Transmembrane organization of the Na+,K(+)-ATPase molecule. The organization of the hydrophobic domain of the Na,K-ATPase in the E1 and E2 form of the enzyme has been studied by labelling with two hydrophobic photoactivable reagents 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine ([125I]TID) and 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl) diazirinyl] phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphorylcholine ([3H]PTPC/11). The incorporation of the reagents into the alpha-subunit but not into the beta-subunit in the E1-conformation was shown to be lower than that in the E2 form. This indicated the structural rearrangement of the alpha-subunit, which resulted in a change in the accessibility of the membrane-bound fragments for the hydrophobic labels. The set of the [125I]TID-labelled peptides of the alpha-subunit was shown to be the same for the E1 and E2 form of the enzyme: Asp68-Lys142, Ile265-Lys341, Val545-Lys589, Ser770-Lys826, Leu842-Arg880, Asn936-Arg972 and Met973-Arg999, which points to the different level of modification of the same fragments. The first results of molecular modelling of the spatial organization of the intramembrane part of Na+,K(+)-ATPase are also presented.


Structural modelling of P-type ion pumps. Over forty sequences of P-type ion pumps have been determined. They fall into five families showing between 20% and 50% identity in sequence. The conserved residues are concentrated in several regions which are found in all the pumps. All the defined functional sites are associated with conserved segments and provide a basis for subdivision into domains, to which tentative secondary and tertiary structures can be assigned. The domains have been assembled into a structure consisting of a conserved core with variable loops and deletions on the surface, which accommodates site mutants, affinity labels and known epitopes. This model has been correlated with the results of an electron crystallographic study of the Ca++ pump. Two types of crystal have been examined in negative stain and in amorphous ice; thin plates which diffract to 4A and long helical tubes which diffract to 15A. The plates have given a 6A projection map, some of which can be interpreted by difference from the map of the negatively stained crystal, as transmembrane helices. Three dimensional interpretation will require a tilt series. In the meantime, analysis of the tubes has given a 14A 3D map which clearly defines the cytoplasmic domains and their relation to the transmembrane region (Stokes and Toyoshima in preparation). Although it is not yet possible to assign specific functions to the cytoplasmic lobes, the structure at this resolution is consistent with the model.


Identification of the cation binding domain of Na/K-ATPase. This paper summarises results and conclusions from experiments with renal Na/K-ATPase, utilising proteolytic digestion to define minimal peptide structures involved in cation occlusion and chemical modification with dicyclohexylcarbodiimide (DCCD) to investigate the role of carboxyl groups and location of K (Rb) and/or Na binding residues. Extensive digestion with trypsin or non-selective proteases in the presence of Na or Rb and absence of divalent cations reveals an essential C-terminal 19Kd fragment of the alpha chain (N-terminal asn 830) and indicates that occlusion sites of Na or K ions must reside within transmembrane segments. The bulk of the beta chain is not involved. Kinetics of inactivation of Rb or Na occlusion and covalent labelling with DCCD indicate that each of two Rb(K) or Na sites contains a carboxyl group. The third Na site may contain only neutral ligating groups. One carboxyl group is located on the 19Kd fragment and the other on tryptic fragment of about 9Kd. When cyanogen bromide was used to digest labelled alpha chain, glu 953 was found to be labelled in a Rb-protectable fashion. In tryptic "19Kd-membranes", fragments containing all putative transmembrane segments of the alpha chain have been identified (i.e. 19, 10.9, 8.7 and 8.0 Kda respectively). The cation occlusion "cage" is apparently composed of ligating groups from different trans-membrane segments, including segments of the 19Kd fragment. Construction of models is hampered by the fact that the number of the transmembrane segments is still uncertain, particularly in the crucial C-terminal domain. Alternative ways of arranging the tryptic fragments across the membrane are discussed.


