Dithiotreitol specifically inhibits metabotropic responses of glutamate and depolarizing agents in rat brain synaptoneurosomes.

Dithiotreitol (DTT), a sulfhydryl reducing agent inhibits in a dose-dependent manner the inositol phosphates (IPs) accumulation responses evoked by glutamate and potassium without affecting that of carbachol in rat forebrain synaptoneurosomes. Furthermore, DTT neither provokes a depolarization of the membrane, nor increases the internal calcium concentration. Depolarization and internal calcium rise are known to stimulate IPs production. Moreover, DTT does not modify the depolarizing effect and the calcium rise elicited by glutamate and potassium. In addition, the antioxidant compounds 2-aminoethylisothiouronium bromide (AET) and ascorbic acid have no effect on the basal and stimulated IPs accumulation. Thus, it is concluded that: (1) two distinct transduction pathways exist, one stimulated by glutamate and depolarizing agents and the other one by cholinergic agonists; (2) DTT produces its inhibition by reducing disulfide bridges likely at the level of proteins of the phosphoinositide transduction mechanism.


Changes in content of neuroactive amino acids and acetylcholine in the rat hippocampus following transient forebrain ischemia.

Changes in content of selected neuroactive amino acids [glutamic acid, aspartic acid, glycine, gamma-aminobutyric acid (GABA) and taurine] and acetylcholine (ACh) in the rat hippocampus following transient forebrain ischemia were investigated using male Wistar rats. Rats were allowed to survive for 1 or 5 days following 10 or 20 min of 4-vessel occlusion, and killed by a focused microwave irradiation. A significant reduction in all neuroactive amino acids examined except GABA was noted in the hippocampus on the fifth day. One day after the 4-vessel occlusion for 10 min, no significant effect on the content of neuroactive amino acids in all brain areas was observed. gamma-Aminobutyric acid content in the hippocampus was only significantly reduced on the fifth day after the occlusion for 20 min. Similarly, a significant decrease in ACh content in the hippocampus was observed on the fifth day after the occlusion for 20 min. Considering the data that a significant loss of neuronal cells in the hippocampus (delayed neuronal death) was detected only 5 days after the 4-vessel occlusion, it can be said that the alterations in the hippocampus of neuroactive amino acids such as glutamic acid, aspartic acid, glycine and taurine are more sensitive than those in GABA and ACh against cerebral ischemia. A possible correlation of these changes of neuroactive amino acids in the occurrence of delayed neuronal death in the hippocampus is also suggested.


Involvement of Ca2+ entry and inositol trisphosphate-induced internal Ca2+ mobilization in muscarinic receptor-mediated catecholamine release in dog adrenal chromaffin cells.

Catecholamine (CA) release from adrenal medulla evoked by muscarinic receptor stimulation has been studied using isolated perfused adrenal gland and cultured chromaffin cells from dogs. Muscarine and oxotremorine (1-100 microM), and bethanechol (0.1-1 mM) dose-dependently stimulated CA release. Muscarine-evoked CA release was antagonized with M1-antagonist, pirenzepine and, to a lesser extent, with atropine; and was reduced either by removal of extracellular Ca2+ or treatment with Ca2+ channel blockers. Muscarine caused an increase of 45Ca uptake and 22Na uptake. Tetrodotoxin (TTX) did not affect muscarine-evoked increase of 22Na uptake and CA release. Under the absence of extracellular Ca2+, muscarine stimulated a 45Ca efflux. Muscarine-induced CA release was attenuated by treating the cells with 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate-HCl (TMB-8) which blocks Ca2+ release from the intracellular store. A phospholipase C inhibitor, neomycin, markedly reduced muscarine-induced CA release but not nicotine- and high K(+)-evoked release. Cinnarizine, a Ca2+ channel blocker, attenuated muscarine-evoked but not caffeine-induced CA release and 45Ca efflux in the absence of extracellular Ca2+. Muscarine caused an increase in intracellular free Ca2+ concentration ([Ca2+]i) in the presence of extracellular Ca2+. It caused a similar increase, but to a lesser extent, in the absence of extracellular Ca2+. The increase of [Ca2+]i induced by muscarine without extracellular Ca2+ was reduced by neomycin and cinnarizine. Polymixin B and retinal, which reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CA release, had little effect on muscarine-induced CA release. Muscarine increased cellular Ins(1,4,5)P3 production, and atropine inhibited this increase.(ABSTRACT TRUNCATED AT 250 WORDS)


The search for Y chromosome polymorphism is extended to negroids.

DNA samples of Negroid and Caucasoid origin were used to screen for Y-specific RFLPs. A total of 7 Y chromosome probes and 13 restriction enzyme digests were used to examine a conservative estimate of 20000bp. No new Y-specific polymorphisms were revealed. The paucity of polymorphism on the Y appears to be unrelated to possible bottleneck effects during raciation.


Chromosome 4q DNA rearrangements associated with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder which maps to chromosome 4qter, distal to the D4S139 locus. The cosmid clone 13E, isolated in a search for homeobox genes, was subsequently mapped to 4q35, also distal to D4S139. A subclone, p13E-11, detects in normal individuals a polymorphic EcoRI fragment usually larger than 28 kilobases (kb). Surprisingly, using the same probe we detected de novo DNA rearrangements, characterized by shorter EcoRI fragments (14-28 kb), in 5 out of 6 new FSHD cases. In 10 Dutch families analysed, a specific shorter fragment between 14-28 kb cosegregates with FSHD. Both observations indicate that FSHD is caused by independent de novo DNA rearrangements in the EcoRI fragment detected by p13E-11.


[Interrelation of plasma volume, fluid metabolism and neurohormonal activation after ultrafiltration in congestive heart failure].

Ultrafiltration improves the clinical condition of patients with congestive heart failure (CHF) through a reduction of excessive body water. We investigated the relationships among intra and extravascular fluids, hemodynamics and neurohumoral pattern following plasma water subtraction. In 55 patients with CHF (35 in NYHA class IV, Group A, and 20 in NYHA class II-III, Group B), removal of 3242 +/- 201 ml and 1741 +/- 119 ml of plasma water acutely reduced plasma volume (calculated from hematocrit changes) by -20.7% and -12.9% in Group A and in Group B, respectively. Plasma volume returned to baseline values within 48 hours. Body weight and ventricular filling pressures also lowered and remained so for 2 days. After ultrafiltration urinary output increased and norepinephrine, renin activity and aldosterone plasma levels decreased in Group A, while a fall of diuresis and a rapid rise of plasma levels of the 3 hormones were observed in Group B. Two days after ultrafiltration the persistence of reduced body weight with recovery of plasma volume indicates a shift of fluid from the extravascular to the intravascular compartment. The different behaviour of hemodynamics, urinary output and neurohumoral pattern changes observed in the 2 groups after ultrafiltration, suggest that in severe heart failure (Group A) the physiological responses to intravascular volume depletion are unsettled while are preserved in less severe stages of the disease (Group B).


[Effect of alpha-adrenergic receptor blockade on peripheral vasoconstriction induced by the cold pressor test. Evidence for functional integrity of alpha 1 and alpha 2 adrenergic receptors in patients with congestive heart failure].

Central sympathetic stimulation results in direct vascular vasoconstriction mediated by activation of alpha-adrenergic receptors. However, it is unknown whether this vasoconstriction is mediated by alpha 1 or alpha 2-adrenoceptor subtypes. In patients with congestive heart failure (CHF), both circulating and neuronally released catecholamines produces vasoconstriction via alpha-adrenergic stimulation. This vasoconstriction produces detrimental hemodynamic effect in CHF patients since it increases right and left ventricular filling pressures and pulmonary and systemic vascular resistances. While the myocardial alpha 1-adrenoceptors seems not to be down-regulated in heart failure, the functional integrity of vascular alpha 1 and alpha 2-adrenoceptors in CHF remains to be elucidated. Accordingly, the present study was designed to assess whether the limb vascular response to alpha 1 or alpha 2-adrenoceptor stimulation is impaired in patients with CHF. We studied 25 control subjects and 19 patients with CHF due to primary dilated cardiomyopathy. Forearm blood flow was measured by venous occlusion plethysmography. Sympathetic stimulation was produced by cold. The intraarterial pump-infusions of BHT 933 (a selective alpha 2-adrenoceptors agonist agent, 0.1, 1, 10 micrograms/kg/min for 5 min) produced the same peripheral vasoconstriction in CHF and in control subjects: 32 +/- 23.9%, 47.3 +/- 20% and 55 +/- 26.1% respectively at I, II, and III dose in CHF and 28.7 +/- 38.6%, 36 +/- 14.5%, and 57 +/- 13.8% respectively at I, II, and III dose in control subjects. The dose response to BHT 933 was not different in the 2 groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Down-regulation of ras and myc expression associated with mdr-1 overexpression in adriamycin-resistant tumor cells.

The murine melanoma tumor cells, B16-BL6, are a recognized model for experimental and spontaneous metastasis. B16-BL6 cells express a lower metastatic phenotype upon acquisition of resistance to adriamycin. Using this novel system, the role of ras, c-myc, and multidrug-resistant gene (mdr1) expression in the metastatic and drug-resistant phenotype was examined. The metastatic cells expressed a high level of c-Ki-ras and c-myc, whereas down-regulation of both proto-oncogenes was observed in the adriamycin-resistant cells. The mdr1 gene, which encodes P-glycoprotein of the drug-resistant superfamily gene, was overexpressed in drug-resistant melanoma cells. These results suggest that altered expression of genes that regulate cellular proliferation and growth may be a determinant of metastasis and drug sensitivity of tumor cells.


[Observation on the early development of inoculated Brugia malayi microfilariae in mosquitoes].

This paper reports the early development and variation of inoculated Brugia malayi microfilariae in 3 groups of mosquitoes, i. e. young Anopheles sinensis, young Culex quinquefasciatus and old Cx. quinquefasciatus. Significant differences were observed among these groups. Sixty hours after inoculation, the percentage of normally developing filarial larvae in young An. sinensis was 72.3%; the percentage of melanized microfilariae in young Cx. quinquefasciatus was 75.4% and the percentage of filarial larvae which developed abnormally or remained in diapause status in old Cx. quinquefasciatus was 67.1%, each being much higher than that in the other groups. It was suggested that the immune competence of the mosquitoes had influence upon the filarial development. The intensity of the immune response not only varied with the species, but also diminished with the aging of mosquitoes.


Management of variceal haemorrhage.

