstructed group.(ABSTRACT TRUNCATED AT 250 WORDS)


Cleavage and deprotection of peptides on MBHA-resin with hydrogen bromide. Dilute hydrogen bromide in trifluoroacetic acid containing pentamethylbenzene and thioanisole was used in the cleavage and deprotection of peptides on MBHA-resin. Particular attention was paid to potential applicability of the method to kilogram scale synthesis of thymosin alpha 1. In the HPLC purification of the peptides, acetonitrile was replaced by relatively nontoxic isopropanol. The change should be economically and environmentally very attractive.


Sixty cases of primary hepatocellular carcinoma in one year. A preliminary appraisal. We studied all the 70 cases of liver cancer referred to us in 1988. Sixty (85.71%) were primary hepatocellular carcinoma (HCC), the other 10 (14.29%) were metastatic liver cancer. Of the 60 HCC, 48 were males and 12 females. Peak age incidence was between 20-40 years. Forty-five (75%) had tumours in both lobes. Of the remaining 15, seven had tumours in the right lobe while eight were in the left. More detailed assessment identified eight as Child's C. Seven (11.66%) had laparotomy, and two were inoperable. Three (60%) died shortly after resection, leaving two survivors. One of the survivors lived for 24 months, and the other had recurrent tumour after 26 months. The majority had cirrhosis of the liver, were positive for markers of hepatitis B virus, and showed elevated serum alpha fetoprotein (AFP). Palliative treatment was disappointing, and all were dead within 15 months. Prognosis of HCC in our environment is poor. Considering the advances made in liver surgery in recent years, our mortality and morbidity figures can improve. Notwithstanding, preventive measures should be intensified.


The value of selective mediastinoscopy in predicting resectability of patients with bronchogenic carcinoma. From 1980 to 1990, 1505 patients underwent thoracotomy as definitive treatment for non small cell lung cancer. Computed tomography (CT) of the chest has been used routinely since 1984 for assessment of mediastinal lymph node involvement. A total of 235 cervical mediastinoscopies and 71 anterior parasternal mediastinotomies were performed on the same patient population as preoperative staging when CT scan demonstrated mediastinal lymph nodes larger than 1 cm. Radical resections showed a constant increase in number from 70.1% in the period 1980-84 to 82.7% in the period 1985-90. Exploratory thoracotomies and thoracotomies with residual tumor showed a parallel reduction: 14.5% in 1980-84 to 7.4% in 1985-90 for the former, 15.4% in 1980-84 to 9.8% in 1985-90 for the latter. The percentage of N2 disease to the total number of thoracotomies decreased from 23.6% in 1980-81 to 11.2% in 1989-90. We conclude that a selective use of cervical mediastinoscopy and anterior parasternal mediastinotomy, based upon the results of CT scan, may have contributed to reduce the number of exploratory thoracotomies and thoracotomies with residual tumor.


Management of cancer of the opposite breast following breast preservation. Breast preservation has been shown to be a good alternative to mastectomy in selected patients with breast cancer. The purpose of this study was to evaluate the characteristics of cancer developing in the opposite breast to determine if breast preservation should be attempted in that breast as well. From 1979 to 1988, 172 women underwent tylectomy, axillary dissection and irradiation for carcinoma of the breast. All had follow-up mammogram. Mean age was 55 years. Mean follow-up time was 50 months. Thirteen patients (7.6%) developed cancer in the opposite breast. Three cancers were carcinoma in situ, nine were stage I, and one was stage IIa. Nine of 13 patients had breast preservation therapy, and four had mastectomies. Ten patients are alive with no evidence of disease, two are alive with disease and one died with disease. Breast preservation for bilateral breast cancer is a safe alternative if patients can be followed closely.


Cytomegalovirus infection as a complication of OKT3 therapy in kidney transplant recipients. We compared the incidence of clinical CMV illness in 25 renal transplant recipients treated with OKT3 for steroid resistant cellular rejection with 88 renal transplant patients treated only with conventional immunosuppression (cyclosporin A and steroids). Nine (36%) patients in the OKT3 group developed CMV illness compared to (2.3%) amongst those treated conventionally (p < 0.0005). Patients who received OKT3 were divided into four groups according to the CMV antibody status of the donor and recipient. Six of the 9 episodes of CMV infection occurred in patients not previously exposed to CMV, who received a kidney from a CMV positive donor. Three (12%) of the patients treated with OKT3 died of CMV disease. A further 2 patients died of other causes giving an overall mortality in the OKT3 treated group of 20%. We concluded that when OKT3 therapy is used in association with donor/recipient CMV mismatch it is associated with a high CMV morbidity and mortality.


Evaluation by somatosensory evoked potentials of the neurotoxicity of cisplatin alone or in combination with glutathione. The use of high doses of cisplatin (DDP) in the treatment of different solid tumors is often prevented by the onset of a disabling sensory neuropathy. In an attempt to minimize DDP-induced neurotoxicity different schedules of DDP administration have been tested. Moreover, during the past few years some putative neuroprotective drugs have been reported as reducing DDP neurotoxicity. In this prospective, randomized study we evaluated in a series of 33 patients affected by relapsing ovarian cancer the effect on the sensory pathway of a non-conventional schedule of DDP administration as monochemiotherapy or in combination with one of the neuroprotective drugs (i.e. glutathione). The results of the neurophysiologic examinations performed before and immediately after chemotherapy suggest that these schedules besides being safe and effective in the treatment of the ovarian cancer, have an extremely low peripheral neurotoxicity.


An analysis of peripheral type benzodiazepine receptors on blood mononuclear cells during high dose steroid treatment of multiple sclerosis. We report here a study of peripheral type benzodiazepine receptors (pBZr) in mononuclear cells (MNC) from blood of patients with multiple sclerosis (MS) during periods of stable and active disease and from normal controls. Most active MS patients were retested in a longitudinal study, both during a treatment with high dose steroids and while medication free. Active MS produces a significant decrease of receptor density compared with the controls whereas remission of the disease shows no effect. Four weeks of steroid treatment restore binding density to normal levels, and two weeks of drug withdrawal result in a small, but significant increase in number of the binding sites compared with the control value. We suggest that the number of pBZr in blood MNC might change during the clinical course and steroid therapy of MS.


Cooperative transcription activation between Ad1, a CRE-like element, and other elements in the CYP11B gene promoter. We previously reported the presence of six different cis-acting elements (Ad1 to Ad6) in the promoter region of the bovine CYP11B gene. Although the Ad1 site (TGACGTGA) was similar to a palindromic CRE (TGACGTCA), two other upstream sequences, Ad3 and Ad4, were identified as the cAMP response sequences of the gene. We analyzed the functional relationship between the Ad1 site and the upstream elements. Mutation analyses of the Ad1 site indicated that the 5' half of the site (TGACG) was important for the transcription of the gene in vitro. In Y-1 cells, a plasmid with a mutated Ad1 showed no response to cAMP. The effect of the mutation at the Ad1 site on the cAMP response was almost the same as that of the deletion of Ad3 and Ad4, although the role of each element seemed to be different. These results indicated that both the Ad1 site and the upstream elements, Ad3 and Ad4, were necessary for the full response to cAMP of the CYP11B gene. When the Ad1 site in the promoter region was replaced with a palindromic CRE, elevated transcription activity was detected both in vitro and in vivo. Two kinds of CREBs (43 and 47 kDa) purified from a HeLa cell nuclear extract bound to the Ad1 site. The binding of the palindromic CRE to the nuclear factor(s) was stronger than that of Ad1.