Chemomechanical coupling in the gastric H,K ATPase. The gastric H,K ATPase is investigated in terms of its secondary structure by analysis of the binding sites of the extracytoplasmic inhibitors and by tryptic cleavage of intact, inside-out gastric vesicles. The inhibitors affect phosphorylation and other partial reactions of the ATPase that depend on cytoplasmic conformational changes. The K competitive imidazopyridine, SCH28080 binds to the first pair of transmembrane segments, M1/M2, probably at phe124 and asp136. Omeprazole which generates a cationic sulfenamide in acid spaces binds to either cys813 or cys822 at one site and cys892 at the other. These cysteines are located at the membrane spanning pairs, M5/M6 and at M7/M8. Tryptic cleavage of intact inside out vesicles followed by labelling with fluorescein-5-maleimide provides direct evidence for 8 membrane spanning segments between positions 104/162 (M1/M2), 291/358(M3/M4), 776/835 (M5/M6),853/946 (M7/M8). Evidence is lacking so far for M9/M10, postulated on the basis of hydrophobicity for the Ca ATPase. Conformational studies suggest that there is interaction between the cytoplasmic loop between M4 and M5 (ATP domain) and the extracytoplasmic domain of the enzyme at the inhibitor binding sites.


Functional domains of Na,K-ATPase; conformational transitions in the alpha-subunit and ion occlusion. The purified membrane-bound Na,K-ATPase from the outer renal medulla crystallizes in different forms with monomeric or dimeric unit cells, but the alpha beta-unit is the minimum asymmetric unit in P1-crystals, and cation binding sites and occlusion cavities for Na+ or K+(Rb+) are formed within the structure of the alpha beta-unit. Domains have been identified that engage in structural transitions mediating energy transduction between ATP or phosphorylation sites in the cytoplasmic protrusion and cation sites in the membrane. The purpose of this study has been to examine the relationship between ion binding and occlusion, and transitions between two conformations of the alpha-subunit, the Na-form (E1) or the K-form (E2) that are defined by the kinetics of inactivation of Na,K-ATPase and K-phosphatase activities and a specific sequence of tryptic (E1: T2(Lys30)-T3(Arg262); E2: T1(Arg438)-T2(Lys30)) or chymotryptic (E1: C3(Leu266)) cleavage of the alpha-subunit. In E1-forms stabilized in an oligomycin complex, three sites for Na+ occlusion were found per alpha-subunit. In E2-forms stabilized as the phosphorylated ouabain complex, two Na+ or two Rb+ were occluded per alpha-subunit, the E2[2Rb] conformation having a higher affinity. Selective chymotryptic cleavage (Leu266) in the second cytoplasmic loop completely blocks E1-E2 transition, ion- and charge translocation without affecting ligand binding to the protein. Structural transitions of this segment reflect changes in capacity and orientation of cation sites that are part of the ion translocation process in the alpha-subunit of the Na,K-pump.


Phosphoinositides and cell growth.

Phosphoinositides and cell growth. The findings described above illustrate how the src kinase can influence several new pathways of inositol phosphate metabolism, both at the membrane level with the production of novel D-3 phosphoinositides and the activation of PI-3 kinase, and at the cytosolic level by altering the expression of certain inositol polyphosphates, in particular Ins(1,4,5,6)P4. At present, it is difficult to speculate on the role these phenomena play in cellular transformation by src, since the functions of D-3 phosphoinositides and most inositol polyphosphates are unclear. There is evidence, however, that these new pathways of phosphoinositide metabolism occur in response to other types of cellular stimulations besides src transformation. Novel D-3 phosphoinositides are expressed in a variety of nonneoplastic cells, including human platelets treated with thrombin, smooth muscle cells and stimulated neutrophils. In addition, unusual InsP4 isomers such as D/L-Ins(1,4,5,6)P4 are found in chicken erythrocytes, murine macrophages, AR4-2J rat pancreatoma cells and adrenal glomerulosa cells, to name only a few. Recently, associations have been reported between PI-3 kinases and cytoskeletal elements in thrombin- stimulated platelets, and between activated ras proteins in rat liver epithelial cells. The latter discovery is particularly intriguing since GTP-binding proteins such as ras are known to influence cell shape and serve as downstream effector proteins in the signal transduction pathways of numerous growth factor receptors. Thus, one function of novel phosphoinositides and their metabolites may lie at the level of cytoskeletal and cell shape regulation. Clearly, additional roles for phosphoinositides exist in cells besides their traditional use as precursors for the generation of Ins(1,4,5)P3 and diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)