Variceal bleeding is the most important complication of portal hypertension. Mortality due to the first variceal bleeding is very high (50%) and of those surviving a variceal bleeding episode, up to 80% may rebleed. Proper management of the acute variceal bleeding episode, the prevention of rebleeding and primary prophylaxis for variceal haemorrhage are therefore mandatory in order to improve the morbidity and mortality of cirrhotic patients with variceal bleeding. Injection sclerotherapy would be the treatment of choice for acute variceal bleeding. Drug treatment in the form of either a combined vasopressin-nitroglycerin regimen or somatostatin may be used as an alternative. Patients not responding to these treatments should be referred for surgery. For the prevention of variceal rebleeding, non-selective beta-blockers should be tried first, reserving long-term injection sclerotherapy for patients with contraindications or intolerance to beta-blockers or in whom beta-blocker therapy has failed. Surgical rescue in the form of either shunt surgery or lever transplantation should be considered if either treatment fails. A new technique, transjugular intrahepatic portosystemic stent-shunt (TIPSS) may replace shunt surgery in the future. Beta-blockers is the treatment of choice for primary prophylaxis of variceal haemorrhage and has a role in preventing acute and chronic bleeding from congestive gastropathy. However, the above sequential approach from the least invasive to the more invasive therapeutic options may not be appropriate for all cirrhotic patients with variceal bleeding.


[How do cancers resist to chemotherapy?].

Resistance is often defined as a lack of therapeutic response. Cellular resistance involves a decrease in intracellular levels of the antitumor agent due to a variety of mechanisms. These mechanisms are active in tumors with initial resistance as well as in those which respond initially but fail to be completely destroyed by chemotherapy. Although acquired forms of resistance seem to be the result of selection, some studies suggest that antitumor agents may induce resistance. Four main mechanisms of resistance are currently being investigated: 1) multidrug resistance, involving expression of a membrane P-glycoprotein, responsible for resistance to hydrophobic cationic agents; 2) detoxification of hydrophilic agents by the enzyme glutathione-S-transferase; 3) increased production of enzymes targeted by antimetabolites; 4) mutation or decreased synthesis of topoisomerases I and II which are the targets of very recent antitumor agents. New data were presented at the 1992 symposium of the American Association for Cancer Research; expression of P-glycoprotein is controlled by the mutant protein P53, the oncogene ras and differentiation agents. Physiological effects of this molecule are related to the chloride pump. Bone marrow stem cells from transgenic mice obtained by transfection of the gene MDR1 in germ cells exhibit resistance. Many agents can reverse P-glycoprotein-related resistance. Results from three phase I trials with Cyclosporin A as reversion agent were reported. It is essential to conduct clinical trials in order to assess the true value of these new data which hold promise for improving the performance of antitumor agents.


[Cancer of the breast].

Breast cancer remains a key concern for oncologists. The possibility of tamoxifen treatment to prevent breast cancer in high-risk women was one of the central topics discussed for the 1992 ASCO edition. The rationale for the studies being developed in the US and Europe rests on experimental data and results of adjuvant hormone therapy trials. Decreased risks of cancer in the opposite breast, of cardiovascular disease, and of osteoporosis are effects that make tamoxifen extremely attractive for breast cancer prevention trials in postmenopausal women. In premenopausal women, however, preventive tamoxifen should be viewed with special caution because increased incidence of second cancers have been reported, although with dosages higher than those suggested for preventive therapy, and also because of difficulties with defining familial forms. The value of anthracyclines for adjuvant therapy has been demonstrated by several studies. Furthermore, a dose-response relationship has been reported with anthracyclines used as adjuvant therapy or in metastatic disease. New dose-limiting toxic effects, including thrombocytopenia and mucitis, develop when dosages are increased, with concomitant rG-CSF therapy. In patients with metastases, taxol seems to be a promising drug. Ongoing phase I trials seek to determine the optimal dosage and administration modalities for the taxol-doxorubicin combination.


Necrotising arteritis in amoebic colitis.

Massive intestinal haemorrhage rarely occurs in amoebic colitis. We report a case of caecal amoebic ulcer in a 61 year old diabetic male who presented with massive lower intestinal haemorrhage requiring blood transfusion and emergency surgical intervention. Histologically, trophozoites of Entamoeba histolytica were seen invading the wall of the submucosal arteries, causing necrotising arteritis. Rupture of a necrosed artery probably caused massive haemorrhage.


[Advances in lung cancer].

Sessions about lung cancer of the 28th annual meeting of ASCO approached especially non small cell lung cancer (NSCLC). Undoubtedly the outstanding facts for this pathology was first the acknowledgment of the importance of a correct staging system, in particular in stage IIIA for which resection is a prognostic factor different for tumors T3N0, T3N1 as for N2. Two important topics have been discussed on therapy: stage IIIA treatment, in particular neoadjuvant therapy before surgery, and new drugs like Navelbine, taxol, and campthotecine which suggest the possibility of improvements for chemotherapy of NSCLC in the next few years. For small cell lung cancer (SCLC), different trials were designed to improve dose intensity with growth factors, fractionation of therapy, autologous bone marrow transplantation.


Endocrine ophthalmopathy: a re-evaluation of the association with thyroid autoantibodies.

Autoantibodies to the three major thyroid autoantigens, the TSH-receptor (TSH-R), thyroid peroxidase (TPO) and thyroglobulin (TG), have been investigated in 63 Graves' patients with severe endocrine ophthalmopathy. In agreement with other studies, TSH-R antibodies were detectable in 88% of patients and dominated the autoantibody spectrum. TPO antibodies were detectable in 60% of patients and TG antibodies in only 25% of patients. The prevalences, as well as the amounts, of all three thyroid autoantibodies were not significantly different from the values in 51 Graves' patients without clinically significant ophthalmopathy. However, in the subgroup of patients with TG antibodies, the ophthalmopathy patients displayed a shift towards IgG4 TG antibodies. Furthermore, in the same TG antibody positive subgroup, the amount of TSH-R antibody was significantly higher in the ophthalmopathy patients than in Graves' patients without ophthalmopathy. These qualitative differences in thyroid autoantibodies in patients with and without ophthalmopathy raise the possibility that further qualitative differences, such as thyroid autoantibody epitopes, may exist in patients with ophthalmopathy. Our observations, combined with recent evidence for the presence of TSH-R specific mRNA in retro-orbital tissue, suggest that it may be premature to dismiss the potential pathogenetic or diagnostic value of thyroid autoantibodies, particularly TSH-R antibodies, in Graves' ophthalmopathy.


HLA-DR/DQ gene variation in nongoitrous autoimmune thyroiditis at the serological and molecular level.

The etiology of autoimmune diseases is multifactorial with genetic factors being an important prerequisite. There are two clinical manifestations of autoimmune thyroiditis: the goitrous form (Hashimoto's thyroiditis) and the atrophic variant, which is characterized by hypothyroidism (primary myxoedema). Different genetic markers were assumed to be predisposing factors for the distinct clinical presentation. In the present study, we determined HLA A,B,C,DR,DQ alloantigens serologically and HLA-DQ by gene analysis in patients with nongoitrous autoimmune thyroiditis and randomly chosen controls. To verify the exact classifications, thyroid volume (median 5.85 ml) was measured by ultrasonography. HLA-DR5 was found in 16 of 36 (44%) patients with nongoitrous autoimmune thyroiditis and in only 26 of 175 controls (15%) (Pc = 0.0018). There was a tendency towards a lower frequency of HLA-DR7 with 6% positivity in patients vs. 29% in controls (Pc = 0.052). Regarding HLA-DQ, DQ7 was found in 17 of 35 patients (48%) vs. 21 of 98 controls (21%) (Pc = 0.028) (relative risk 3.5). No other association was found with HLA-A,B,C and HLA-DR and -DQ. Our data indicate that the genetic susceptibility to autoimmune nongoitrous thyroiditis is closely associated to HLA-DR5 and DQ7 and not distinct from goitrous disease. We conclude that factors other than genetic ones explain the different immunological and clinical manifestation of chronic lymphocytic thyroiditis.


Polymorphism study of TCR alpha and gamma genes in insulin dependent diabetes mellitus (IDDM) multiplex families.

T-cell receptor (TCR) alpha and gamma genes polymorphisms were analysed by Restriction Fragment Length Polymorphism (RFLP) in 10 Insulin Dependent Diabetes Mellitus (IDDM) multiplex families. TCR alpha and gamma alleles distribution does not significantly differ between affected and non affected children. Furthermore there was no excess of C alpha or V gamma allele sharing in affected sib pairs. Therefore the T-cell receptor alpha and gamma chain alleles studied do not seem to affect IDDM susceptibility per se.


Antiamnesic properties of some neuropeptides and the involvement of neurotransmitters in the rats.

The effects of certain neuropeptides on electroconvulsive (ECS) shock-induced amnesia were studied in rats. The ECS was applied immediately after the learning a one-trial passive avoidance paradigm. The peptides--rat atrial natriuretic peptide (rANP 1-28, ANP), porcine brain natriuretic peptide (pBNP 1-32, BNP), rat calcitonin gene-related peptide (CGRP) and bombesin--were injected into the lateral brain ventricle 30 min after the ECS. In order to study the possible role of neurotransmitters in mediating the action of the peptides on amnesia, the animals were treated immediately after the ECS with, doses which themselves did not modify either the learning itself or the amnesic action of the ECS. All the above peptides attenuated the ECS-induced amnesia, but the transmitters involved in these actions differed. The anticonvulsive action of ANP was prevented by haloperidol (10 micrograms/kg i. p.), propranolol (10 mg/kg i. p.) and atropine (2 mg/kg i. p.). Phenoxybenzamine (2.0 mg/kg i. p.), bicuculline (1 mg/kg i. p.), methysergide (5 mg/kg i. p.) and naloxone (0.3 mg/kg i. p.) had no effect. Besides alpha-adrenergic and cholinergic receptor blockers the beta-adrenergic blocker propranolol was also effective in preventing the antiamnesic action of the BNP. As concerns the action of bombesin, only haloperidol was effective. Alpha- and beta-adrenergic and cholinergic receptor blockers and opiates are involved in the antiamnesic properties of CGRP. The results showed that, despite the fact that the studied, peptides attenuated the ECS-induced amnesia, different transmitters are involved in their action.


Purification and properties of the cellular prion protein from Syrian hamster brain.