1H-NMR study of Ca(2+)-and Mg(2+)-dependent interaction between troponin C and troponin I inhibitory peptide (96-116). The Ca(2+)-and Mg(2+)-dependence of the interaction between rabbit skeletal muscle troponin C (TnC) and a 21 residue peptide corresponding to 96-116 of troponin I (denoted as CN4) was examined by means of 1H-NMR spectroscopy. The spectral changes of TnC with 4 mol of Ca2+ (Ca4TnC) and TnC with 4 mol of Mg2+ (Mg4TnC) were observed as a function of CN4 concentration. As CN4 was added to Ca4TnC, resonances of the following residues changed in chemical shift: Tyr10, Phe23, Phe72, Ala106, Gly108, Tyr109, Ile110, His125, Gly144, Ile146, Phe102 or Phe151, and Phe148 located in the N- and C-domains of Ca4TnC. Such CN4-induced change was also observed for resonances of Phe19, 26, and 75 in the N-domain of Ca4TnC by means of NOESY and HOHAHA experiments. The presence of CN4 increased the native-to-unfolded transition temperature of the N-domain of Ca4TnC. On the basis of these results, we conclude that CN4 binds to both the C- and N-domains of Ca4TnC ([CN4]:[TnC] = 1:1) and stabilizes the structure of the N-domain. The CN4-binding constant was estimated to be 1.1 x 10(5) M-1. As CN4 was added to Mg4TnC, chemical shift change was observed for resonances of Phe99, Tyr109, and Ile110 in the C-domain, while no change was observed for resonances arising from the N-domain. The presence of CN4 did not change the thermal stability of the N- and C-domains of Mg4TnC. The CN4-binding constant of Mg4TnC was obtained as 0.9 x 10(4) M-1, which is one-tenth of that of Ca4TnC.(ABSTRACT TRUNCATED AT 250 WORDS)


Further studies on the phosphorylation and kinetics of rat liver fructose-1,6-bisphosphatase: importance of the three-dimensional structure of a substrate to protein kinase A. Rat liver fructose-1,6-bisphosphatase was phosphorylated by cAMP-dependent protein kinase to 2.6 mol phosphate/mol subunit but not by Ca2+/phospholipid-dependent and Ca2+/calmodulin-dependent protein kinases. It was demonstrated that phosphorylation of Ser-341 and Ser-356, and to a much lower extent, Ser-338, was dependent on the presence of intact arginine residues. This observation implicates that the intact three-dimensional structure of the substrate is necessary for phosphorylation of Ser-356 since the closest arginine is located at a six amino acid residue distance.


Growth requirements and expression of LDL receptor and HMG-CoA reductase in Hep G2 hepatoblastoma cells cultured in a chemically defined medium. A serum-free chemically defined medium (CDM) has been developed which sustains the growth in culture of the highly differentiated human hepatoma cell line Hep G2. Unlike rodent hepatoma lines, Hep G2 cells in serum-free medium have an absolute requirement for lipoprotein lipids (either low density lipoprotein (LDL) or high density lipoprotein (HDL)) for growth. In the presence of LDL (or HDL) growth was further enhanced by insulin, triiodo-L-thyronine, 17 alpha-ethinylestradiol but not by epidermal growth factor (EGF). On type I collagen gels cells cultured in CDM were contact inhibited and formed monolayers. This contrasted with the pattern of growth of cells cultured in the presence of serum on type I collagen gels and cells cultured on tissue-culture plastic in either CDM or medium containing serum which formed foci of multilayered cells. Expression of the LDL receptor and HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase genes was comparable in Hep G2 cells cultured in CDM and serum-containing medium. Furthermore, the binding and internalisation of 125I-LDL at 37 degrees C was modulated by hormones that have previously been shown to affect LDL receptor levels in liver in vivo or in hepatocytes cultured in serum-containing medium in vitro. The culture system described provides a basis for studying the regulation of hepatocyte-specific functions by soluble factors (either plasma- or cell-derived) and cell-substratum interactions in a human liver cell line.


Role of protein kinase C in the phosphatidylserine-induced inhibition of DNA synthesis in blood mononuclear cells. The mechanism of immunosuppressant activity of phosphatidylserine has been studied in peripheral blood mononuclear cells depleted or not of monocytes. After the addition of phosphatidylserine, mass determinations and uptake of labeled compound demonstrate its transfer into the cells. Phosphatidylserine incorporation causes a 2.5-fold increase of membrane-bound protein kinase C activity. The activation of translocated enzyme is indicated by the inhibition of phosphoinositide hydrolysis, and early feedback effect induced by activated protein kinase C. This action of phosphatidylserine is reproduced by tetradecanoylphorbolacetate and is prevented by the protein kinase C inhibitor, staurosporine. Consistently, phosphatidylserine (8 nmol/10(6) cells) decreases by 46% the production of inositol phosphates in cells responding to phytohemagglutinin. The decrease of phosphoinositide signal pathway as well as the inhibition of mitogen-induced DNA synthesis are produced at the same phosphatidylserine concentration and are equally manifest in total mononuclear cells or in preparations depleted of monocytes. However, only in the presence of monocytes does tetradecanoylphorbolacetate enhance the action of phospholipid, decreasing its IC50 from 13-15 microM to 7 microM. Thus, the data suggest that a reaction driven by protein kinase-C and a factor released by activated monocytes are involved in the phosphatidylserine-induced inhibition of lymphocyte DNA synthesis.


Cell division in Aspergillus.

Cell division in Aspergillus. Amenable to sophisticated genetic and molecular analysis, the simple filamentous fungus Aspergillus nidulans has provided some novel insights into the mechanisms and regulation of cell division. Mutational analysis has identified over fifty genes necessary for nuclear division, nuclear movement and cytokinesis. Molecular and cellular analysis of these mutants has led to the discovery of novel components of the cytoskeleton as well as to clarifying the role of established cytoskeletal proteins. Mutations leading to defects in the kinases (i.e. p34cdc2) and phosphatases (i.e. cdc25 and PP1), which are known to regulate mitosis in other eukaryotes, have been identified in Aspergillus. Additional, as yet novel, mitotic regulatory molecules, encoded by the nimA and bimE genes, have also been discovered in Aspergillus.


Altered glycosylation and cell surface expression of beta 1 integrin receptors during keratinocyte activation. We studied the mechanism by which cell adhesiveness becomes activated when keratinocytes are removed from skin and placed into cell culture. Our results suggest that activation involves altered beta 1 integrin subunit glycosylation accompanied by an increase in cell surface beta 1 integrin receptors. Activated keratinocytes contained two forms of the beta 1 integrin subunit, approximately 93 kDa and approximately 113 kDa. As shown by pulse-chase experiments, the smaller represented the cytoplasmic precursor of the larger, and only the 113 kDa mature form was detected in integrin receptors expressed at the cell surface. Pre-activated keratinocytes contained beta 1 integrin subunits ranging from approximately 97 to 110 kDa. These beta 1 subunits had been processed through the Golgi, based on resistance to endoglycosidase-H treatment, and were not converted to 113 kDa subunits during subsequent cell culture. Experiments with endoglycosidase-F showed that differences in the apparent sizes of beta 1 integrin subunits observed in pre-activated and activated keratinocytes could be attributed to differences in subunit glycosylation. Smaller beta 1 subunits found in pre-activated keratinocytes, like the precursor beta 1 subunits of activated cells, appeared to be less efficient in reaching the cell surface. Overall, a approximately 10-fold increase in the level of cell surface integrin receptors occurred concomitant with the increased proportion of 113 kDa beta 1 subunits found in activated cells. Endoglycosidase-F experiments also indicated that there were changes in keratinocyte alpha subunits associated with beta 1. In related experiments, keratinocytes cultured in low Ca2+, serum-free MCDB medium for 4 days proliferated but their adhesiveness did not become activated. Therefore, keratinocyte proliferation and activation of adhesion are regulated separately. Finally, substantial activation of keratinocytes was observed when serum was added to cells cultured in MCDB with serum, indicating a role for serum factors in the activation process.


Adhesion of Golgi cisternae by proteinaceous interactions: intercisternal bridges as putative adhesive structures. We have investigated the nature of the component(s) responsible for holding the cisternal membranes of the Golgi complex into a stacked unit. Isolated Golgi complexes were treated with a variety of agents to induce the separation of intact Golgi stacks into single cisternal elements, i.e. "unstacking", and the effects were analyzed and quantitated by electron microscopy. In control experiments, isolated, intact Golgi stacks were stable at 4 degrees C and 20 degrees C for > or = 1 h; however, some unstacking occurred at 32 degrees C. Treatment of intact Golgi stacks with a variety of proteolytic enzymes resulted in a time- and dose-dependent unstacking of the cisternae, although stacks were resistant to various other proteases. Following liberation from the stack, single cisternae remained flattened with dilated rims. The integrity of intact Golgi stacks was unaffected by treatment with various concentrations and combinations of monovalent and divalent cations, or chelators of divalent cations. Electron microscopic observations of tannic acid- or negatively stained Golgi complexes, revealed the presence of highly structured, intercisternal "bridges". When seen within intact Golgi complexes, these bridges were only consistently found between closely apposed cisternae and were not observed on dilated rims or secretory vesicles. These bridges, on both intact stacks and physically disrupted cisternae, were rectangular, being approximately 8.5 nm in width, approximately 11 nm in height. Treatment with proteases under conditions that resulted in the with proteases under conditions that resulted in the unstacking of intact complexes also removed these bridge structures. These data show that proteinaceous components are responsible for holding Golgi cisternae together into a cohesive, stacked unit, and identify a candidate bridge structure that could serve this purpose.