Advanced non-small-cell lung cancer (NSCLC) treated with folinic acid (F), fluorouracil (FU), vincristine (O), and mitomycin-C (Mi), (F-FOMi). Thirty-one patients with advanced non-small-cell lung cancer (NSCLC) were treated with a combination of folinic acid, fluorouracil, vincristine, and mitomycin (F-FOMi). Eight partial responses (26%), eight stable disease (26%), and 15 progressive disease (48%) were obtained. Patients with performance status (PS) 0-1 had a significantly better response rate than those with PS 2-3. Overall actuarial survival was 10 months. Toxicity was mild and mainly gastrointestinal with mucositis and diarrhea. F-FOMi seems to be comparable to regimens more widely used in the treatment of NSCLC.


Oncogenes and cell growth.

Oncogenes and cell growth. In this short overview of oncogenes and cell growth, the protein products have been divided into two classes, proto-oncogenes and oncogenes. Proto-oncogenes can be activated by point mutations and deletions. Two classes exist: the dominant, which leads to cell growth and the suppressor, which by definition suppresses growth. The mechanism of action is multiplex--duplication of hormone action, resemblance to receptors, or kinases and DNA binding proteins. It is clear that the regulation of cell growth and differentiation is very complex and that the products of proto-oncogenes play important roles in this regulation. Their functions appear to be at two levels. The first level is that of transduction of signals to the nucleus where the signals can be acted upon. The second is at the level of specific gene regulation, where incoming signals are turned into a response by the cell through activation of specific genetic programs. Nuclear proto-oncogene products play intimate roles in activation of these programs. The nature of the specific target genes regulated in response to these oncogene and proto-oncogene products however, remains a critical area of intensive research.


A comparison of alcohol and drug treatment clients: are they from the same population? As part of the Alcohol Research Group's Community Epidemiology Laboratory, representative samples of clients from a northern California county's public alcohol and drug treatment systems were interviewed. This paper compares clients in alcohol treatment agencies (N = 381) with those in drug treatment agencies (N = 307). In the United States the traditional separation of the two systems is in question, as there is a belief that most clients of both systems have dual alcohol and drug problems. This study found that while large numbers of clients reported use of both alcohol and other drugs, important differences were found between the two treatment systems. Alcohol treatment clients reported significantly higher alcohol consumption rates and lower drug use than did drug treatment clients. However, a higher proportion of alcohol treatment clients reported consuming alcohol with other drugs and attributing problems to both alcohol and drugs than did drug treatment clients. Discriminant function analysis found sociodemographic characteristics, problems attributed to alcohol, and problems attributed to drugs to provide significant contributions in predicting membership in the alcohol versus the drug treatment system.


Normal high-resolution karyotypes in 26 unrelated individuals with hereditary colorectal neoplasia. High-resolution karyotype analysis was performed on peripheral blood cultures from 26 patients with hereditary colorectal neoplasia. The aims of this study were: first, to determine the frequency of cytogenetically visible chromosome 5q deletions in familial adenomatous polyposis and, thus, whether routine karyotype analysis should be included in screening regimens for affected families; and, second, to search for chromosomal abnormalities in hereditary nonpolyposis colorectal cancer that might assist in localizing the gene or genes responsible. No cytogenetic abnormalities were detected among 21 unrelated patients with familial adenomatous polyposis and five with hereditary nonpolyposis colorectal cancer. We conclude that cytogenetic analysis is of no value in the management of families with typical familial adenomatous polyposis or Gardner's syndrome, and should be confined to those families with atypical features such as mental retardation or facial dysmorphism.