The cellular prion protein (PrPC) is encoded by a chromosomal gene, and its scrapie isoform (PrPSc) features in all aspects of the prion diseases. Prior to the studies reported here, purification of PrPC has only been accomplished using immunoaffinity chromatography yielding small amounts of protein. Brain homogenates contain two PrPC forms designated PrPC-I and -II. These proteins were purified from a microsomal fraction by detergent extraction and separated by immobilized Cu2+ ion affinity chromatography. PrPC-II appears to be generated from PrPC-I by limited proteolysis of the N-terminus. Fractions enriched for PrPC-I were purified further by cation-exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Greater than 90% of the final product migrated as a broad band of M(r) 33-35 kDa as judged by silver staining after SDS-PAGE. Digestion of PrPC-I with peptide-N-glycosidase (PNGase) compressed the band and shifted its mobility giving an M(r) of 27 kDa. The protocol described should be amenable to large-scale preparation of PrPC, enabling physical comparisons of PrPC and PrPSc.


[Correlation of proliferation of pancreatic cancer cells with both saturation index and cell membrane insulin receptors].

Exogenous epidermal growth factor (or somatostatin) can stimulate (or inhibit) proliferation of pancreatic cancer cells, with a corresponding decrease (or increase) of the saturation index of the cell membrane and up-regulation (or down-regulation) of insulin receptor expression. By measuring the saturation index and insulin receptor numbers on the cell membrane it may be possible to evaluate the curative effects of treatment on pancreatic cancer at an early time.


X-ray structure of Glu 53 human lysozyme.

The three-dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 A resolution by X-ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild-type HL. The root mean square difference for the superposition of equivalent C alpha atoms is 0.141 A. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild-type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 A between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild-type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction.


[Re-evaluation of ultrasonographic localization in primary hyperparathyroidism (report of 55 cases)].

In 1983-1991, 55 cases of primary hyperparathyroidism confirmed by pathology and surgery were screened by ultrasonography. Its frequency was 3.5 or 7.5 MHz. The ultrasonographic results showed as follows. The appearance rate of the foci in the normal position of the parathyroid was 95%, including adenoma and hyperplasia; its size of pathology was bigger than that determined by US; the sensitivity of US was 55.3%, specificity 96.2%, accuracy 81.8%, positive predictive value 81.5%, and negative predictive value 86.5%. The results improving its level of ultrasonographic diagnosis were proved as follows. (1) knowing the anatomy of parathyroids, and preventing from false positive and false negative; (2) differentiating from primary and secondary hyperparathyroidism; (3) grasping the features of multiple endocrine neoplasia; (4) careful and repeated examination was key to early and correct diagnosis.


[Evaluation of treatments with beta-blockaders after myocardial infarction].

Beta-blockers have been used after myocardial infarction since 1965: however, it was not until the beginning of the 80s that the large multicentre clinical trials published results showing clearly their beneficial effects and leading to their widespread usage: betablockers significantly reduce the medium term (1 to 3 years) risk of death (-22 to -24%), especially sudden death (-32%) and the frequency of recurrent infarction (-27%). The cardio-protection so obtained is multifactorial, essentially related to their antiarrhythmic, antiischemic and antihypertensive effects. It has been established that beta-blockade should be instituted as soon as possible in the hours following the infarct (intravenous relayed by oral administration) and may be useful associated with aspirin. Although the large scale clinical trials did not determine the optimal dosage or the duration for which treatment should be administered, they did show that the groups of high risk patients were those to benefit the most from this therapy. Beta-blockers are usually well tolerated. However, it must be pointed out that 18% of patients were excluded from the two principal trials (only 25 to 30% of infarct patients were included) because of contraindications to beta-blockers and that 25 to 30% of the patients initially included had to interrupt the treatment because of side effects.


[Ventricular anti-arrhythmic treatments during postinfarction].

Antiarrhythmic agents may be prescribed in the post-infarction period either as systematic therapy to prevent sudden death or as prophylactic treatment against recurrences of documented life-threatening arrhythmias. Systematic therapy or even the treatment of symptomatic ventricular extrasystoles by Class IC anti-arrhythmics is associated with an increased risk, especially in patients with a low risk of sudden death at the outset. Betablockers are effective on symptoms: they are not always effective on the arrhythmia but at least they do not aggravate the mortality of these patients. However, for high risk patients with post-infarction left ventricular dysfunction, betablockers are the only drugs which have a proven efficacy: they should therefore be prescribed, especially those whose efficacy has been demonstrated, at the same dosages as those used in clinical trials. Preventive treatment of sustained ventricular tachycardia should be chosen with respect to the patient's hemodynamic status. When the ejection fraction is under 40%, amiodarone and betablockers are the drugs of first intention, with controls of their efficacy by the inability to induce or the slowing of the tachycardia rhythm during endocavitary electrophysiological studies.


[Enzymatic and molecular studies in a case of hepato-erythropoietic porphyria. Homozygote form of type familial cutaneous porphyria].

Porphyrias are either hepatic or erythroid, depending on the principal site of the specific enzymatic defect. Homozygous uroporphyrinogen decarboxylase deficiency, known as hepato-erythropoietic porphyria (HEP), can involve several mutations. A young man, aged 20 years, had gradually developed photosensitivity since the age of 1 year, leading to hypertrichosis and sclerodermoid changes in sun-exposed areas of skin. He displayed high urinary uroporphyrin and 7-carboxylic porphyrins, and elevated fecal and red blood cell iso-coproporphyrin and coproporphyrin. Erythrocyte uroporphyrinogen decarboxylase activity of the patient was reduced to 18% of normal control values, while those of his grandmother and his half-brother were 62-65% of normal. Amplification of the genomic DNA by PCR and hybridization with allele-specific oligonucleotides (ASOs) demonstrated the presence of a Gly 281-->Glu mutation in the patient and in his grandmother and half-brother. Enzymatic studies and details of the familial lineage are important for precisely classifying this type of porphyria. Molecular biology studies are necessary before considering any future gene therapy.


Na+/K(+)-ATPase regulation by neurotransmitters.

A long period of experimental work has led to the conclusion that Na+/K(+)-ATPase is the enzymatic version of the Na+/K+ pump. This enzymatic system is in charge of various important cell functions. Among them cationic equilibrium and recovering of resting membrane potential in neurons is relevant. A tetrameric ensemble of peptides conform the system known as alpha and beta subunits. The alpha subunit is subdivided in alpha 1, alpha 2 and alpha 3, according to different location and properties. Regulatory factors intrinsic to the Na+/K(+)-ATPase system are: ATP, Na+ and Mg2+ concentrations inside the cell, and K+ outside. The enzyme activity is also regulated by extrinsic factors like some hormones (insulin and thyroxine). Induction of gene expression or post-translational modifications of the preexisting pool of the enzyme are the basic mechanisms of regulation proposed. Other extrinsic factors that seem to regulate the enzyme activity are some neurotransmitters. Among them the most extensively studied are catecholamines, mainly norepinephrine (NE) and lately serotonin (5-HT). The mechanism suggested for NE activation of the enzyme seems to involve specific receptors or a non-specific chelating action related to the catechol group that would relieve the inhibition by divalent cations. Another possibility is that NE removes an endogenous inhibitory factor present in the cytoplasm. The Na+/K(+)-ATPase is activated also by 5-HT. In vivo pharmacological and nutriological manipulations of brain 5-HT are accompanied by parallel responses of Na+/K(+)-ATPase activity. Serotonin agonists do activate the enzyme and antagonists neutralize the activation. In vitro there is a different dose dependent activation, according to the brain region. The mechanism involved seems to implicate a specific receptor system. Serotonin-Na+/K(+)-ATPase interaction in the rat brain is probably of functional relevance because it disappears in amygdaloid kindling. Also it seems to influence the ionic regulation of the pigment transport mechanism in crayfish photoreceptors. In relation to other neurotransmitters, a weak response to histamine was observed with acetylcholine, GABA and glutamic acid, the results were negative.


Quantitative autoradiographic study of somatostatin and neurotensin binding sites in medulla oblongata of SIDS.

Quantitative autoradiography analysis of neurotensin (NT) and somatostatin (SS) binding sites was performed on coronal sections of the medulla oblongata from 2 fetuses, 6 controls and 7 victims of Sudden Infant Death Syndrome (SIDS). Throughout the first postnatal year, mean SS binding site density was similar in controls and SIDS in all structures of the medulla oblongata. The density of neurotensin binding sites was significantly higher in the nucleus of tractus solitarius (NTS) of SIDS than in controls, but there was no significant differences in the other areas of the medulla oblongata. Our findings suggest an immature developmental pattern of increased NT binding sites the NTS of SIDS. This alteration may be related to an abnormal central cardiorespiratory and arousal control which is thought to be present in SIDS.


Na+/K(+)-ATPase as an effector of synaptic transmission.

Evidence is presented in support of the hypothesis that transmitter monoamines can exert their post-synaptic effects by stimulation or inhibition of Na+/K(+)-ATPase in neuronal or glial cell plasma membranes. Stimulation of electrogenic sodium pumping, causing a hyperpolarization with an increase in membrane resistance, could account for the depression of neuronal spontaneous firing and the signal/noise enhancing actions of these amines. Conversely, inhibition of an electrogenic sodium pump in neuronal plasma membranes would lead to depolarization and enhanced excitability.


Time-resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: conformational analysis of intermediates and transition-state complexes.

Single tryptophan-containing mutants of low adenylylation state Escherichia coli glutamine synthetase have been studied by frequency-domain fluorescence spectroscopy in the presence of various substrates and inhibitors. At pH 6.5, the Mn-bound wild-type enzyme (wild type has two tryptophans/subunit) and the mutant enzymes exhibit heterogeneous fluorescence decay kinetics; the individual tryptophans are adequately described by a triple exponential decay scheme. The recovered lifetime values are 5.9 ns, 2.6 ns, and 0.4 ns for Trp-57 and 5.8 ns, 2.3 ns, and 0.4 ns for Trp-158. These values are nearly identical to the previously reported results at pH 7.5 (Atkins, W.M., Stayton, P.S., & Villafranca, J.J., 1991, Biochemistry 30, 3406-3416). In addition, Trp-57 and Trp-158 both exhibit an ATP-induced increase in the relative fraction of the long lifetime component, whereas only Trp-57 is affected by this ligand at pH 7.5. The transition-state analogue L-methionine-(R,S)-sulfoximine (MSOX) causes a dramatic increase in the fractional intensity of the long lifetime component of Trp-158. This ligand has no effect on the W158S mutant protein and causes a small increase in the fractional intensity of the long lifetime component of the W158F mutant protein. Addition of glutamate to the ATP complex, which affords the gamma-glutamylphosphate-ADP complex, results in the presence of new lifetime components at 7, 3.2, and 0.5 ns for Trp-158, but has no effect on Trp-57. Similar results were obtained when ATP was added to the MSOX complex; Trp-57 exhibits heterogeneous fluorescence decay with lifetimes of 7, 3.5, and 0.8 ns. Decay kinetics of Trp-158 are best fit to a nearly homogeneous decay with a lifetime of 5.5 ns in the MSOX-ATP inactivated complex. These results provide a model for the sequence of structural and dynamic changes that take place at the Trp-57 loop and the central loop (Trp-158) during several intermediate stages of catalysis.


Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins.

In vitro experiments employing the soluble proteins from Escherichia coli reveal that about half of them, in their unfolded or partially folded states, but not in their native states, can form stable binary complexes with chaperonin 60 (groEL). These complexes can be isolated by gel filtration chromatography and are efficiently discharged upon the addition of Mg.ATP. Binary complex formation is substantially reduced if chaperonin 60 is presaturated with Rubisco-I, the folding intermediate of Rubisco, but not with native Rubisco. Binary complex formation is also reduced if the transient species that interact with chaperonin 60 are permitted to progress to more stable states. This implies that the structural elements or motifs that are recognized by chaperonin 60 and that are responsible for binary complex formation are only present or accessible in the unfolded states of proteins or in certain intermediates along their respective folding pathways. Given the high-affinity binding that we have observed in the present study and the normal cellular abundance of chaperonin 60, we suspect that the folding of most proteins in E. coli does not occur in free solution spontaneously, but instead takes place while they are associated with molecular chaperones.


Renaturation of citrate synthase: influence of denaturant and folding assistants.

Citrate synthase (CS), which has been denatured in either guanidine hydrochloride (GdnHCl) or urea can be assisted in its renaturation in a variety of ways. The addition of each of the assistants--bovine serum albumin (BSA), oxaloacetate (OAA), and glycerol--promotes renaturation. In combination, the effect of these substances is additive with respect to the yield of folded CS. The report of Buchner et al. (Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F.X., & Kiefhaber, T., 1991, Biochemistry 30, 1586-1591) that refolding of CS is facilitated by the GroE system (an Escherichia coli chaperonin [cpn] that is composed of GroEL [cpn60] and GroES [cpn10]) has been confirmed. However, we observed substantially higher yield of reactivated CS, 82%, and almost no reactivation in the absence of GroES, < 5%, whereas Buchner et al. reported 28% and 16%, respectively. In addition, we find that GroE-assisted refolding is more efficient for CS denatured in GdnHCl than for CS denatured in urea. This result is discussed in light of the known difference in the denatured states generated in GdnHCl and urea. Because GroEL inhibits the BSA/glycerol/OAA-assisted refolding, this system will be useful in future studies on the mechanism of GroE-facilitated refolding.


Theory of chaperonin action: inertial model for enhancement of prokaryotic Rubisco assembly.

We have performed a computational simulation of the aggregation and chaperonin-dependent reconstitution of dimeric prokaryotic ribulose bisphosphate carboxylase/oxygenase (Rubisco), based on the data of P. Goloubinoff et al. (1989, Nature 342, 884-889) and P. V. Viitanen et al. (1990, Biochemistry 29, 5665-5671). The aggregation is simulated by a set of 12 differential equations representing the aggregation of the Rubisco folding intermediate, Rubisco-I, with itself and with aggregates of Rubisco-I, leading up to dodecamers. Four rate constants, applying to forward or reverse steps in the aggregation process, were included. Optimal values for these constants were determined using the ellipsoid algorithm as implemented by one of us (Ecker, J.G. & Kupferschmid, M., 1988, Introduction to Operations Research, Wiley, New York, pp. 315-322). Intensive exploration of simpler aggregation models did not identify an alternative that could simulate the data as well as this one. The activity of the chaperonin in this system was simulated by using this aggregation model, combined with a model similar to that proposed by Goloubinoff et al. (1989). The model assumes that the chaperonin can bind the folding intermediate rapidly, and that the chaperonin complex releases the Rubisco molecule slowly, permitting time for its spontaneous folding while interacting with the chaperonin. This is followed by self-association of the folded Rubisco monomer to yield the active dimeric Rubisco. A modification of the model that simulates temperature effects was also constructed. The most important results we obtained indicate that the chaperonin-dependent reconstitution of Rubisco can be simulated adequately without invoking any catalysis of folding by the chaperonin. In addition, the simulations predict values for the association rate constant of Rubisco-I with the chaperonin, and other variables, that are subject to experimental verification.


Scar formation in the cardiac conduction system of a patient with Takayasu's arteritis.

An autopsied case of Takayasu's arteritis associated with complete atrioventricular (AV) block is described for the first time. The findings of scar formation and diffuse infiltration of lymphocytes into the cardiac conduction system, particularly the AV node, were similar to those in patients with connective tissue diseases or congenital complete heart block. The degree of AV block progressed with aggravation of the disease. These findings suggest that complete AV block may have been induced by acquired autoimmunity involving the cardiac conduction system.


Transglutaminase changes in intestinal mucosa after experimental small bowel resection in the rat.

The low serum transglutaminase found in various intestinal disorders (celiac disease and IBD) suggested to us to study the serum and mucosal transglutaminase behaviour in an experimental model of small intestine resection in rats to reduce cellular mass and induce enterocyte hyperproliferation in the proximal part left in continuity. Transglutaminase activity in the intestinal mucosa was significantly higher in resected rats than in control and sham operated animals from days 4 (121 +/- 10 v basal 94 +/- 3 mU/g protein, p < 0.01) to 10 (165 +/- 37 mU/g protein, p < 0.05) after surgery; no significant difference was observed at days 12 and 15 (110 +/- 15 and 105 +/- 23 respectively). Both serum alkaline phosphatase activity (partly produced in enterocytes) and serum transglutaminase were significantly lower in resected rats at each time-point beginning at day 6 (208 +/- 34 v 557 +/- 125 UI and 1.55 +/- 0.11 v 3.78 +/- 0.70 mU/ml, p < 0.001 respectively). These data suggest an involvement of transglutaminase in enterocyte proliferation and confirm the association between reduced intestinal mass and low levels of the enzyme in serum.


Principles and methods of medicamentous protection of myocardial cellular membranes for prevention of cardiac reperfusion syndrome.

A model of regional transitory coronary insufficiency was employed in experiments on 300 male albino rats under urethan anaesthesia (13.4 mmol/kg b.m.). The periods of myocardial ischaemia lasted 10, 40 and 120 min, followed by reperfusion lasting 40 min. The alteration of cellular membrane apparatus proved to be one of the most important mechanisms responsible for myocardial lesion during transitory coronarogenic ischaemia and for the development of cardiac reperfusion syndrome. The damage to cardiomyocyte membrane and intensity of reperfusion syndrome can be substantially reduced by using the following measures: inhibition of lipoperoxidation by antioxidative preparations, esp. myofedrin and verapamil; suppression of the intensity of phospholipase reactions activated by Ca2+; reduction of calcium ion accumulation in cardiomyocytes by substances lowering intracellular calcium content, esp. by verapamil.


Primary and secondary changes in the brains of suckling Balb/c mice with experimental hemorrhagic fever.

To testify whether primary changes caused by the virus with its related factors and secondary changes caused by hypotension in the brains of patient with epidemic hemorrhagic fever (EHF) could be repeated in the animal model, suckling Balb/c mice were inoculated IP with 100 LD50/0.05 ml of Chen strain of hemorrhagic fever virus. After the onset of the disease, paraformaldehyde and glutaraldehyde were used for fixation by perfusion through left ventricle. Sections stained with HE and MAb against EHF virus by immunocytochemical method (4-step PAP) showed diffuse viral antigen deposition. All brains were diffusely scattered with single cell acidophilic necrosis which are believed to be caused primarily by the virus. 33.3% of the brains also showed symmetrical distribution of cerebral infarct-like necrosis which are believed to be caused secondarily by hypotension. This result supports our previous study on the autopsy of brains of EHF patients.


[Susceptibility of thirty-one species of mosquitoes to Romanomermis yunanensis].

A new species of Mermithidae was found parasitizing the larvae of Culex tritaeniorhynchus and Culex fatigans in Henan, China and then named Romanomermis yunanensis. Thirty-one species of Mosquitoes involving six genera have been tested for susceptibility to R. yunanensis and Culicinae mosquito have consistently been highly susceptible. At a 1:5 ratio of mosquito larvae to nematode juveniles, the parasitisms of Aedes aegypti, Ae. albopictus, Culex fatigans, Culex tritaeniorhynchus, Anopheles sinensts, Psorophora columbiae and Culesta inornata were 98.2%, 98.5%, 98.6%, 97.1%, 0%, 98.8%, and 99.0% respectively. R. yunanensis presented a phenomenon of retarted development in Anopheles maculatus, which remained at the parasitic stage both in the larvae and pupa of An. maculatus. The parasitism of An. dirus was 89.7%, but most (88.8%) parasitizing mermithids underwent melanization and died. The comparative susceptibility of some North American mosquitoes to R. yunanesis and R. culicivorax at a 1:5 infective ratio showed that both percentage infection and intensity of infection statistically supported the generalization that, under laboratory conditions, R. yunanesis is a more vigorous and aggressive parasite of Culicinae.


Effects of casein and soy-protein on alpha-linolenic acid metabolism in rats.

In order to study the effects of different proteins on alpha-linolenic acid (alpha-LnA) metabolism, rats were given the diet added respectively with milk casein and soy-protein isolate (SPI) as sources of proteins and perilla oil as a source of lipid. The results obtained are as follows. The ratio of (C20:3 + C20:4)/C18:2 in liver microsomal PL, liver PE fraction, and kidney PE and PC fractions was significantly lowered by the SPI treatment when compared to the casein treatment, similarly to the already established results. In the liver microsomal PL and PE and PC fractions of liver and kidney in rats treated with SPI, there was also observed a significant decrease or a decrease tendency in the (C20:4 + C20:5)/C18:3 ratio. A similar tendency was again shown in the sigma (n-3)M/C18:3 ratio indicating metabolic conversion from C18:3(n-3) to C22:6. On the other hand, contrary to the ratios of (C20:3 + C20:4)/C18:2, sigma (n-3)M/C18:3, and (C20:3 + C20:5)/C18:3, the (C22:5 + C22:6)/C20:5 ratio which is the parameter for metabolic conversion of C20:5(n-3) was elevated in the PE and PC fraction of liver, heart and kidney in the SPI group compared to the casein group. Then, further analysis of the metabolic process from C20:5 to C22:6 showed that the C22:5/C20:5 ratio increased while the C22:6/C22:5 ratio decreased in the SPI group compared to the casein group. Based on these results, it is assumed that the metabolic process from C18:3(n-3) to C20:5(n-3) and from C22:5 to C22:6 is affected by SPI but that the elongation process from C20:5(n-3) to C22:5(n-3), on the contrary, is rather accelerated by SPI.