In situ hybridisation of EBV DNA-DNA hybrids using wet heat in polypropylene containers. To explore procedures designed to optimise DNA-DNA in situ hybridisation, using cells infected with Epstein-Barr virus (EBV) and tissues and subfragments of the EBV DNA as probes. The denaturation step occurred in a polypropylene container, using wet heat generated by a hot water container, the pressure cooker, or the microwave oven, without coverslips, reaching a temperature of 121 degrees C or more in these two last systems. Two different visualisation systems were used. Fixed cells and tumours harbouring a high and medium to low copy number (a few hundreds to 33 copies per cell), were clearly labelled, using a simple reiterated subfragment (BamW) of the EBV DNA, and fresh frozen cells, harbouring a very low copy number (one to two on average) labelled using BamW as well as BamH (single non-reiterated 6 kilobase subfragment). This is a valuable alternative technique for DNA-DNA ISH that can be used in fresh frozen samples as well as fixed samples.


Comparative molecular neuroanatomy of cloned GABAA receptor subunits in the rat CNS. gamma-Aminobutyric acidA (GABAA) receptors in the mammalian central nervous system (CNS) are members of a family of ligand-gated ion channels consisting of heterooligomeric glycoprotein complexes in synaptic and extrasynaptic membranes. Although molecular cloning studies have identified 5 subunits (with approximately 40% amino acid homology) and isoforms thereof (approximately 70% homology), namely alpha 1-6, beta 1-4, gamma 1-3, delta, and rho, the subunit composition and stoichiometry of native receptors are not known. The regional distribution and cellular expression of GABAA receptor messenger RNAs (mRNAs) in the rat CNS have now been investigated by in situ hybridization histochemistry with subunit-specific 35S-labelled oligonucleotide probes on adjacent cryostat sections. Whereas alpha 1, beta 2, and gamma 2 transcripts were the most abundant and ubiquitous in the rat brain--correlating with the radioautographic distribution of GABAA receptors revealed by an ionophore ligand--others had a more restricted expression while often being abundant. For example, alpha 2 transcripts were found only in the olfactory bulb, cerebral cortex, caudate putamen, hippocampal formation, and certain lower brain stem nuclei; alpha 3 only in the olfactory bulb and cerebral cortex; alpha 5 in the hippocampal formation; and alpha 6 only in cerebellar granule cells. In addition, beta 1, beta 3, gamma 1, and delta mRNAs were also uniquely expressed in restricted brain regions. Moreover, in the spinal cord, alpha 1-3, beta 2,3, and gamma 2 mRNAs were differently expressed in Rexed layers 2-9, with alpha 2, beta 3, and gamma 2 transcripts most prominent in motoneurons of layer 9. Although differential protein trafficking could lead to the incorporation of some subunits into somatic membranes and others into dendritic membranes, some tentative conclusions as to the probable composition of native proteins in various regions of the CNS may be drawn.(ABSTRACT TRUNCATED AT 400 WORDS)


Electron microscopic localization of photoaffinity-labelled delta opioid receptors in the neostriatum of the rat. The distribution of delta opioid receptors, selectively labelled in vitro with the photoaffinity probe monoiodo azido-DTLET ([D-Thr2,pN3Phe4, Leu5]enkephaly-Thr6), was analyzed by light and electron microscopic radioautography in sections from rat neostriatum. Preliminary experiments indicated that up to 65% of specific 125I-azido-DTLET binding to rat striatal sections was still detectable following prefixation of the brain with 0.5% glutaraldehyde. These experiments also showed that up to 20-30% of the specifically bound radioactivity was covalently linked following ultraviolet irradiation and was thereby retained in tissue during subsequent postfixation and dehydration steps. Accordingly, the topographic distribution of the covalently attached azido-DTLET molecules was similar to that seen in fresh frozen sections and characteristic of that previously described for delta sites. Light and electron microscopic examination of the label in prefixed, striatal sections irradiated with ultraviolet light revealed that a significant proportion of specifically bound 125I-azido-DTLET molecules was intraneuronal. Specifically, 16% of the labelled binding sites were found in dendrites, 12% in perikarya and 4% in axon terminals. These results suggest that an important proportion of delta opioid binding sites labelled in the neostriatum correspond to receptors that are undergoing synthesis, transport and/or recycling. They also imply that a major fraction of delta sites are associated with intrastriatal neurons, as opposed to afferent axons. Approximately 44% of the labelled binding sites were associated with neuronal plasma membranes. Although most of these were found at the level of axodendritic (20%) and dendrodendritic (7%) appositions, comparison of the labelling incidence of these two compartments with their frequency of occurrence in tissue suggested that delta sites are fairly widely dispersed along neuronal plasma membranes. Only a small proportion (smaller than that of mu or kappa sites labelled in the same region) was associated with synaptic specializations. These results support the concept that delta receptors correspond to molecular entities that are distinct from mu and kappa sites and suggest that delta ligands act primarily nonjunctionally on the plasma membrane of striatal neurons.


Hyperthermia in sauna is unable to increase the plasma levels of ACTH/cortisol, beta-endorphin and prolactin in cocaine addicts. In order to establish possible different reactions between normal subjects and cocaine addicts to short term exposure to heat, thermal, cardiovascular and pituitary hormonal responses to hyperthermia in sauna were measured in 8 male cocaine addicts (studied after 14 days of abstinence) and in 8 age and weight matched normal men. Subjects sat for 30 min in a sauna room, where the temperature was 90 C and the relative humidity 10%. Physiological and hormonal parameters were measured just before and after sauna and after 30 min of rest at normal (21 C) room temperature. Significant and comparable increments in systolic and diastolic blood pressure, pulse rate and sublingual temperature were observed in the two groups at the end of sauna. All these parameters decreased to normal values after 30 min of rest at normal room temperature. Before sauna, ACTH, cortisol and beta-endorphin levels were similar in the two groups, whereas plasma prolactin concentrations were significantly higher in cocaine addicts. All examined hormones rose significantly in the normal controls at the end of sauna. All hormones, except cortisol, returned to the basal levels after 30 min at normal room temperature. In contrast, no significant hormonal responses to hyperthermia were observed at any time point in cocaine addicts. These data do not provide evidence of alterations in the cardiovascular and thermal adaptive responses to hyperthermia in cocaine abusers. On the other hand, the results show an impairment of the ACTH/cortisol, beta-endorphin and prolactin responses to hyperthermia in cocaine addicts.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatitis C antibodies in patients with alcoholic liver disease commonly have an identifiable risk factor. Hepatitis C virus (HCV) antibodies detected by radioimmunoassay (RIA) and immunoblot assay were determined in 139 patients with alcoholic liver disease (ALD). Risk factors for exposure to HCV were recorded. Anti-HCV was detected by RIA in 38 (27%), but only 18 (13%) were confirmed with an immunoblot assay. One hundred seven (77%) had risk factors. Of these 107, anti-HCV was detected in 36 by RIA and in 17 by immunoblot assay. Only 2 of 32 patients without risk factors had anti-HCV detected by RIA (1 was reactive with immunoblot, p < 0.05 vs. patients with risk factors). Parenteral drug use and multiple heterosexual contacts were significantly associated with anti-HCV. Age, sex, duration of alcoholism, race, and birthplace did not correlate with detectable HCV antibodies.


Changes in ligand binding to GABAA receptor sites in pacific salmon (Oncorhynchus) brain during spawning migration and "aging". When several years old, pacific salmon return to the site of birth, to spawn. At this time, a rapid aging process begins and the fish die within a few weeks after reproducing. Age-related changes of high and low affinity GABA binding sites were studied in salmon brains at three different phases of the spawning migration, i.e. shortly after returning to the natal stream, at the time of spawning, and thereafter. High affinity GABA binding slightly increased while the fish deteriorated. The low affinity component showed a remarkable decrease in density and a concomitant increase in affinity during this final episode of salmon life.


Synchronous subungual glomus tumours in adjacent digits. In the hand, glomus tumours usually present as a single lesion comprising one or more discrete masses affecting the subungual area of one digit. We report a case in which typical glomus tumours occurred simultaneously in the subungual area of two adjacent digits.