Lack of evidence for the heterosexual transmission of hepatitis C. To determine the potential for sexual transmission of the hepatitis C virus (HCV), we specifically studied a cohort of 42 young adults (median age, 39 yr) with chronic HCV infection and their stable sexual partners. All HCV assays were supplemented with the four-antigen recombinant immunoblot assay, and 39 of 42 partners were tested for HCV RNA by the nested polymerase chain reaction. Ninety percent of the partners reported frequent and unprotected sexual intercourse with the index patients. Two of 42 partners tested positive for the anti-HCV antibody and both were HCV RNA positive; one had independent risk factors for viral hepatitis. Therefore, one of 41 partners, (2.4%; 95% CI, 0.6-12.9%) without independent risk factors for HCV was anti-HCV positive. This woman was one of five partners (20%; 95% CI, 1-66%) who reported frequent razor-sharing with the index patient. The partner frequently sustained skin lacerations, with bleeding, secondary to this shared razor blade. We conclude that heterosexual transmission of hepatitis C is extremely uncommon, despite frequent and unprotected sexual intercourse.


Combined liquid chromatography/electron paramagnetic resonance spectrometry/electrospray ionization mass spectrometry for radical identification. Electron paramagnetic resonance (EPR) spectrometry and mass spectrometry (MS) have been coupled together on-line with liquid chromatography (LC)/UV detection. These combined techniques have been applied to the determination of spin-trapped radical adducts, including phenyl, 2-, 3-, and 4-chlorophenyl, and 2-bromophenyl radicals trapped with alpha-(1-oxo-4-pyridyl)-N-tert-butylnitrone (4-POBN), and phenyl radicals trapped with 2-methyl-2-nitrosopropane (MNP), alpha-phenyl-N-tert-butylnitrone (PBN), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Oxidized and reduced forms of the radical adducts were also detected by the on-line LC/EPR/MS system.


Neuronal plasticity as an adaptive property of the central nervous system. This short review presents examples of plasticity in the brains of vertebrates including man. The basic ability of the nervous system to make functionally relevant adaptations to functional challenges of various kinds during development and adulthood is called plasticity. Enucleation of the eyes or lesioning of the lateral geniculate body during development lead to the generation of a new architectonic area within the nonhuman primate and human primary visual cortex. The enucleation of one eye in rats at various postnatal stages causes profound plastic changes in the callosal system of the visual cortex. The central representation of the periphery in the adult cerebral cortex (somatotopy) can also be altered by adaptive processes. Naturally occurring nerve cell death during pre- and early postnatal development can be manipulated by impairing normal development of neuro-transmission. These findings argue for an important role of transmitter receptors in brain plasticity. The number of receptors shows, for most brain regions and receptor types, an overshoot of growth during ontogeny. After lesions have damaged the adult geniculo-cortical and septo-hippocampal systems, receptors can exhibit plastic changes such as upregulation of the number of binding sites (visual cortex) and modifications in the coupling of receptors, transducer proteins (G-proteins) and second messengers (hippocampus).


No evidence for spumavirus or oncovirus infection in relapsing-remitting multiple sclerosis. Polymerase chain reaction analysis was used to investigate the possible role of human spumaretrovirus and oncoretroviruses (human T-cell lymphotropic virus types I [HTLV-I] and II [HTLV-II]) in multiple sclerosis. Eleven patients with relapsing-remitting multiple sclerosis in exacerbation and 11 normal blood donors were included in the study. Cerebrospinal fluid cells, peripheral blood mononuclear cells, and plasma were cocultured with allogeneic mononuclear cells for 6 weeks. Cultured cells were subjected to polymerase chain reaction analysis with primers selected for the pol and gag (human spumaretrovirus), pol and env (HTLV-I), and pol (HTLV-II) genes. Polymerase chain reaction was negative in all patient and blood donor control samples, whereas positive controls were consistently reactive with high sensitivity. No culture exhibited cytopathic effects and supernatants were negative for reverse transcriptase activity. Thus, our results do not support a role for these retroviruses in the pathogenesis of multiple sclerosis.


[Contribution of immunohistochemistry and in situ hybridization techniques in diseases caused by or associated with Epstein-Barr virus]. Epstein-Barr virus is associated with many diseases. Today, the pathologist may study either by immunohistochemistry or in situ hybridization on tissue sections: EBV genome, EBV messenger RNA, EBV latent and replicative proteins. Several technics can be performed on fixed paraffin-embedded tissue to demonstrate the presence of EBV DNA, EBER-1 RNA, LMP-1 protein. Frozen tissues are required for the study of EBNA-2, ZEBRA and replicating proteins expression. The results, obtained during the study of benign and malignant proliferations always or often associated with EBV, such as infectious mononucleosis, Burkitt's lymphomas, AIDS associated lymphomas, lymphoproliferations in immunocompromised patients, Hodgkin's disease, and some epithelial proliferations, are summarized.