Effects of thienodiazepine derivatives, etizolam and clotiazepam on the appearance of Fm theta.

The effects of new thienodiazepine anxiolytics, etizolam and clotiazepam, on the appearance of frontal midline theta activity (Fm theta) were studied by the double-blind crossover method. The results were as follows; 1) Both clotiazepam and placebo tended to increase the Fm theta appearance in all subjects, but etizolam showed no such tendency. 2) Clotiazepam significantly increased the Fm theta appearance as compared with placebo in subjects with a high neurotic tendency (N-scale of MPI above 19). 3) Clotiazepam tended to increase the Fm theta appearance as compared with placebo and etizolam in subjects with a high anxiety level (MAS score above 14). 4) Apparently more subjects complained of drowsiness after the administration of etizolam than after clotiazepam or placebo.


(-)Deprenyl (selegiline) is devoid of amphetamine-like behavioural effects in rats.

Central effects of high dose (-)deprenyl (50-100 mg/kg sc) was compared to that of (+)deprenyl and (+/-)amphetamine (AM). (+)Deprenyl and AM induced stereotyped behaviour, increased spontaneous motility in smaller and decreased it in higher doses, inhibited escape behaviour and shuttle-box avoidance. (-)Deprenyl failed to possess any of these effects, providing further evidence that AM-type metabolites have no share in the action of (-)deprenyl.


Regulation of protein kinase C after stimulation of alpha 1-adrenoceptors in rat hippocampus.

Endogenous inhibitor of protein kinases (type II inhibitor, GABA-modulin) blocks the phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) as a competitive inhibitor of substrate proteins when histone is used as a substrate. Moreover, type II inhibitor blocks the phosphorylation of endogenous membrane proteins by PKC. Stimulation of alpha 1-adrenoceptors induced rapid redistribution of PKC from cytosol to membrane fraction which lasted at least 3 h, accompanied by rapid and short-lasting translocation of type II inhibitor from membrane to cytosol fraction. The cytosol content of type II inhibitor reached maximal level 10 and 20 min and became normal again 40 min after i.p. administration of methoxamine. The above actions of methoxamine were completely blocked by pretreatment with prazosin. It seems that short-lasting redistribution of type II inhibitor from membrane to cytosol fraction allows the effective phosphorylation of membrane proteins by PKC after stimulation of alpha 1-adrenoceptors.


Cross reactivity among the ligands of the 7 helix family.

"Specific" drugs for receptors of the 7 helix family inhibited in high doses other receptors of the same family, as well. Atropine inhibited [3-H]-clonidine binding, beta-adrenergic drugs: propranolol, betoxalol, ICI 118.551 inhibited labelled QNB binding to all the 3 main subtypes of muscarinic receptors. Authors propose the existence of common features in the ligand binding sites of the receptors of 7 helix family.


Anxiolytic profile of girisopam and GYKI 52,322 (EGIS 6775). Comparison with chlordiazepoxide and buspirone.

The anxiolytic action of two 2,3-benzodiazepines: girisopam: GYKI 51,189 (EGIS 5810): (1-(3-chlorophenyl)-4-methyl-7,8-dimethoxy-5H-2,3-benzodiazepine), and GYKI 52,322 (EGIS 6775): (1-(4-aminophenyl)-4-methyl-7,8-dimethoxy-5H-2,3-benzodiazepine) was investigated in comparison to chlordiazepoxide and buspirone using three different animal models of anxiety: the lick conflict, the elevated plus maze and the open field methods in rats. Both 2,3-benzodiazepines exerted anxiolytic effect in all three tests used, however their pharmacological profile differs considerably from that of either chlordiazepoxide or buspirone. Using the animal models mentioned above the order of potency was GYKI 52,322 (EGIS 6775) > chlordiazepoxide > girisopam > buspirone.


Inhibition of hippocampal field potentials by GYKI 52466 in vitro and in vivo.

GYKI 52466 is a specific antagonist of the neuronal excitation mediated by the non-NMDA type excitatory amino acid receptors, at several sites in the central nervous system. The experiments presented here show that the drug has a dose-dependent, slowly developing, long-lasting and reversible inhibitory action on the field potentials recorded from the CA1 region of the rat hippocampus, in vitro. Its action is similar to that of the well-known non-NMDA receptor blocker, CNQX. When the stimulus intensity-dependence of the population spikes was investigated, both drugs shifted the input-output curves in a parallel manner, while the maximum responses were only slightly depressed at the doses applied. With i.v. application, GYKI 52466 also inhibited the hippocampal field potentials recorded from the CA1 region of anesthetized rats dose-dependently. The inhibition was relatively weak compared to the effect found in earlier studies in the spinal cord, by the same doses. Four mg/kg i.v., a doses which is able to block spinal reflexes completely, caused an only about 20% depression of the recorded responses in the hippocampal CA1 area.


Gln-41 is intermolecularly cross-linked to Lys-113 in F-actin by N-(4-azidobenzoyl)-putrescine.

The bifunctional reagent N-(4-azidobenzoyl)-putrescine was synthesized and covalently bound to rabbit skeletal muscle actin. The incorporation was mediated by guinea pig liver transglutaminase under conditions similar to those described by Takashi (1988, Biochemistry 27, 938-943); up to 0.5 M/M were incorporated into G-actin, whereas F-actin was refractory to incorporation. Peptide fractionation showed that at least 90% of the label was bound to Gln-41. The labeled G-actin was polymerized, and irradiation of the F-actin led to covalent intermolecular cross-linking. A cross-linked peptide complex was isolated from a tryptic digest of the cross-linked actin in which digestion was limited to arginine; sequence analysis as well as mass spectrometry indicated that the linked peptides contained residues 40-62 and residues 96-116, and that the actual cross-link was between Gln-41 and Lys-113. Thus the gamma-carboxyl group of Gln-41 must be within 10.7 A of the side chain (probably the amino group) of Lys-113 in an adjacent actin monomer. In the atomic model for F-actin proposed by Holmes et al. (1990, Nature 347, 44-49), the alpha-carbons of these residues in adjacent monomers along the two-start helices are sufficiently close to permit cross-linking of their side chains, and, pending atomic resolution of the side chains, the results presented here seem to support the proposed model.


Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins.

Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb Hb S, beta 6 Glu-->Val) have been obtained from X-ray and electron microscopic studies. Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers. Site-directed mutagenesis with expression of the Hb S beta subunits in Escherichia coli provides the experimental tools to test these models. For Hb S, the beta 6 Val residue is intimately involved in a specific lateral contact, at the donor site, that interacts with the acceptor site of an adjacent molecule composed predominantly of the hydrophobic residues Phe 85 and Leu 88. Comparing natural and artificial mutants indicates that the solubility of deoxyHb decreases in relation to the surface hydrophobicity of the residue at the beta 6 position with Ile > Val > Ala. We also tested the role of the stereospecific adjustment between the donor and acceptor sites by substituting Trp for Glu at the beta 6 location. Among these hydrophobic substitutions and under our experimental conditions, only Val and Ile were observed to induce polymer formation. The interactions for the Ala mutant are too weak whereas a Trp residue inhibits aggregation through steric hindrance at the acceptor site of the lateral contact. Increasing the hydrophobicity at the axial contact between tetramers of the same strand also contributes to the stability of the double strand. This is demonstrated by associating the beta 23 Val-->Ile mutation at the axial contact with either the beta 6 Glu-->Val or beta 6 Glu-->Ile substitution in the same beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)


Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129.

Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.


A series of point mutations reveal interactions between the calcium-binding sites of calmodulin.

Calmodulin is a member of the "EF-hand" family of Ca(2+)-binding proteins. It consists of two homologous globular domains, each containing two helix-loop-helix Ca(2+)-binding sites. To examine the contribution of individual Ca(2+)-binding sites to the Ca(2+)-binding properties of CaM, a series of four site-directed mutants has been studied. In each, the glutamic acid at position 12 in one of the four Ca(2+)-binding loops has been changed to a glutamine. One-dimensional 1H-NMR has been used to monitor Ca(2+)-induced changes in the mutant proteins, and the spectral changes observed for each mutant have been compared to those for wild-type CaM. In this way, the effect of each mutation on both the mutated site and the other Ca(2+)-binding sites has been examined. The mutation of glutamate to glutamine at position 12 in any of the EF-hand Ca(2+)-binding loops greatly decreases the Ca(2+)-binding affinity at that site, yet differs in the overall effects on Ca2+ binding depending on which of the four sites is mutated. When the mutation is in site I, there is only a small decrease in the apparent Ca(2+)-binding affinity of site II, and vice versa. Mutation in either site III or IV results in a large decrease in the apparent Ca(2+)-binding affinities of the partner C-terminal site. In both the N- and C-terminal domains, evidence for altered conformational effects in the partners of mutated sites is presented. In the C-terminus, the conformational consequences of mutating site III or site IV are strikingly different.


Electrostatic interactions in the association of proteins: an analysis of the thrombin-hirudin complex.

The role of electrostatic interactions in stabilization of the thrombin-hirudin complex has been investigated by means of two macroscopic approaches: the modified Tanford-Kirkwood model and the finite-difference method for numerical solution of the Poisson-Boltzmann equations. The electrostatic potentials around the thrombin and hirudin molecules were asymmetric and complementary, and it is suggested that these fields influence the initial orientation in the process of the complex formation. The change of the electrostatic binding energy due to mutation of acidic residues in hirudin has been calculated and compared with experimentally determined changes in binding energy. In general, the change in electrostatic binding energy for a particular mutation calculated by the modified Tanford-Kirkwood approach agreed well with the experimentally observed change. The finite-difference approach tended to overestimate changes in binding energy when the mutated residues were involved in short-range electrostatic interactions. Decreases in binding energy caused by mutations of amino acids that do not make any direct ionic interactions (e.g., Glu 61 and Glu 62 of hirudin) can be explained in terms of the interaction of these charges with the positive electrostatic potential of thrombin. Differences between the calculated and observed changes in binding energy are discussed in terms of the crystal structure of the thrombin-hirudin complex.


[Neurological manifestation of Takayasu's arteritis].