Histological changes during progression of adenoid cystic carcinoma. Eight cases of adenoid cystic carcinoma were reviewed to determine whether and how the histological features of the tumour vary with the progress of the disease. The tumours were classified by their histological patterns as tubular, cribriform, trabecular or solid. The relative amount of each pattern seen in routine light microscopic sections was calculated histomorphometrically and compared in the primary tumour and recurrent and/or metastatic lesions in the same case. In the early stage, the tubular pattern predominated. Later, the highest percentage shifted to the cribriform, then to the trabecular and finally, in the late stage, to the solid pattern. There was no reverse direction of histological transformation from the solid to the tubular pattern. These results may help to explain previous reports that the tubular pattern usually represents a favourable prognosis, the solid pattern a poor prognosis and the cribriform pattern an intermediate prognosis.


Differential effects of saturated fatty acids on low density lipoprotein metabolism in the guinea pig. Studies have shown that dietary fat saturation affects guinea pig plasma low density lipoprotein (LDL) levels by altering both LDL receptor-mediated catabolism and flux rates of LDL (Fernandez et al. 1992. J. Lipid Res. 33: 97-109). The present studies investigated whether saturated fatty acids of varying chain lengths have differential effects on LDL metabolism. Guinea pigs were fed 15% (w/w, 35% calories) fat diets containing either palm kernel oil (PK), 52% lauric acid/18% myristic acid; palm oil (PO), 43% palmitic acid/4% stearic acid; or beef tallow (BT), 23% palmitic acid/14% stearic acid. Plasma LDL cholesterol levels were significantly higher for animals fed the PK diet (P < 0.001) with values of 83 +/- 19 (n = 12), 53 +/- 8 (n = 12) and 44 +/- 16 (n = 10) mg/dl for PK, PO, and BT diets, respectively. The relative percentage composition of LDL was modified by fat type; however, LDL diameters and peak densities were not different between diets, indicating no effect of saturated fatty acid composition on LDL size. ApoB/E receptor-mediated LDL fractional catabolic rates (FCR) were significantly lower in animals fed the PK diet (P < 0.01) and LDL apoB flux rates were reduced (P < 0.01) in animals fed the BT diet. A correlation was found between plasma LDL levels and receptor-mediated LDL catabolism (r = -0.66, P < 0.01). A higher apoB/E receptor number (Bmax), determined by in vitro LDL binding to guinea pig hepatic membranes, was observed for animals fed BT versus PK or PO diets and Bmax values were significantly correlated with plasma LDL levels (r = -0.776, P < 0.001). These results indicate that saturated fatty acids of varying chain length have differential effects on hepatic apoB/E receptor expression and on LDL apoB flux rates which in part account for differences in plasma LDL cholesterol levels of guinea pigs fed these saturated fats.


Conservative surgery and radiation therapy for intraductal carcinoma of the breast. Nineteen women with intraductal carcinoma of the breast were treated with conservative surgery and radiotherapy from 1982 to 1990. All underwent excisional biopsy or wide local excision of the primary tumor. Definitive irradiation consisted of 4500 cGy in 180 cGy fractions given through tangential fields followed by a breast boost to the primary site to a total dose of 5900-6500 cGy. No patient received regional node irradiation. Median follow-up was 38 months. The five year actuarial rate of local failure was 9%. One patient failed with an infiltrating ductal carcinoma in the treated breast 31 months after initial treatment. Salvage mastectomy was performed. She remains without evidence of disease 43 months after initial treatment. Metastatic breast carcinoma has not developed in any of the patients. Cosmetic result was good to excellent in all patients. With short-term follow-up, conservative surgery and radiotherapy appear to be an acceptable alternative to mastectomy in carefully selected patients with ductal carcinoma in situ. As retrospective and randomized trials mature, the natural history of these lesions treated with conservative surgery and irradiation will be further defined.


Nutrient-induced activation of trehalase in nutrient-starved cells of the yeast Saccharomyces cerevisiae: cAMP is not involved as second messenger. Starvation of Saccharomyces cerevisiae cells for specific nutrients such as nitrogen, phosphate or sulphate causes arrest in the G1 phase of the cell cycle at a specific point called 'start'. Re-addition of different nitrogen sources, phosphate or sulphate to such starved cells causes activation of trehalase within a few minutes. Nitrogen-source- and sulphate-induced activation of trehalase were not associated with any change in the cAMP level, but in the case of phosphate there was a small transient increase. When nitrogen-source-activated trehalase was isolated by immuno-affinity chromatography from crude extracts, the purified enzyme showed the same activity profile as in the original crude extracts, indicating that post-translational modification is responsible for the activation. In the yeast mutants cdc25-5 and cdc35-10, which are temperature sensitive for cAMP synthesis, incubation at the restrictive temperature lowered but did not prevent nitrogen-, phosphate- or sulphate-induced activation of trehalase. Since under these conditions the cAMP level in the cells is very low, it is unlikely that cAMP acts as a second messenger in this nutrient-induced effect. Nitrogen-source-induced activation of trehalase requires the presence of glucose at a concentration similar to that able to stimulate the RAS-adenylate cyclase pathway. This indicates that the same glucose-sensing system might be involved in both phenomena. Nitrogen-starved cells fractionated according to cell size all showed nitrogen-source-induced activation of trehalase to the same extent, indicating that the nitrogen-induced signalling pathway involved is not dependent on the well-known cell size requirement for progression over the start point of the cell cycle.


An analysis of the effect of changes in growth temperature on proteolysis in vivo in the psychrophilic bacterium Vibrio sp. strain ANT-300. In the psychrophilic bacterium Vibrio sp. strain ANT-300, the rate of protein degradation in vivo, measured at fixed temperatures, increased with elevation of the growth temperature. A shift in growth temperature induced a marked increase in this rate. Dialysed cell-free extracts hydrolysed exogenous insulin, globin and casein (in decreasing order of activity) but did not hydrolyse exogenous cytochrome c. Cells contained at least seven protease separated by DEAE-Sephacel chromatography, one of which was an ATP-dependent serine protease. The ATP-dependent proteolytic activity in extracts of cells incubated for 3 h at 16 degrees C after a shift-up from 0 degrees C increased to a level 36% and 17% higher than that of cells grown at 0 degrees C and 13 degrees C, respectively. A shift-down to 0 degrees C from 13 degrees C induced only a slight increase in the proteolytic activity. Extracts of all cells, whether exposed to temperature shifts or not, showed the same temperature dependence with respect to both ATP-dependent and ATP-independent protease activity. In all the extracts these proteases also exhibited the same heat lability. The ATP-dependent protease was inactivated by incubation at temperatures above 25 degrees C. There was an increase in ATP-independent protease activity during incubation at temperatures between 25 and 30 degrees C, but a decrease at 35 degrees C and higher. These results suggest that the marked increases in proteolysis in vivo, caused by a shift in temperature, may result not only from increases in levels of ATP-dependent serine protease(s) but also from increases in the susceptibility of proteins to degradation.


Filamentation promotes F'lac loss in Escherichia coli K12. The stability of plasmid F'lac in Escherichia coli strain SP45 (a temperature conditional mutant which grows as spherical cells at 42 degrees C and as a rod at 30 degrees C) was studied. F'lac elimination was demonstrated when bacteria exposed to subinhibitory concentrations of various chemicals were induced to form filaments. No plasmid loss was found when spherical cells were subjected to the same treatments. Plasmid loss was also observed in dnaA46 and lexA41 mutants when cell filamentation was induced at 42 degrees C, but not when they were cultured at 30 degrees C. Nalidixic acid promoted F'lac elimination at 0.25 micrograms ml-1 in a recA13 mutant and at 1.5 micrograms ml-1 in the recA+ counterpart. A marked difference was found in the rate of F'lac elimination from thermosensitive DNA gyrase mutants [gyrA43(Ts) and gyrB41(Ts)] between rods and their spherical (rodA51) derivatives growing at semipermissive temperature (36.5 degrees C). Plasmids carrying the ccd segment of F in DNA gyrase mutants were lost after 2.5 generations from rods and after 6 generation from spherical cells. Plasmid segregation into non-viable minicell-like elements was found after induction of filaments. These data suggest that plasmid stability is correlated with cell shape and that curing is more easily achieved when bacteria can elongate normally.