Characterization of Trypanosoma cruzi isolates from Paraguay, using restriction enzyme analysis of kinetoplast DNA. Eleven Paraguayan strains of Trypanosoma cruzi, from Chagas' disease patients and the bug vectors, were examined by restriction endonuclease analysis of kinetoplast DNA using Hae III, Msp I, Eco RI, HinfI, Taq I and Rsa I. Four schizodeme-profile groups were identified. Group 1 had much simpler profiles than groups 2, 3 and 4 and, although there were homogeneous profiles in the latter three groups, each group could be distinguished from the others. The profiles of group 1 could not be matched with any of the standard strains from Brazil, Chile and Columbia included in the schizodeme comparison. The profiles of groups 3 and 4 shared most features with those standards of the Brazilian Z2 zymodeme.


Antibody to hepatitis C virus in patients with chronic schistosomiasis. To clarify the effect of hepatitis C virus (HCV) infection in patients with chronic schistosomiasis, 96 patients with schistosomiasis and 137 patients with chronic liver disease without schistosomal infection were analysed by domination of antibody to HCV (anti-HCV). In 45 of 96 schistosomiasis patients, the serum alanine aminotransferase (ALT) level was continuously elevated, and the positive rate of anti-HCV was 52.9%, which is almost the same prevalence rate as in patients with chronic liver disease (48.9%). In contrast, in the remaining 51 schistosomiasis patients, serum ALT level was continuously within the normal range and the positive rate of anti-HCV was 0%. Histological investigation showed that the positive rate of anti-HCV in HBsAg-negative schistosomiasis patients was 14% for hepatic fibrosis, 71% for chronic hepatitis, 80% for liver cirrhosis and 56% for hepatocellular carcinoma. In all anti-HCV-positive patients, serum ALT level was continuously elevated. The serum transaminase levels in anti-HCV-positive patients were higher than those in anti-HCV-negative patients. These data suggest that in patients with chronic schistosomiasis, HCV infection accelerates the derangement of liver function, and may be a major aetiological factor in the development of chronic hepatitis and liver cirrhosis, supporting a causative association between HCV infection and hepatocellular carcinoma.


Radical laser segmentectomy for T1 N0 lung cancer. Over the last 40 months, 18 lung cancer patients with T1 N0 non-small cell lung carcinoma have been treated with radical laser segmentectomy. This innovative operative method consists of a combination of anatomical or nonanatomical segmentectomy by neodymium:yttrium-aluminum garnet laser parenchyma sparing with complete hilar lymph node dissection. Although the median follow-up period is too short, there is no local recurrence and no cancer deaths. There have been no major complications. Even deep-seated tumors can be resected with a clear safety margin using this method. Radical laser segmentectomy may be a useful adjunct to preserve normal lung tissue and to perform very radical resection.


Dose ranging efficacy and safety of subcutaneous sumatriptan in the acute treatment of migraine. US Sumatriptan Research Group. Sumatriptan, a specific serotonin1-like receptor agonist, was studied in the acute treatment of migraine. Two hundred forty-two adult migraineurs participated in a randomized, double-blind study in which one dose of 1, 2, 3, 4, 6, or 8 mg of subcutaneous sumatriptan succinate was evaluated in sequential ascending fashion. At each dose level, a placebo group was included. Efficacy was defined as reduction of moderate or severe pain to mild or no pain, without the use of rescue medication. Headache relief rates showed an approximate dose-response relationship and at 1 hour were as follows: placebo, 24%; 1 mg, 43%; 2 mg, 57%; 3 mg, 57%; 4 mg, 50%; 6 mg, 73%; and 8 mg, 80%. Relief of nausea and improvement in clinical disability were also approximately dose related. Adverse events were dose related; the most common types were injection site reactions and tingling. The 6-mg dose was as effective as the 8-mg dose but was associated with fewer adverse effects and so is optimal.