38 cases of Takayasu's arteritis were reported. The mean age of onset was 23.3 years with a female: male ratio of 1:1.7. The median delay between first symptom and time of diagnosis was 12.2 years. Headache was the most common symptom of neurologic manifestations (55%). Major neurologic events occurred in 52.7% patients in this group, including TIA, cerebral infarction, hypertensive encephalopathy, lacunar infarct, seizure, paraplegia, watershed infarct, cerebral hemorrhage, Moyamoya phenomenon, and confusion in the order of frequency. A variety of mechanisms that must be taken into account in explaining this neurologic events were proposed. The secondary hypertension and cardiac complications play an important role in causing neurologic symptoms. The formation of anastomotic networks has "Jekyll and Hyde" effect on brain both in preventing or limiting the ischemic injury and in producing some special symptoms and signs, that further widen the clinical spectrum of brain involvement.


Restriction endonuclease digestion patterns of harvest mice (Reithrodontomys) chromosomes: a comparison to G-bands, C-bands, and in situ hybridization.

Constitutive heterochromatin of a karyotypically conserved species of harvest mouse was compared to that of three karyotypically derived species of harvest mice by examining banding patterns produced on metaphase patterns produced by two of these restriction endonucleases (EcoRI and MboI) were compared to published G- and C-banded karyotypes and in situ hybridization of a satellite DNA repeat for these taxa. The third restriction endonuclease (PstI) did not produce a detectable pattern of digestion. For the most part, patterns produced by EcoRI and MboI can be related to C-banded chromosomes and in situ hybridization of satellite DNA sequences. Moreover, digestion with EcoRI reveals bands not apparent with these other techniques, suggesting that restriction endonuclease digestion of metaphase chromosomes may provide additional insight into the structure and organization of metaphase chromosomes. The patterns produced by restriction endonuclease digestion are compatible with the chromosomal evolution of these taxa, documenting that in the highly derived taxa not only are the chromosomes rearranged but the abundance of certain sequences is highly variable. However, technical variation and difficulty in producing consistent results even on a single slide with some restriction endonucleases documents the problems associated with this method.


Fixed implant-supported prostheses with welded titanium frameworks.

Eighty-six edentulous patients provided with Brånemark implants were randomly selected for fixed prostheses with modified framework designs. This study describes alternative laboratory techniques in which premachined titanium components are welded together to form the framework. Clinical experience, following the patients for 1 year after placement, indicates that it is a predictable technique with a similar pattern of complications as experienced by patients with cast frameworks supported by implants. The prostheses are considered to be slightly more bulky than cast frameworks, but seem to have, on a clinical level, a better fit to the implants.


[Main drug interactions at a hospital emergency department. Analysis of 100 cases].

We prospectively reviewed clinical charts of 100 consecutive patients that were admitted during the first trimester 1991 to the Emergency Department of a general hospital in order to determine the more frequent prescribed drugs and their interactions using a computer program (Drug Interaction Program), emphasizing in those drugs used to treat peptic ulcers. Number of drugs prescribed to each patient was 4.20 +/- 1.39. Antacids (39%) and cimetidine (35%) occupied the third and fourth place. There were interactions in 79 patients and in 66 of them (84%) they were important. Antacid and cimetidine were similarly prescribed, but of 35 patients who received cimetidine only 3 (8.5%) had a primary indication for its use (Gastrointestinal bleeding). Significant clinical interactions of cimetidine with other medications are analyzed. Our results indicate that drug interactions are a permanent risk in our hospitals. We suggest to use a computer program on drug interactions or an updated chart of medications in the emergency rooms of our hospitals.


[The withdrawal syndrome and lipid peroxidation during the chronic administration of narcotic analgesics to rats].

Diverse behavioral disorders and the intensity of lipid peroxidation (LPO) of biological membranes were estimated in different rat tissues after the 7-day administration and subsequent withdrawal of morphine or promedol. 24 hours after the withdrawal of the analgetics the demonstrated a high initial level of motor activity in the open field. Naloxone, an antagonist of opiate receptors, potentiated motor activity and the intensity of withdrawal syndrome (by 160%) in rats with morphine rather than promedol dependence. The behavioral disorders in dependent animals were accompanied by LPO activation in liver and brain membranes.


[The effect of propranolol and flusoxolol on the lysosomal enzyme activity of the rat ventricular myocardium].

The nonselective beta-blocker propranolol and the selective beta 1-adrenoblocker flusoxolol were tested for their effects on the activities of acid phosphatase, acid DNAase, cathepsin D, beta-glucosidase and beta-galactosidase in intact rat ventricular myocardial homogenates. The two drugs were found to have the most noticeable effect on the activity of three enzymes under study: acid phosphatase, beta-glucosidase and beta-galactosidase. They were able to stabilize lysosomal membranes during long-term homogenate preincubation at 37 degrees S. It is suggested that the mechanism of action of the drugs on intact rat ventricular myocardial lysosomes under the conditions of the study involves the binding of both propranolol and flusoxolol to beta-adrenoceptors on the lysosomes.


[The absence of reproducible effects from anxiolytics and anxiogens on the behavior of different strains of mice in a dark-light chamber].

Experiments on 1,200 male mice (150 groups) of three strains (SHR bred from Swiss, C57BL/6 and CC57BR) were made during 4 years (1986-1989) to test five anxiolytics (diazepam, phenazepam, chlordiazepoxide, phenibut, buspiron) and ethanol, three anxiogens (pentylenetetrazol, caffeine, yohimbine) and three putative endogenous anxiogens (beta-phenylethylamine, kynurenine and quinolinic acid) using a dark-light chamber. None of the drugs administered within a wide dose range produced any stable reproducible effect on the number of transitions between two compartments or on the dark preference. Thus, the reported data on an increase in the number of transitions induced by benzodiazepine anxiolytics were not confirmed. The data obtained demonstrate that the dark-light chamber is not a reliable system for simulating anxiety in mice.


[A hypothesis of the possible mechanism of the action of analgesic agents at the neuronal level].

The opiate analgetic promedol and non-opiate analgetics analgin, clonidine, baclofen, tolibut, vasopressin and calcitonin given in adequate doses block the inward electrosensitive sodium transmembrane ionic current of neurons. Like some drugs which do not exhibit any analgetic effect, promedol, analgin, clonidine, vasopressin and calcitonin also block the electrosensitive delayed potassium current. It is assumed that the blocking of the sodium ionic current may be one of the potential analgetic mechanisms at the neuronal level.


[Memory disorders in schizophrenia].

The current interest in memory disorders in schizophrenia results from the way perceptions of schizophrenia--whose organic origin is becoming increasingly evident--and memory--according to which there exist not one, but several memories--have developed. Memory disorders in the schizophrenic cannot be considered in isolation from knowledge accumulated in other areas of the cognitive and neuro-sciences; a more detailed understanding of these disorders requires a comparison of the different cognitive approaches, both with each other and with the neurobiological and clinical approaches, so that they can be integrated. Despite numerous methodological and conceptual difficulties, it now appears to have been established that the schizophrenic's memory deficit should be seen in the context of a wider cognitive deficit, that the memory tasks are not all disturbed and that the memory deficit cannot be identified with one specific form of memory. Thus, iconic formation, short-term memory in the traditionally accepted sense and implicit memory are hardly, if at all, affected; in contrast, the early processing of information, working memory and explicit memory are disturbed, probably to the extent that they require the implementation of strategies to organise the information to be memorized. Finally, in certain tasks, such as those evaluating latent inhibition or negative priming, schizophrenics perform better than normal subjects, suggesting that schizophrenics' cognitive deficit is localised. This profile of memory disorders is compatible with a dysfunction predominating in the frontal and temporo-hippocampal regions. Neuroleptics and anticholinergics have opposite effects on cognitive and mnesic performance, which is improved by the former and aggravated by the latter. The influence of clinical symptoms, positive or negative, institutionalisation of patients and chronic tardive dyskinesia is unclear. Among the theoretical proposals put forward to account for the observed disorders, those relating to a disturbance of the action planning process and to that of the internal representation of context are compatible with the observed memory disorders. All the clinically derived data and those produced by the cognitive and neurosciences indicate a need to reformulate the links between memory, selective attention and evaluation of the relevance of a stimulus, to develop a general model of the reciprocal interactions between cognition and affectivity and to look for the origin of a pathology as complex as schizophrenia, not in a local lesion in an isolated cerebral structure but in a disturbance of the dynamic interactions within a functional, parallel and distributed network of broadly interconnected regions.


[Neurobiology of memory].

A first part of the paper describes the neuro-anatomical structures involved in the processes of memory and their connections, as demonstrated by the recent hodological methods. The classical view of the Papez's limbic circuitry represents only one part of the reality. Several other circuits do exist, including those relaying in the amygdaloid complex and in the septal area. Actually, all these structures can be understood as the nodal points of a set of cortico-subcortical networks, where information is distributed and processed according to the cognitive demand and finally stored in the cortical layers. In a second part, the various mechanisms at the cellular level in relation with the processes of learning, are discussed (i.e. plasticity of synaptic transmission, dynamic neuronal changes at the morphological, electrophysiological and metabolic levels). The role played by humoral neuromodulation is examined, although it remains largely unknown at present (i.e. acetylcholine, catecholamines, GABA, glutamate, neuropeptides). Unfortunately, therapeutical implications from these fundamental data stay mostly frustrating up to now. Finally, relationships between sleep and memory are considered. The abundant experimental work performed on animals mainly stresses the REM-sleep. However, data from the human pathology are much less conclusive, and no clear evidence of a specific interaction between one aspect of sleep and one kind of memory emerges. To conclude, a few remarks are warranted on the intimal links existing between the anatomical-functional set of memory and the neurobiological bases of behavioural responses and anticipatory processes.


[Anticancer substances of vegetable origin. Spindle poisons: vincaleukoblastine, leurocristine and navelbine; taxol and taxotere].

The biological activity of spindle poisons can easily be measured using an in vitro assay based on the interaction of these substances with their cellular "receptor": tubulin. The use of this assay led us to select Navelbine and Taxotere as antimitotic substances. These compounds, as well as their natural parents: vincaleucoblastine, leurocristine and taxol respectively, have been obtained by semi-synthesis using relatively abundant natural precursors as starting materials. This paper summarizes the preparation of these important anticancer drugs.


[Determination of colonization factor antigens CFA in diagnosis of enterotoxigenic strains of Escherichia coli].