The insertion sequence IS200 fingerprints chromosomal genotypes and epidemiological relationships in Salmonella heidelberg. In Salmonella heidelberg the copy number of the Salmonella-specific insertion element IS200 was found to vary from four to six. All strains tested contained at least one common insertion site which was serovar specific, and most strains contained three common sites. Concurrent analysis of plasmids indicated that all insertion sequence copies were chromosomally located, and also supported the equivalence of an IS200 fingerprint and clonality. Seven intra-serovar clonal lines were thereby identified. One of these was associated with human infections, including septicaemias. Another was associated with chicken as a host: all these strains also carried a unique plasmid of 23 MDa, which was typed as a member of the IncX group. The chromosomal fingerprint of a third clone showed it to be a descendant of the chicken line marked by a single IS200 transposition. One or two representatives of four other clonal lines were identified. These lines of S. heidelberg could be related by divergent evolution, and the most recent relatives conformed to a continuous branching process model of IS200 transposition. This insertion sequence provided a highly discriminatory molecular marker of the S. heidelberg chromosome, and two of the seven clonal lines so identified were associated with distinct clinical/epidemiological contexts.


Diffusion of beta-lactam antibiotics into proteoliposomes reconstituted with outer membranes of isogenic imipenem-susceptible and -resistant strains of Enterobacter cloacae. The influence of outer membrane (OM) permeability on carbapenem susceptibility was studied in strains of Enterobacter cloacae, a species in which carbapenem resistance depends upon the conjunction of overproduction of the chromosomal cephalosporinase and reduction of OM permeability. Relative trans-OM diffusion rates were measured using the liposome swelling assay. Proteoliposomes were reconstituted with OM from the members of an isogenic set of E. cloacae strains, selected in vivo or in vitro, which produced either porins F and D (wild-type), or F or D only, or neither. For all but one mutant, and compared with the wild-type strain, the respective increases in MICs and decreases in trans-OM diffusion of carbapenems were: nil and 13 to 18%; 4- to 32-fold and 33 to 50%; > or = 64-fold and > or = 90%. Our results suggest (i) that carbapenems (and other beta-lactam antibiotics) diffuse through porins F and D, but more rapidly through porin F, and (ii) that OM permeability is the critical factor in determining the level of MICs of carbapenems for cephalosporinase-overproducing strains of E. cloacae. The OM of one particular low-level carbapenem-resistant and porin F- and D-deficient mutant was at least five times more permeable to carbapenems than the similarly porin-deficient high-level resistant mutants. We infer from this observation the possible existence of an alternative carbapenem penetration pathway which could be associated with two as yet uncharacterized overproduced OM proteins of about 22 and 47 kDa.


The catalase-peroxidase of Mycobacterium intracellulare: nucleotide sequence analysis and expression in Escherichia coli. The activation of catalase genes in response to oxidative stress may contribute to the intracellular survival of mycobacteria. In this report, the nucleotide sequence of a mycobacterial catalase gene is described. The deduced protein sequence of this Mycobacterium intracellulare gene (MI85) was 60% identical to the Escherichia coli hydroperoxidase I (HPI) protein, 59% identical to the Salmonella typhimurium (HPI) catalase, and 47% identical to a Bacillus stearothermophilus peroxidase. The MI85 protein, expressed in E. coli, has also been shown to have peroxidase and catalase activities. Furthermore, Southern blot hybridizations, which demonstrated that a MI85 gene probe hybridizes with chromosomal DNA from thirteen different strains of mycobacteria, suggest that this catalase-peroxidase gene is prevalent in the mycobacterial genus. The availability of catalase gene probes should permit an evaluation, at the molecular level, of the role of catalase in mycobacterial pathogenesis.


Siderophore-specific induction of iron uptake in Pseudomonas aeruginosa. Pseudomonas aeruginosa has two siderophore-based high-affinity iron-uptake systems utilizing pyoverdin and pyochelin. Using strain IA1, a mutant deficient in production of both siderophores, we have shown that addition of purified siderophore to the growth medium induces expression of specific iron-regulated outer-membrane proteins and increases 55Fe-siderophore transport. Addition of pyoverdin from the parent strain PAO1 or from a clinical strain 0:12 induced expression of an 85 kDa IROMP and increased the rate of 55Fe-pyoverdin transport. Transport rates for 55Fe-PAO1 pyoverdin increased from 1.27 to 3.57 pmol Fe min-1 per 10(9) cells. Addition of purified pyochelin induced expression of a 75 kDa IROMP accompanied with increased 55Fe-pyochelin uptake without affecting 55Fe-pyoverdin transport. 55Fe-pyochelin transport increased from 0.3 to 10.6 pmol min-1 per 10(9) cells. Addition of pyoverdin from the parent strain or a chromatographically distinct pyoverdin caused increased reactivity with an anti-85 kDa mAb in Western blotting, indicating that the same receptor is being induced. These results suggest that P. aeruginosa can respond specifically to the presence of siderophore and moreover that not only can the pyoverdin receptor transport its cognate ferri-pyoverdin but also different ferri-pyoverdins, albeit at a reduced rate.


Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate. To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.


Impact of chromium ions on nucleoside triphosphates and nucleic acids. Chromatography on hydroxyapatite, polarography, and spectroscopy have been used to evaluate the interactions of Cr(III) and Cr(VI) with DNA and nucleoside triphosphates. Cr(III) catalyzes the removal of pyrophosphate from nucleoside triphosphate (NTP) molecules. It also causes extensive aggregations of DNA through DNA-DNA cross-linking. Cr(VI) does not affect the DNA structure unless reduced to lower oxidation states.


Total hip arthroplasty complicated by a malignant fibrous histiocytoma. A case report. Tumors associated with total hip arthroplasty are uncommon. The authors report the eighth case of a malignant fibrous histiocytoma associated with a total joint arthroplasty and discuss its etiology and significance.


Antiviral (RNA) activity of selected Amaryllidaceae isoquinoline constituents and synthesis of related substances. A series of 23 Amaryllidaceae isoquinoline alkaloids and related synthetic analogues were isolated or synthesized and subsequently evaluated in cell culture against the RNA-containing flaviviruses (Japanese encephalitis, yellow fever, and dengue viruses), bunyaviruses (Punta Toro, sandfly fever, and Rift Valley fever viruses), alphavirus (Venezuelan equine encephalomyelitis virus), lentivirus (human immunodeficiency virus-type 1) and the DNA-containing vaccinia virus. Narciclasine [1], lycoricidine [2], pancratistatin [4], 7-deoxypancratistatin [5], and acetates 6-8, isonarciclasine [13a], cis-dihydronarciclasine [14a], trans-dihydronarciclasine [15a], their 7-deoxy analogues 13b-15b, lycorines 16 and 17, and pretazettine [18] exhibited consistent in vitro activity against all three flaviviruses and against the bunyaviruses, Punta Toro and Rift Valley fever virus. Activity against sandfly fever virus was only observed with 7-deoxy analogues. In most cases, however, selectivity of the active compounds was low, with toxicity in uninfected cells (TC50) occurring at concentrations within 10-fold that of the viral inhibitory concentrations (IC50). No activity was observed against human immunodeficiency virus-type 1, Venezuelan equine encephalomyelitis virus, or vaccinia viruses. Pancratistatin [4] and its 7-deoxy analogue 5 were evaluated in two murine Japanese encephalitis mouse models (differing in viral dose challenge, among other factors). In two experiments (low LD50 viral challenge, variant I), prophylactic administration of 4 at 4 and 6 mg/kg/day (2% EtOH/saline, sc, once daily for 7 days, day -1 to +5) increased survival of Japanese-encephalitis-virus-infected mice to 100% and 90%, respectively. In the same model, prophylactic administration of 5 at 40 mg/kg/day in hydroxypropylcellulose (sc, once daily for 7 days, day -1 to +5) increased survival of Japanese-encephalitis-virus-infected mice to 80%. In a second variant (high LD50 viral challenge), administration of 4 at 6 mg/kg/day (ip, twice daily for 9 days, day -1 to +7) resulted in a 50% survival rate. In all cases, there was no survival in the diluent-treated control mice. Thus, 4 and 5 demonstrated activity in mice infected with Japanese encephalitis virus but only at near toxic concentrations. To our knowledge, however, this represents a rare demonstration of chemotherapeutic efficacy (by a substance other than an interferon inducer) in a Japanese-encephalitis-virus-infected mouse model.