Neurologic manifestations of progressive systemic sclerosis. Neurologic involvement in progressive systemic sclerosis is considered uncommon. We retrospectively examined the prevalence and nature of neurologic complications in 50 patients with progressive systemic sclerosis. In 20 (40%), neurologic abnormalities were detected, with a total of 28 neurologic manifestations. All levels of the central and peripheral nervous system were affected: muscle (22%), peripheral nerve (18%), spinal cord (8%), and brain (6%). Of note were the presence of myelopathy in four patients and inclusion-body myositis in two. In 10 patients (20%), no definable cause of the neurologic dysfunction could be identified, apart from progressive systemic sclerosis. Thus, neurologic presentations of progressive systemic sclerosis are much more common than previously reported and may be due to direct involvement of the nervous system by a primary pathologic process in a significant number of patients.


Menopause related changes of adrenocortical steroid production. To elucidate changes of adrenocortical steroid production with age, reproductive age (n = 14), peri-menopausal (n = 12) and post-menopausal (n = 13) women were studied using basal hormone levels and the results of a rapid ACTH stimulation test. Peripheral serum levels of pregnenolone, 17 alpha-hydroxypregnenolone, 17 alpha-hydroxyprogesterone, dehydroepiandrosterone and androstenedione were measured by RIA. The basal levels of all steroids except pregnenolone decreased with age. A significant increase in the levels of all steroids after ACTH stimulation was observed in all subjects. Although, there was an age related decrease in the response of C19-steroids to ACTH, the response of pregnenolone and 17 alpha-hydroxyprogesterone to ACTH did not alter with age. The preserved response of 17 alpha-hydroxyprogesterone to ACTH in post-menopausal women suggests that 17 alpha-hydroxyprogesterone production plays a role in the age independency of cortisol. In the present paper, the changes in the capacity of the post-menopausal adrenal cortex to produce androgens and C21-steroids were demonstrated.


Detection of human papillomavirus DNA in the normal cervices of Japanese women by the dot-blot (Vira Pap) method. Exfoliated cervical cells obtained from 9 patients with cervical intraepithelial neoplasia (CIN) I and II, 11 with CIN III, 22 with cervical carcinoma, and 599 with cytologically normal cervices (including 63 pregnant women) were evaluated for the presence of human papillomavirus (HPV) by the dot-blot (Vira Pap) method. HPV infection was detected in 33.3% of the CIN I and II cases, in 54.5% of the CIN III cases, in 68.2% of invasive cervical carcinoma cases, and in 4.2% of the normal group. The rate of detectable HPV infection increased in exfoliated cervical cells during pregnancy. These results suggested that a few Japanese women with cytologically normal cervices were infected with HPV.


Characterization of hypervariable regions in the putative envelope protein of hepatitis C virus. We previously identified two hypervariable regions [HVR1 (27 amino acids) and HVR2 (7 amino acids)] in the putative envelope glycoprotein (gp70) by comparison of the amino acid sequences of many isolates of the HCV-II genotype. To understand the functional features of these HVRs, using the polymerase chain reaction we analyzed the rate of actual sequence variability in the region including HVR1 and HVR2 of HCV isolated successively at intervals of several months from two patients with chronic C-type hepatitis. In both patients, the amino acid sequence of HVR1, but not HVR2, was found to change dramatically during the observation period (about one amino acid per month). However, no alteration of the amino acid sequence of HVR1 of HCV was observed in a patient in the acute phase of chronic hepatitis. Restriction digestion analysis of sequence diversity showed that a HCV genome with a newly introduced mutation in HVR1 often became the predominant population at the next time of examination. Alterations of amino acids in HVR1 occurred sequentially in the two patients in the chronic phase. These findings suggest that mutations in HVR1 are involved in the mechanism of persistent chronic HCV infection.