This study was aimed at establishment whether preliminary determination of colonization factor antigens CFA may be useful in selection of potentially pathogenic strains of Escherichia coli with serological types belonging to ETEC and 750 isolates of E. coli from children with symptoms of diarrhoea. Enterotoxigenicity of strains was evaluated by suckling mice test and culture of Y1 cell tissue. Colonization factor antigens CFA were evaluated on the basis of slide agglutination and agar gel immunodiffusion with application diagnostic sera prepared for this study. Ability of enterotoxin production was found in 25% strains of E. coli with serological types belonging to ETEC. In 90% these strains were isolated from cases of epidemic diarrhoea. ETEC strains were found in 11% of hospitalized children and in 5% who were treated outside of hospital because of diarrhoea. MRHA adhesins occurred on 80% of ETEC strains were all diagnosed as CFA/I. CFA/II were not found and in only three strains non-fimbrial CFA/IV was present. Preliminary determination of CFA during selection of ETEC strains presents as a very sensitive method (97%) and is also highly specific (99%). Application of this method will result in significant increase of affectivity of biological tests directed toward determination of E. coli enterotoxigenicity.


[Occurrence of P.fimbrii in strains from selected genera of Enterobacteriaceae].

This study was aimed at recognition of frequency of occurrence of P fimbriae in strains of Escherichia coli isolated from samples of feces of children with symptoms of diarrhoea and at search of these adhesions in strains representing other than Escherichia genera of Enterobacteriaceae strains. One hundred forty laboratory strains were investigated. They belonged to genus Citrobacter, Enterobacter, Hafnia, Klebsiella, Morganella, Proteus, Providencia, Salmonella, Shigella, and Yersinia. Also were tested 1277 colonies of enteric rods isolated from the MacConkey's medium inoculated with samples of feces from 163 children with symptoms of diarrhoea. Mannose-resistant active hemagglutination test was performed with human group O erythrocytes and guinea pig erythrocytes stabilized with glutaraldehyde. Presence of P fimbriae was detected by the slide latex test with latex covered by P1 glycoprotein. Among 140 laboratory strains of Enterobacteriaceae in 21 strains (3-E. cloaceae, 2-Hafnia, 13-K. pneumoniae, 2-P. rettgeri and in one strains of Providencia) presence of MRHA adhesins was demonstrated. Nine of these strains (2-Hafnia, 5-K. pneumoniae and 2-P. rettgeri) reacted specifically in the latex test. Among 1142 colonies of E. coli isolated from children with symptoms of diarrhoea, 326 colonies belonged to 13 EPEC serotypes. With 118 (36.2%) of EPEC colonies a positive result of MRHA reaction was found with human erythrocytes and 34 (10.4%) with guinea pig erythrocytes. Positive latex test was obtained with 77 (23.6%) colonies. All these colonies possessed MRHA adhesins. Remaining 816 colonies of E. coli strains did not represent microorganisms belonging to serotypes accepted as enteropathogenic. From 112 (13.7%) colonies out of 816 not belonging to EPEC, positive results was obtained in the MRHA test with human erythrocytes and this was the case also with 41 (5.0%) colonies in MRHA reaction with application of guinea pig erythrocytes. The latex test was positive in 65 (7.9%) colonies of E. coli. From remaining 135 colonies other than E. coli, positive result of latex test of presence of P fimbriae was obtained with 54 (40.0%) colonies, including 14 colonies of E. cloacae, 23-K. pneumoniae and 17-K. oxytoca. In all these strains presence of MRHA adhesins was demonstrated. These investigations demonstrated that among EPEC strains significantly more frequently, than not belonging to these serotypes of E. coli, MRHA adhesins, including P fimbriae was observed.(ABSTRACT TRUNCATED AT 400 WORDS)


[Root resorption].

Root resorption can be divided into two main categories: internal root resorption and external root resorption. Internal root resorption is a pathology that can lead to tooth destruction in the short term and must consequently be stopped as soon as possible by applying adequate canal treatment. Until now, despite many research studies, very little is known about its causes and the way this kind of resorption appears. There are many different forms of external root resorption and it has a very diverse etiology. An external root resorption can thus appear in case of orthodontic treatment or due to the pressure brought by cysts, tumours or impacted teeth. A trauma or an infection may also lead to the development of an external root resorption. Because they are so diverse, each of these forms of external root resorption requires a specific treatment. It should also be noted that internal root resorptions, and some forms of external resorption too, often respond favourably to a calcium hydroxide treatment.


[The pharmacological regulation of neutrophil activity by cholinotropic preparations, barbiturates and fenazepam].

The in vitro experiments with human blood cells and in vivo experiments on Wistar rats have demonstrated that stimulation of neutrophils enhances their phagocytic metabolic activity. Barbiturates (benzonal and phenobarbital) and phenazepam in low concentrations (doses) increase neutrophilic function evaluated by the NBT test, but in high ones cause opposite effects.


Analysis of cytogenetic damage induced in CHO cells by the pyrethroid insecticide fenvalerate.

A synthetic pyrethroid insecticide, fenvalerate, was tested for its ability to induce chromosome aberrations (CA) and sister chromatid exchanges (SCE) in CHO cells. Fenvalerate was assayed both in the presence and in the absence of a rat liver activation system (S9-mix). Our results indicate that fenvalerate in the presence of S9-mix is able significantly to increase the frequency of CA, while in the SCE test this increase occurred both in the presence and in the absence of S9-mix. In addition, fenvalerate affected the cell cycle, causing a decrease in the mitotic index (MI) and in the proliferative rate index (PRI).


[The GABA-ergic component of the anxiolytic action of 1-(2-pyrimidinyl)-piperazine derivatives].

The 1-(2-pyrimidinyl)-piperazine derivatives campirone, campironine, levopironine) evoked hyperpolarizing responses of rat dorsal root ganglion neurons mediated by 5-hydroxytryptamine(1A) receptor activation and, like chlordiazepoxide, potentiated neuronal responses evoked by GABA-depolarizing receptor activation. The drugs studied in the lighted space and threatening situation avoidance tests showed an anxiolytic effect. Picrotoxin was found to be effective in inhibiting the anxiolytic effect of chlordiazepoxide, levopironine and campironine, but it failed to affect the antianxious action of campirone. Whether the GABA-ergic mechanisms may contribute to the anxiolytic action of 1-(2-pyrimidinyl)-piperazine derivatives.


[The role of the genotype in the cataleptogenic effect of neuroleptics].

The cataleptogenic effects of three neuroleptics from one chemical group was investigated in 8 mice strains (CBA, A/He, C57B1/6, C3H/He, BALB/c, AKR, DD, and CC57Br. Despite significant interstrain, differences in the action of the drugs, haloperidol (0.5 mg/kg) and trifluperidol (0.5 mg/kg) produced much greater cataleptogenic action than fluspirilene (2 mg/kg). At the same time the intensity of catalepsy in various mice strains after haloperidol was not coincident with that after trifluperidol (r = 0.22): CBA mice displayed the maximum catalepsy, but AKR, DD and CC57Br mice, the minimum when haloperidol was given; with trifluperidol, the maximum catalepsy was observed in AKR mice, but absent in DD mice. Fluspirilene induced catalepsy only in CBA and A/He mice. Thus, the presence of catalepsy, a side effect of most neuroleptics, is largely predetermined by hereditary factors.


Valproic acid induced abnormal development of the central nervous system of three species of amphibians: implications for neural tube defects and alternative experimental systems.

Embryos of Ambystoma mexicanum, Xenopus laevis, and Hyperolius viridiflavus taeniatus were exposed to various concentrations of valproic acid (VPA: 0.1, 1.5, 10 mM) from blastula stage (S) 9 on up to advanced gastrulation of control embryos (S 11 1/2-12). At 10 and 5 mM VPA early development was affected in all species tested. However, the most pronounced effects occurred in Ambystoma: the neural folds appeared delayed and showed a flattened and wavy shape; the neural tube was not formed and embryos successively died. In Xenopus and Hyperolius (10, 5 mM VPA) the beginning of gastrulation was delayed up to neurulation of control embryos. In Xenopus many of the embryos completed neurulation, whereas some embryos exposed to 10 mM VPA showed neural tube defects (NTDs) of different type and degree (open neural tube at different regions of the dorsum). In Hyperolius neural folds arose around the blastoporus and fused later on (earlier in embryos treated with 5 mM VPA), but the shape of these embryos was abnormal and the development was not continued (pronounced effect at 10 mM VPA). Comparing the three species, Xenopus proved to be the least sensitive species (at 5 mM VPA 14.2% NTDs of total malformations compared to 100% in the other species). The most sensitive species, Ambystoma, developed head-oedema at 1 mM VPA, whereas the anurans were not affected. Our results suggest a similar mechanism of VPA-induced NTDs in mammals and amphibians.


Effect of acyl derivatives of 4,4,4-trifluoro-1-phenyl-1,3-butanedione on 2-acetylaminofluorene-induced glutathione S-transferase positive foci in the rat liver.

Acyl derivatives of 4,4,4-trifluoro-1-phenyl-1,3-butanedione (TFPB), 1-benzoyl-2-trifluoromethyl-2-acetoxyethene (BTAE), and 1-benzoyl-2-trifluoromethyl-2-(4-methylthio)benzoyloxyethene (BTME), were synthesized and investigated for inhibition of tRNA binding by N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), and induction of glutathione S-transferase placental form (GST-P) positive foci in the rat liver by 2-acetylaminofluorene (2-AAF). Male F344 rats were given BTAE or BTME intraperitoneally and 2-AAF by intragastric intubation. Two weeks following the treatment, the rats were maintained on the diet containing 0.05% phenobarbital for an additional 6 weeks and then killed. Development of GST-P positive foci was not affected by concomitant treatment with BTAE or BTME. These two compounds inhibited the in vitro binding of N-AcO-AAF to tRNA. Thus, although these diacylmethane derivatives had the in vitro inhibitory activity, they did not inhibit tumor-initiating activity of 2-AAF in the rat liver.


Analysis of carcinogenic activity of some pesticides in a medium-term liver bioassay in the rat.

Eight pesticides were tested in a medium-term bioassay based upon the induction of preneoplastic lesions in the liver. Rats were initially given diethylnitrosamine intraperitoneally at a dose of 200 mg/kg body weight and 2 weeks later were treated with the pesticides for 6 weeks and then killed; all rats had a partial hepatectomy at week 3. Hepatocarcinogenic potential was assessed by comparing the number and area of glutathione S-transferase placental form positive foci in the liver with those of controls given diethylnitrosamine (DEN) alone. Positive results were seen with p,p-DDT and Triadimefon. Permethrin (mixture of 39% cis form and 61% trans form) showed borderline results. Permethrin (25/75), Deltamethrin, Cypermethrin (52/48), while Trimorphamide and Propineb gave negative results. Our findings provide experimental evidence to indicate that compounds active in this assay have a potential for liver carcinogenicity in rodents.


A morphologic basis postulated for valproic acid's embryotoxic action in rats.