Effects of miocamycin and erythromycin on polymorphonuclear cell function. Previous studies have shown that erythromycin can enter phagocytic cells, stimulating their functional activity. In this work we compared the effects of erythromycin and another newer macrolide antibiotic, miocamycin, on a series of in vitro tests aimed at evaluating their influence on polymorphonuclear cell (PMN) functions. Results indicate that erythromycin induces an increase in leukotriene B4 production in PMNs, while chemotaxis, killing of Candida albicans and respiratory burst are not influenced, at least at the doses used in this study. On the contrary, all these activities are significantly enhanced following incubation with miocamycin, and the response varies according to the antibiotic concentration.


Piperacillin/tazobactam plus gentamicin as empirical therapy for febrile neutropenic patients with haematological malignancy. The efficacy of piperacillin/tazobactam (PIPC/TBT) in combination with gentamicin was assessed as empirical therapy in 44 febrile neutropenic patients with haematological malignancy. A favourable response to therapy was seen in 67% patients overall and in 57% of patients with microbiologically documented infection. PIPC/TBT demonstrated good clinical and in vitro activity against isolated pathogens, particularly Gram positive cocci such as Staphylococcus epidermidis. The MIC of both Gram positive and Gram negative pathogens to PIPC was reduced in the presence of TBT. PIPC/TBT plus gentamicin is a safe and effective combination for empirical therapy in febrile neutropenic patients, even in a unit with a predominance of Gram positive infections.


The inactivating potassium currents of hair cells isolated from the crista ampullaris of the frog. 1. A-type outward currents were studied in sensory hair cells isolated from the semicircular canals (SCC) of the leopard frog (Rana pipiens) with whole-cell voltage- and current-clamping techniques. 2. There appear to be two classes of A-type outward-conducting potassium channels based on steady-state, kinetic, pharmacological parameters, and reversal potential. 3. The two classes of A-type currents differ in their steady-state inactivation properties as well as in the kinetics of inactivation. The steady-state inactivation properties are such that a significant portion of the fast channels are available from near the resting potential. 4. The inactivating channels studied do not appear to be calcium dependent. 5. The A-channels in hair cells appear to subserve functions that are analogous to IA functions in neurons, that is, modulating spike latency and Q (the oscillatory damping function). The A-currents appear to temporally limit the hair cell voltage response to a current injection.


Potassium currents contributing to action potential repolarization and the afterhyperpolarization in rat vagal motoneurons. 1. Intracellular recordings were made from neurons in the dorsal motor nucleus of the vagus (DMV) in transverse slices of rat medulla maintained in vitro at 30 degrees C. Neurons had a resting potential of -59.8 +/- 1.4 (SE) mV (n = 39) and input resistance of 293 +/- 23 M omega (n = 44). 2. Depolarization elicited overshooting action potentials that were blocked by tetrodotoxin (TTX; 1 microM). In the presence of TTX, two types of action potentials having low and high thresholds could be elicited. The action potentials were blocked by cobalt (2 mM) indicating they were mediated by calcium currents. 3. Under voltage clamp, depolarization of the cell from membrane potentials negative of the resting potential activated a transient potassium current. This current was selectively blocked by 4-aminopyridine (4-AP) (5 mM) and catechol (5 mM) indicating that it is an A-type current. This current inactivated with a time constant of 420 ms and recovered from inactivation with a time constant of 26 ms. 4. When calcium currents were blocked by cadmium or cobalt, the rate of action potential repolarization was slower. In the presence of tetraethylammonium (TEA; 200-400 microM) or charybdotoxin (CTX; 30 nM) a small "hump" appeared on the repolarizing phase of the action potential that was abolished by addition of cadmium. These results indicate that a calcium-activated potassium current (IC) contributes to action potential repolarization. 5. Actions potentials elicited from hyperpolarized membrane potentials repolarized faster than those elicited from resting membrane potential. This effect could be blocked by catechol, indicating that voltage-dependent potassium currents (IA) can also contribute to action-potential repolarization. In the presence of catechol and calcium channel blockers, action potentials still had a significant early afterhyperpolarization suggesting that another calcium independent outward current is also active during repolarization. This fast afterhyperpolarizations (AHP) was not blocked by TEA. 6. Action potentials were followed by prolonged AHPs, which had two phases. The initial part of the AHP was blocked by apamin (100 nM) indicating that it results from activation of SK type calcium-activated potassium channels. The slow phase was selectively blocked by catechol suggesting that it is due to activation of IA. 7. It is concluded that a TTX-sensitive sodium current and two calcium currents contribute to the action potential in rat DMV neurons. At least three different currents contribute to action-potential repolarization: IC, IA, and a third unidentified calcium-insensitive outward current.(ABSTRACT TRUNCATED AT 400 WORDS)


EPR study of melanin-protein interaction: photoinduced free radicals and progressive microwave power saturation. The photo-induction of free radicals in synthetic L-dihydroxyphenylalanine (L-DOPA) melanin in the presence of bovine serum albumin (BSA) was studied by electron paramagnetic resonance (EPR) spectroscopy. By monitoring the signal intensities and progressive microwave power saturation it was shown that L-DOPA melanin in solution behaves as a single macromolecule, interacting with BSA and molecular oxygen. In the absence of oxygen, the EPR signal of L-DOPA melanin was homogeneously broadened; the magnetic interaction with oxygen induced inhomogeneous broadening. In aqueous solution, the presence of BSA decreased the accessibility of oxygen to paramagnetic centres in the melanin. On UV-visible illumination, the presence of BSA modified the rates of formation and decay of photoinduced free radicals, resulting in a net enhancement of the EPR signal compared with that observed in pure L-DOPA melanin.


Bacterial concentration fluorescence immunoassay (BCFIA) for the detection of periodontopathogens in plaque. A bacterial concentration fluorescence immunoassay (BCFIA) was developed to rapidly detect periodontopathic bacteria in human plaque samples. The BCFIA utilized fluorescent-tagged monoclonal antibodies (MAbs) directed against the lipopolysaccharide of selected Gram-negative bacteria. Microorganisms identified in plaque using the BCFIA included Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum. The immunoassay procedure involved combining a patient's plaque sample with a species-specific fluorescein isothiocyanate-labeled MAb and then incubating the mixture in a specialized microtiter plate allowing the MAb to bind to its homologous bacteria. Bound and unbound fluorescent-tagged MAbs were separated by filtration and total bound bacterial fluorescence was determined with a fluorimeter. The relative number of a bacterial species in a given plaque sample was estimated by reference to a standard curve carried through the BCFIA. The BCFIA had a lower detection limit of near 10(4) specific bacterial cells in a mixed bacterial preparation or plaque sample. When compared to cultivable flora procedures in detecting the 4 periodontopathogens, the BCFIA had high levels of statistical sensitivity, 97% to 100%, while statistical specificity ranged between 57% and 92%. There was a 71% to 82% agreement between BCFIA and DNA probe methodology in detecting periodontopathogens in plaque. The BCFIA, when compared to cultivable flora, offers the advantage of evaluating both live and dead bacterial cells in plaque. This may in part, if not fully, explain the lower specificity values of the BCFIA when compared to cultivable flora. Screening plaque samples for periodontopathic bacteria is considerably faster and results in a greater frequency of detection with BCFIA than cultivable flora based methods.


DNA probe and enzyme analysis in periodontal diagnostics. Recent research in periodontology has indicated that a finite number of bacterial species are associated with periodontal disease. This has generated the hypothesis that periodontal disease is essentially an infection due to one or more of the putative periodontopathogens; i.e., the specific plaque hypothesis (SPH). This microbial specificity paradigm has already changed the way researchers and some clinicians view periodontal disease. The clinician must heed several cautions signs, however, if he is to use the SPH to provide treatment options that could enhance the delivery of care. There are several diagnostic technologies that can be used to detect and semi-quantitate those bacterial species that have been identified as periodontopathogens. This paper discusses diagnostic tests based upon the detection in plaque of DNA segments or enzyme activity(ies) that are unique for one or more of the suspected periodontopathogens.


Characteristics and utilization of antibody measurements in clinical studies of periodontal disease. The detection and quantitation of immune responses to infections have long been used as a diagnostic tool in medical infections. Recently, increasing evidence has supported that active, specific antibody responses to selected members of the subgingival microbiota are noted in periodontitis patients. This report describes the various specificities of this antibody as they relate to periodontitis classification and prognosis. The functional aspects of the serum antibody have come under increasing scrutiny to understand better the potential immunologic mechanisms acting in the periodontium. Data are available that describe opsonizing potential, complement fixing ability, blocking functions, and anti-toxic capacity for the antibody. Longitudinal alterations in specific antibody levels are shown to relate to infection and accompany changes in the burden of a specific microorganism in the subgingival plaque. Thus, these antibody changes could be useful indicators of altered host-parasite interactions that presage a disease-active episode. Finally, studies were designed to examine the ability of antibody to reflect the effects of treatment on the disease. The results indicated that specific antibody levels change with mechanical, antimicrobial, and anti-inflammatory treatments. The findings described in this report suggest that evaluation of the level and specificity of serum antibody can be a beneficial adjunct in designing and implementing clinical studies delineating the initiation, progression, and treatment of periodontitis.