Inhibition of insulin secretion by KN-62, a specific inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase II. The effects of KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CamPKII), on insulin secretion and protein phosphorylation were studied in rat pancreatic islets and RINm5F cells. KN-62 was found to dose-dependently inhibit autophosphorylation of CamPKII in subcellular preparations of RINm5F cells (K0.5 = 3.1 +/- 0.3 microM), but had no effect on protein kinase C or myosin light chain kinase activity. KN-62, but not the inactive analogue KN-04, dose-dependently inhibited glucose-induced insulin release (K0.5 = 1.5 +/- 0.5 microM) in a manner similar to the inhibition of CamPKII autophosphorylation. KN-62 (10 microM) inhibited carbachol (in the presence of 8 mM glucose) and potassium-stimulated insulin secretion from islets by 53% and 59%, respectively. These results support a role of CamPKII in glucose-sensitive insulin secretion.


Frequent amplification of the c-met gene in scirrhous type stomach cancer. Amplification of the c-met gene, that encodes hepatocyte growth factor receptor, was examined on human esophageal, gastric and colorectal carcinomas. Six (55%) of the 11 gastric carcinoma cell lines and 15 (23%) of the 64 advanced gastric carcinomas showed the c-met gene amplification. Among them, c-met amplification was detected in 5 gastric cancer cell lines, derived from scirrhous gastric carcinoma and in 5 (38%) of 13 scirrhous gastric carcinoma tissues. Furthermore, patients of gastric carcinoma with c-met amplification showed significantly advanced tumor stage and poorer prognosis than those without the amplification. Conversely, no amplification was detected in any of the esophageal and colorectal carcinoma cell lines as well as carcinoma tissues except one colonic carcinoma. These results overall suggest that amplification of the c-met gene might participate in carcinogenesis and progression of stomach cancer, especially scirrhous type stomach carcinoma.


Dipeptide inhibitors of ubiquitin-mediated protein turnover prevent growth factor-induced neurite outgrowth in rat pheochromocytoma PC12 cells. Dipeptide inhibitors of the ubiquitin-dependent proteolysis pathway governed by N-terminal recognition (N-end rule) in reticulocyte lysates significantly suppress NGF- and bFGF-induced neurite outgrowth in rat pheochromocytoma PC12 cells, but do not cause retraction of already formed neurites. Peptides which do not inhibit proteolysis are also without effect on PC12 cell differentiation. Suppression of neurite outgrowth is readily reversible upon removal of the inhibitors. These data demonstrate a requirement for specific protein turnover in the process of neuron-like differentiation in PC12 cells and provide the first demonstration of a physiological role for the N-end rule.


Cloning, sequencing, and expression of human gonadotropin releasing hormone (GnRH) receptor. Gonadotropin releasing hormone is a hypothalamic decapeptide that stimulates the release of gonadotropic hormones from the anterior pituitary gland. Therapeutically, the human pituitary GnRH receptor is the target of agonists used in the suppression of prostate cancer. Here we report the isolation of a cDNA representing this receptor. It encodes a protein with a transmembrane topology similar with that of other G protein-coupled, 7-transmembrane receptors. Binding studies of the cloned receptor demonstrate high affinity and pharmacological properties similar with the native human pituitary GnRH receptor. Northern blot and reverse transcriptase/PCR analysis revealed that its mRNA is expressed in pituitary, ovary, testis, breast, and prostate but not in liver and spleen. Availability of a human GnRH receptor cDNA should permit the design of improved analogs for therapeutic applications.


Changes in cell attachment by RSV transformation in rat liver epithelial cells. BRL, a non-malignant rat liver epithelial-like cell line, possessed the ability to adhere through fibronectin to a solid substrate. Oncogenical transformation of these BRL cells with RSV induced a significant decrease in the fibronectin molecules in the extracellular matrix and reduction in its ability to adhere to fibronectin. The alpha 5 and beta 1 subunits of integrin (fibronectin receptor) were quantitatively diminished during RSV transformation in BRL cells. These results suggest that adhesive reduction of BRL cells to a substrate by RSV transformation may be caused by a decrease in cell surface fibronectin and fibronectin receptor molecules.