A tentative 3 phase sequence of pathogenesis is proposed for the embryotoxic action of valproic acid (800 mg/kg) administered orally to rats on day 13 of pregnancy. This is based on histopathological changes in the extraembryonic and embryonic tissues which occurred in the absence of any biologically significant effect on maternal homeostasis. Major events in the first, decidual (or maternal) phase are cells lining the maternal sinusoids in the decidua basalis are necrosed, desquamated, and washed away by the arterial circulation through the afferent channels. The necrosed cells, with their walls still intact, occlude the lumen of these arterial channels at the point of their entry into the giant cell-trophospongial zone. The channel occlusions cause ischemia and homeostasis of the maternal circulation in the labyrinth by reducing the rate of inflow of maternal blood. The embolic occlusion of maternal arterial channels apparently results in a long-term reduction in the number and size of maternal channels that supply arterial blood to the labyrinth. In the second or placental phase, the parenchyma of the labyrinth and connective tissue in the chorionic plate and umbilical cord, which have been deprived of nutrition and oxygen by the ischemia and stasis of maternal blood in the labyrinth, undergo degenerative changes. In the third or embryonic phase, a pleiotropic karyorrhexis in the embryo, initiated as early as 4 h postdosing, appeared aggravated, presumably by the preceding labyrinthine degeneration of the placental phase. The valproic acid-induced embryotoxicity thus seemed to result from a combination of maternal, placental, and embryonic changes.


Effect of striatal lesion with quinolinate on the development of substantia nigra dopaminergic neurons: a quantitative morphological analysis.

We have previously reported that a developmental axon-sparing striatal injury induced by hypoxia-ischemia results in a diminished adult number of substantia nigra (SN) tyrosine hydroxylase (TH)-positive neurons, in the absence of direct nigral injury. To examine the specific role of striatal injury, we made selective striatal lesions in the 7-day-old rat with quinolinic acid (QA), 40, 80, 120 and 480 nmol. Striatal lesions resulted in a decrease in the adult number of TH-positive neurons in the ipsilateral SN. The decrease was correlated with the reduction in striatal area (r = 0.76, p < 0.01); a 25-30% reduction in SN neurons was observed at 50-60% striatal area loss. The area of SN pars compacta (SNpc) in animals with 120- and 480-nmol QA lesions was reduced by 12 and 15%, respectively (p < 0.01) and this reduction also correlated with the loss of striatal area (r = 0.63, p < 0.01). Individual TH-positive neuron size and neuron-packing density were unaltered in SNpc on the experimental side. We conclude that the adult number of SN dopaminergic neurons depends on developmental support by the striatum. We hypothesize that less support by the smaller striatum may result in accentuated developmental cell death in the SNpc.


Release of neurotransmitters and neurosecretory substances during in vitro maturation of mouse hypothalamic cultures.

The maturation of the neurosecretory activity of a hypothalamic nerve cell population grown in vitro, prepared from 10-day-old mice and cultured for 6 days, has been demonstrated in the present report. A low-molecular weight polypeptide of 30-kD was found to be released into the culture media during the 6-day period of incubation, as analyzed by SDS-polyacrylamide gel electrophoresis. Comparative electrophoresis of the in situ hypothalamic, neurohypophysis and cerebral cortex homogenates revealed the presence of a 30-kD protein component in both the hypothalamus and neurohypophysis but not in the cerebral cortex. The release of the 30-kD polypeptide into the incubation media indicates an expression of the neurosecretory activity of the peptidergic neurons of the hypothalamus during in vitro maturation. On the other hand, high pressure liquid chromatography with electrochemical detection showed appreciable quantities of released dopamine (DA), epinephrine and serotonin (5-HT) in the incubation media in which the neurons were allowed to differentiate. There was a steady release of DA during the 6-day incubation period, varying from 0.21 +/- 0.02 to 0.49 +/- 0.05 ng/mg protein. The epinephrine level increased progressively from day 1 to 6 of culture, from 3.73 +/- 0.57 to 12.08 +/- 1.81 ng/mg protein, respectively. The measured 5-HT level was 0.07 +/- 0.001 on day 2 and increased to 0.38 +/- 0.05 ng/mg protein on day 6 of culture. These data demonstrate the functional maturation of catecholaminergic, serotoninergic and peptidergic neurons in these rotary histotypic cultures of the mouse hypothalamus.


[Pulmonary vasoconstrictor responses].

Alterations in the physiological balance to maintain the pulmonary circulation at a normal low pressure level result in an elevation in pulmonary vascular tone. Pulmonary vasoconstrictor responses were analyzed under some experimental conditions, which included microembolism, administration of vasoactive agents, hypoxia, and monocrotaline-induced pulmonary hypertension. It is widely accepted that these responses are highly localized and complex. In the present study, excised canine lung lobes, rat lungs, and pulmonary arterial rings from the rat were employed according to the particular experimental design. The mechanism of the initial rapid elevation followed by a gradual decline in perfusion pressure in microembolism was considered to be related not only to the size of the emboli, but to the degree of mechanical injury of the endothelium. The main sites of constriction of the pulmonary vasculature by several drugs were determined in the pulsatile perfused canine lung lobes, according to the degree of decrease in inflow wave amplitude during antegrade or retrograde perfusion. Further, by applying the same method it was confirmed that the site of hypoxic vasoconstriction is located in the peripheral pulmonary vascular bed between the muscular arteries and veins, which are constricted mainly by serotonin and histamine, respectively. A cross perfusion system was set up, employing two lobes from the same dog, in which normoxic blood was perfused into the hypoxic ventilated lobe and vice versa. As a result, the pulmonary vessels showed a response to ventilation hypoxia that was far more sensitive than that to perfusion hypoxia. The effects of a beta-agonist (isoproterenol) and beta-antagonists (propranolol, pindolol) on hypoxic vasoconstriction were observed. Although pindolol (a vasodilatory beta-blocker) abolished hypoxic pulmonary vasoconstriction, which was similar to the effect of isoproterenol, the mechanism of action of pindolol was suggested to be different from that of isoproterenol. The importance of the K+ channel of vascular smooth muscle and also the endothelium in hypoxic pulmonary vasoconstriction were stressed. In isolated pulmonary artery segments of the monocrotaline-treated rat, the augmentation of sensitivity of the vascular smooth muscle to Ca2+ preceded the occurrence of pulmonary hypertension. Similarly, hyperreactivity to KCl and serotonin was also observed. It was clarified that the hyperreactivity induced by monocrotaline is modified by endothelium-dependent relaxation. Extensive cellular and molecular biological investigations are essential for further progress in this field.


[Pharmacological evidence for the existence of beta 3-adrenergic receptors in canine airway smooth muscle].

There is increasing evidence for the existence of a third atypical beta-adrenergic receptor (beta 3-adrenoceptor) in various tissues including adipocytes, cardiac myocytes and intestinal smooth muscle preparations. In the present study, to determine whether beta 3-adrenoceptors also exist in the airway smooth muscle, we studied isolated bronchial segments from dogs under isometric conditions in vitro. Application of beta-adrenoceptor agonists produced a concentration-dependent relaxation of tissues precontracted with 10(-5) M acetylcholine, the order of potency being isoproterenol (1) > or = salbutamol, a beta 2-selective adrenoceptor agonist (0.95) > or = BRL 37344, a beta 3-selective adrenoceptor agonist, (0.83) >> norepinephrine (0.10). Under the condition in which alpha- and beta 1-adrenoceptors had been blocked by phentolamine and ICI 89406, respectively, the relaxant response to salbutamol was competitively antagonized by the beta 2-adrenoceptor antagonist ICI 118551, and the pA2 value was 7.01 +/- 0.25 (mean +/- SE), whereas the response to BRL 37344 was resistant, with of apparent pA2 value of 5.66. However, cyanopindolol, an antagonist atypical beta-adrenoceptors, antagonized BRL 37344-induced relaxation in a competitive fashion with a pA2 of 6.74 +/- 0.11. This pA2 value was lower than that when salbutamol was used as an agonist (p < 0.05). These results indicate that beta 3-adrenoceptors probably exist in canine bronchial smooth muscle, and that stimulation of this type of receptors produces potent bronchodilation. Therefore, a specific agonist for beta 3-adrenoceptors could be valuable in the treatment of asthma.


Human cytochrome P450IIE1 gene: DraI polymorphism and susceptibility to cancer.

Human cytochrome P450IIE1 (CYP2E) is involved in the metabolic activation of procarcinogens such as N-nitrosodimethylamine, benzene and ethyl carbamate. We screened DNA from 28 individuals for restriction fragment length polymorphisms (RFLPs) is the human P450IIE1 gene and detected an RFLP for the restriction endonuclease DraI. The distribution of the genotypes of this polymorphisms among lung cancer patients (n = 74) differed from that among controls (n = 73) with statistical significance of p < 0.05. In addition, the distribution among patients with cancers of the digestive system (n = 38) was also different from that among controls. Our findings indicate an association between the DraI polymorphism of the IIE1 gene and susceptibility to cancers of the lung and the digestive system.


A possible role of activated macrophages in the adoptive immunotherapy using CD4+ T lymphocytes.

Potent anti-tumor T lymphocytes with CD4+8- phenotype were obtained in peritoneal exudate cells by immunizing mice with irradiated tumor cells and OK-432. These effector cells were used in adoptive immunotherapy for tumor-bearing mice. Admixed administration of effector T cells with irradiated relevant tumor cells resulted in a marked enhancement of anti-tumor activity against local tumor and lymph node metastasis compared with the immunotherapy by effectors alone. The activating state of macrophages inoculated with viable tumor cells had much relevance with the implementation of immunotherapy. Innocent bystander lysis was not observed in this immunotherapy. Interleukin-2 given instead of stimulant tumor cells caused no enhancement, while interleukin-1 emerged stronger enhancement than stimulant tumor. In this case, activating state of macrophages had no relevance with the effectiveness of the therapy. These results suggest that macrophages in tumor play a role to secrete interleukin-1 to enhance anti-tumor activity of specific T cells.


Present status of agonistic and antagonistic analogs of LH-RH in the treatment of advanced prostate cancer.

The methods for treatment of advanced prostate cancer, based on the agonistic analogs of LH-RH were reviewed. New therapeutic approaches utilizing antagonistic analogs of LH-RH such as SB-75 (Cetrorelix) have been described. Analogs of LH-RH chemically linked to various cytotoxic radicals are also being developed. Combinations of LH-RH agonists or antagonists with superactive somatostatin analogues such as Octastatin (RC-160) or with bombesin/GRP antagonists are being investigated in order to delay or prevent the relapse and improve the therapy for prostate cancer.