Effect of adenosine 3',5'-cyclic monophosphate derivatives on acute liver injury induced by carbon tetrachloride. The inhibitory effects of 12 synthetic N6-alkyl cAMPs, 8-substituted cAMPs and cAMP alkylphosphoramidate derivatives (50 or 100 mg/kg, bolus, i.p.) on serum GOT and GPT activities and hepatocyte cytoplasmic vacuolation were examined in male Fischer 344 rats, which were exposed to CCl4 (0.5 mg/kg, p.o.) 30 min prior to the administration of cAMP derivatives. In CCl4-treated rats 6 h later, serum GOT and GPT levels were elevated 10- and 12-fold higher than those of vehicle rats, respectively. Treating CCl4-exposed rats with all cAMP derivatives, except those of alkylphosphoramidate, significantly decreased the levels of serum enzymes. Based on the effects of both serum GOT and GPT elevation, N6-butyl- and N6-heptyl-cAMP were the most potent. It was also observed histopathologically, that both compounds inhibited the occurrence of cytoplasmic vacuolation in CCl4-treated liver cells. This is the first report that cAMP derivatives possess a protective effect in the liver injury model induced by CCl4.


Detection of human papillomavirus DNA in biopsy-proven cervical squamous intraepithelial lesions in pregnant women. This study analyzed cervical squamous intraepithelial lesions (SILs) in pregnant women for human papillomavirus (HPV) using in situ hybridization analysis. HPV DNA was detected in 77% (23/30) of low-grade SILs as compared to 89% of such lesions in nonpregnant women. The detection rate in high-grade SILs was 41% (9/22) compared to 70% in nonpregnant women. Analysis by the polymerase chain reaction showed that the detection rates were similar (96-100%) for low- and high-grade lesions in pregnant and nonpregnant women, which demonstrates that in situ negative tissues indeed contained HPV DNA. Low-grade SILs contained a heterogeneous group of at least 14 different HPV types, whereas most high-grade SILs contained HPV 16. We concluded that cervical SILs in pregnant women are invariably associated with HPV. Further study is needed to determine which of several possible variables, such as the age of the lesion and the viral copy number, may explain the apparent decreased detection rate of HPV by in situ hybridization in SILs during pregnancy.


Analogues of platelet activating factor. 7. Bis-aryl amide and bis-aryl urea receptor antagonists of PAF. A series of bis-aryl amide (13-57 and 66-81) and bis-aryl urea (58 and 85) antagonists of platelet-activating factor (PAF) was prepared that contain, separating the two aromatic rings, linear amide linkages of the form -(CH2)nCONH- (n = 0-2), -OCH2CONH-, and -(CH2)nNHCO- (n = 0-1), branched amide linkages of the form -(CH2)nN(COR)- (n = 1-3, R = CH3 or n-C3H7), and -N(COCH3)CH2-, and urea linkages of the form -NHCONH- and -CH2N(CONHCH3)-. These compounds were examined for their ability to inhibit PAF-induced platelet aggregation of rabbit platelets. These in vitro data were compared to similar data obtained for a number of known PAF antagonists. The compounds were evaluated in vivo, in the mouse, for their ability to prevent death induced by a lethal challenge of PAF. The relationships between the biological activity and the nature, lipophilicity, and position of substituents of the aromatic rings were studied. Best activity was observed for compounds having linkages of the type -CH2CONH-, -CH2N(COR)-, and -CH2NHCO-. Many of these compounds inhibit PAF-induced platelet aggregation with IC50's under 1 microM.


Selective, centrally acting serotonin 5-HT2 antagonists. 1. 2- and 6-substituted 1-phenyl-3-(4-piperidinyl)-1H-indoles. A series of 1-[2-[4-(1H-indol-3-yl)-1-piperidinyl]ethyl]-2-imidazolidinones has been synthesized. The 1-position of the indole is substituted with phenyl groups and in the 2- or 6-positions are additional substituents. An analogous series with the imidazolidinone ring opened to corresponding urea derivatives was also prepared. High potency and selectivity for 5-HT2 receptors (as compared with D2 and alpha 1 receptor affinities) were obtained with medium-large substituents such as 6-chloro, 6-methyl, and 6-trifluoromethyl or a 2-methyl substituent. Larger 6-substituents such as isopropyl considerably reduced activity, while the smaller 6-fluoro substituent afforded unselective compounds. Selective 5-HT2 antagonists were found by combining 6-substitution with both unsubstituted 1-phenyl and substituted 1-phenyl groups (2-F, 4-F, 4-Cl). However, 3-substitution of the phenyl group markedly reduced 5-HT2 receptor affinity, especially with a 3-trifluoromethyl substituent. Introduction of a 3-(2-propyl) substituent in the imidazolidinone ring reduced binding to alpha 1 adrenoceptors with a factor of 3-8. Practically no influence on 5-HT2 and D2 receptor affinities were found by the presence of this substituent compared to the 3-unsubstituted derivatives. Compounds with potent receptor binding also potently inhibited the quipazine-induced head twitch syndrome in rats. The compounds were equally active after oral and subcutaneous administration and they had a long duration of action (> 24 h). Especially urea derivatives were found to be considerably more potent at 24 h than at 2 h after subcutaneous administration. Some of the compounds potently inhibited isolation-induced aggression in mice, an effect which, however, did not correlate to 5-HT2 receptor-mediated activities. On the basis of these structure-activity studies 1-[2-[4-[6-chloro-1-(4-fluorophenyl)-1H-indol-3-yl]-1- piperidinyl]ethyl]-3-(2-propyl)-2-imidazolidinone (Lu 26-042, compound 4c) was selected for further pharmacological and toxicological investigations.


Selective, centrally acting serotonin 5-HT2 antagonists. 2. Substituted 3-(4-fluorophenyl)-1H-indoles. A series of 3-(4-fluorophenyl)-1H-indoles substituted in the 1-position with 4-piperidinyl, 1,2,3,6-tetrahydro-4-pyridinyl, and 4-piperazinyl was synthesized. By variation of the substituents in the benzene part of the indole nucleus in 1-[2-[4-[3-4-fluorophenyl)-1H-indol-1-yl]-1-piperidinyl]-ethyl]-2- imidazolidinones, the highest 5-HT2 receptor affinity and selectivity with respect to dopamine D2 receptors and alpha 1 adrenoceptors were obtained by 5-methyl substitution. Different substituents were introduced in the 1-position of the piperidine ring in the 5-methyl-substituted derivative. Thus replacement of the 2-(2-imidazolidinon-1-yl)ethyl side chain with a 2-(1,3-dimethyl-1-ureido)ethyl or methyl substituent resulted in unchanged affinity and selectivity for 5-HT2 receptors. Replacement with a 2-[3-(2-propyl)-2-imidazolidinon-1-yl]ethyl side chain reduced binding to alpha 1 adrenoceptors with a factor of four, while affinities for 5-HT2 and D2 receptors were retained, compared to the 3-unsubstituted imidazolidinone. Indoles substituted in the 1-position with 4-piperazinyl had generally weaker affinity for both 5-HT2 and D2 receptors compared to corresponding 4-piperidinyl- and 1,2,3,6-tetrahydro-4-pyridinyl-substituted indoles. Introduction of a methyl group in the 2-position of the 5-methyl-substituted indole resulted in further increase of selectivity for the 5-HT2 receptor. Compounds with potent receptor binding also potently inhibited the quipazine-induced head twitch syndrome in rats. The compounds were equally active after oral and subcutaneous administration and showed a long duration of action (> 24 h). In general, the derivatives were found to be considerably more potent at 24 h than at 2 h after the administration. The compounds within this series were prepared as analogues of the previously described 1-(4-fluorophenyl)-3-(4-piperidyl)-1H-indoles by interchange of the C-3 carbon atom and the nitrogen atom in the indole nucleus. The pharmacological results indicate that this isosteric replacement results in higher selectivity for 5-HT2 receptors compared to the former series. The 1-[2-[4-[2,5-dimethyl-3-(4-fluorophenyl)-1H-indol-1-yl]-1- piperidinyl]ethyl]-2-imidazolidinone has high affinity for 5-HT2 receptors (IC50 = 3.4 nM) and extremely low affinity for both dopamine D2 receptors (IC50 = 6900 nM) and alpha 1 adrenoceptors (IC50 = 2300 nM).


Novel cAMP PDE III inhibitors: 1,6-naphthyridin-2(1H)-ones. Two series of medorinone (3) analogs were prepared by modifications at C(2) and C(5). The C(2)-series was prepared from 2-chloro-5-methyl-1,6-naphthyridine (4) by replacement of the chloro group with various nucleophiles. The C(5)-series was prepared from 5-acyl-6-[2-(dimethylamino)-ethenyl]-2(1H)-pyridinone (11), 5-bromo-1,6-naphthyridin-2(1H)-one (17), and 1,3-diketones 19 and 27. 1,6-Naphthyridin-2(1H)-ones are novel inhibitors of cAMP PDE III. Modification of the carbonyl group of 3 or N-methylation at N(1) resulted in a dramatic loss of enzyme activity. Absence of the C(5)-methyl group of medorinone (3) or its shift to C(3) or C(7) also resulted in reduced activity. Substitution at C(3) also diminished activity. However, substitution at C(5) by a wide variety of substituents led to improvement of enzyme activity and several C(5)-substituted analogs were more potent than milrinone.


Stimulation of the active transport of serotonin into mouse platelets by the sulfhydryl oxidizing agent diamide. The purpose of this study was to determine the effects of diamide, a reversible sulfhydryl oxidizing agent, on the transport of serotonin (5-HT) by mouse platelets. Diamide produced a concentration-dependent (10-200 microM) stimulation of 5-HT transport that was rapid and sustained over 0-10 minutes of incubation. When platelets were incubated with diamide (10-200 microM) in the presence of glucose, the content of reduced glutathione was significantly decreased only at a final concentration of 200 microM, while washed platelets incubated with diamide (10-200 microM), in the absence of glucose, had a significant concentration-dependent decrease in their content of reduced glutathione. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked diamide-induced stimulation of 5-HT transport. The kinetics of 5-HT transport showed that diamide caused a marked increase in the maximal rate of transport (Vmax control = 28.4 +/- 1.4 vs. Vmax diamide = 60.9 +/- 4.1 pM/10(8) platelets/4 min) but did not significantly alter the Km values. Ouabain, an inhibitor of platelet Na(+)-K+ ATPase, blocked the stimulation by diamide in a concentration-dependent manner. Dithiothreitol, a disulfide reducing agent, was able to partially reverse the stimulation of platelet 5-HT transport caused by diamide. This study has shown that diamide can stimulate the active transport of 5-HT by mouse platelets and suggests a possible role for free sulfhydryl groups in the regulation of this process.


Constitutional ring chromosomes and tumour suppressor genes. The types of malignancy reported in carriers of constitutional ring chromosomes r(11), r(13), and r(22) are concordant with the chromosomal assignment of tumour suppressor loci associated with Wilms' tumour, retinoblastoma, and meningioma. It is suggested that the somatic instability of ring chromosomes may play a role in this association and that constitutional ring chromosomes may be a source for mapping of tumour suppressor loci with the potential for covering most or all of the human genome. The hypothesis predicts the presence of a locus on chromosome 10 associated with follicular carcinoma of the thyroid, in line with previous cytogenetic findings of rearrangements involving chromosome 10 in thyroid tumours, and a locus on chromosome 22 associated with testicular cancer. Development of neurofibromatoses (NF) that do not fulfil the clinical criteria of neurofibromatosis type 2 (NF2) in carriers with r(22) suggests either the presence of an additional NF locus on chromosome 22 or that ring chromosome mediated predisposition to somatic mutation of a specific tumour suppressor may be associated with atypical development of features usually associated with germline mutations.


Intracellular calcium during photodynamic permeabilization of cardiomyocytes. The purpose of this study was to determine whether an early increase in [Ca2+]i preceding generalized lysis of cardiomyocytes occurred during photodynamic permeabilization. A method was developed which facilitated the simultaneous measurement, in real time, of permeabilization of the sarcolemma to Ca2+ and Mn2+ during photodynamic action. Quin-2 loaded cells were illuminated in the presence of erythrosin B and the change in the fluorescence emission of the calcium-quin-2 complex was used to measure the rate and extent of change in [Ca2+]i. The same system was used in the presence of extracellular Mn2+ to determine how quickly the cardiomyocytes became permeable to either Mn2+ or quin-2. Calcium ions were observed to enter the myocytes prior to permeabilization of the sarcolemma to either Mn2+ or quin-2, and thus before membrane lysis. Lysis of cardiomyocytes did not appear to be dependent upon increases in [Ca2+]i. Controls were performed to rule out fluorescent artifacts. Reperfusion injury and photodynamic therapy involve both the production of free radicals and an early increase in [Ca2+]i. This study demonstrates a direct correlation between the production of reactive oxygen species and prelytic increases in [Ca2+]i in neonatal cardiomyocytes and demonstrates that this phenomenon may be common to many cell types.


Effects of regional ischemia on the ryanodine-sensitive Ca2+ release channel of canine cardiac sarcoplasmic reticulum. In mammalian myocardium, muscle contraction is regulated by the rapid release of Ca2+ ions through ryanodine-sensitive Ca2+ release channels present in the intracellular membrane compartment, sarcoplasmic reticulum (SR). In this study, the effects of regional ischemia on intrinsic SR Ca2+ release channel function were determined by studying the Ca2+ transport and release, and [3H]ryanodine binding properties of whole muscle homogenates and SR-enriched membrane fractions from normal and ischemic myocardium. Measurement of oxalate-supported 45Ca(2+)-uptake rates before and after pretreatment with 1 mM ryanodine, indicated that the SR Ca2+ release channel retained its ability to be effectively closed by the channel-specific probe ryanodine after 15 and 60 min of ischemia. 45Ca2+ efflux from, and high-affinity [3H]ryanodine binding to SR-enriched vesicle fractions indicated retention of regulation of Ca2+ release channel activity by Ca2+, Mg2+ and adenine nucleotide in 15 and 60 min ischemic samples. Further, sodium dodecylsulfate polyacrylamide gel and immunoblot analysis revealed no proteolytic degradation of the M(r) 565,000 SR Ca2+ release channel polypeptide after 15 and 60 min of ischemia. These results suggested a minimal, if any, loss of intrinsic SR Ca2+ release channel function in ischemic hearts.


Decrease in beta 1- and increase in beta 2-adrenoceptors in long-term follow-up after orthotopic cardiac transplantation. Total beta 1- and beta 2- subtype distribution were examined in right ventricular biopsies taken from 100 patients 1-60 months after orthotopic cardiac transplantation and from eight prospective transplant donor hearts serving as controls. The patients were classified into eight groups depending on the time after transplantation that the biopsies were taken: 1-3 (n = 15), 4-7 (n = 15), 8-11 (n = 6), 12 (n = 15), 24 (n = 15), 36 (n = 12), 48 (n = 12) and 60 months (n = 10). The non-selective beta-adrenoceptor antagonist (-)-[125I]-iodocyanopindolol (ICYP) was used as a radioligand to assess total beta-adrenoceptor density. The beta 1- and beta 2-subtype distribution was determined with a beta 1-adrenoceptor saturating concentration of the selective beta 1-adrenoceptor antagonist CGP 20712A (300 nmol/l). In transplant donor hearts the total beta-adrenoceptor density was found to be 70.8 +/- 7.1 fmol/mg protein including a beta 1:beta 2-adrenoceptor ratio of about 80:20%. Until 36 months after cardiac transplantation the total number of beta-adrenoceptors showed no significant change. A slight but insignificant decrease was observed after 48 (16.2%) and 60 (21.2%) months. In contrast, from 12 to 60 months after cardiac transplantation the beta 1:beta 2-adrenoceptor ratio was shifted significantly (66:33% to 61:39%) as compared with transplant donor hearts which was due to an increase in beta 2- and a decrease in beta 1-adrenoceptor number. Thus, the surgically denervated, transplanted human heart exhibits a beta 2-adrenoceptor up-regulation during long-term follow-up. It is suggested that this up-regulation of the beta 2-adrenoceptor subtype could be owing to an increased importance of circulating catecholamines in modulating positive chronotropic and inotropic effects.