Molecular cloning and characterization of the human and porcine transforming growth factor-beta type III receptors. Full-length cDNAs for the transforming growth factor-beta (TGF-beta) type III receptors were isolated from porcine uterus and human placenta cDNA libraries. The human TGF-beta type III receptor coding region encodes a protein of 849 amino acids with a single transmembrane domain and a short stretch of the intracellular domain. Potential glycosaminoglycan attachment sites were found in the extracellular domain. The overall amino acid sequence identities with those of the porcine and rat TGF-beta type III receptors were 83% and 81%, respectively. A high degree of sequence conservation was observed in the transmembrane and intracellular domains, which also have sequence similarity with human endoglin. In addition, two portions with 29 and 52 amino acids in the extracellular domain were found to be substantially similar with human endoglin.


Synergistic stimulation of nitric oxide hemoglobin production in rats by recombinant interleukin 1 and tumor necrosis factor. Nitric oxide (NO) is formed from arginine in Escherichia coli lipopolysaccharide (LPS) treated rat; however, none of specific cytokine inducing NO generation is yet determined. We studied the effect of interleukin 1 (IL-1) and tumor necrosis factor (TNF) on NO production in rats by detecting NO-hemoglobin in their blood, using electron spin resonance. Either IL-1 or TNF alone stimulated NO-hemoglobin formation. Combined administration of IL-1 and TNF markedly enhanced NO-hemoglobin generation, demonstrating the synergistic character of both stimuli on NO production. Further, LPS and TNF in combination were more potent stimulator of NO-hemoglobin production in rats than each alone.


Metabolism of halogenated bisphosphonates by the cellular slime mould Dictyostelium discoideum. Methylenebisphosphonate and its monofluoro-, difluoro- and dichloro- derivatives inhibited growth of amoebae of Dictyostelium discoideum. Dichloromethylenebisphosphonate was the most potent inhibitor of amoebal growth whereas difluoromethylenebisphosphonate was the least potent inhibitor. Each of the bisphosphonates was taken up by the amoebae and incorporated into the corresponding beta, gamma-methylene analogue of adenosine triphosphate. Two of the bisphosphonates were also incorporated into the corresponding analogues of diadenosyl tetraphosphate. No correlation was found between the ability of the bisphosphonates to inhibit amoebal growth and the extent to which they were metabolised.


Phospholipid transverse mobility modifications in plasma membranes of activated platelets: an ESR study. Spin labeled phospholipid analogs were used to directly study changes in aminophospholipid translocase activity in activated platelets. In thrombin-activated platelets, the translocase activity was slightly stimulated, whereas no vesicle formation or proteolysis of cytoskeletal protein occurred. Ca2+ ionophore A23187-mediated activation produced vesiculation and proteolysis. Additionally, the translocase activity was completely inhibited, probably due to a sharp rise the intracellular Ca2+ concentration, as shown when platelets were activated in the presence of various A23187 and Ca2+ concentrations and by the recovery of the translocase activity when Ca2+ was complexed with EGTA. No translocase activity was found in vesicles. Whereas vesiculation and translocase inhibition can occur independently of proteolysis, this later accentuated the shedding phenomenon.


Relation of superoxide generation and lipid peroxidation to the inhibition of NADH-Q oxidoreductase by rotenone, piericidin A, and MPP+. The addition of NADH to submitochondrial particles inhibited by agents which interrupt electron transport from NADH-Q oxidoreductase (Complex I) to Q10 (rotenone, piericidin A, and MPP+) results in superoxide formation and lipid peroxidation. A study of the quantitative relations now shows that oxyradical formation does not appear to be the direct result of the inhibition. Although tetraphenyl boron (TPB) greatly enhances the inhibition by MPP+, it has no effect on O2. formation or lipid peroxidation. When submitochondrial particles completely inhibited by rotenone or piericidin A are treated with bovine serum albumin to remove spuriously bound inhibitor molecules without affecting those bound at the specific inhibition site, NADH-Q activity remains inhibited and lipid peroxidation occurs but superoxide formation ceases. Thus oxyradical formation may be the result of the binding of inhibitors at sites in the membrane other than those related to the inhibition of electron transport.


Amino acid transport systems in the human hepatoma cell line Hep G2. The human hepatoma cell line Hep G2 was used to investigate amino acid transport systems in human liver tissue. The ubiquitous transport systems responsible for the uptake of most neutral amino acids (systems A, ASC and L) were found to be present. Transport system A was predominant for proline uptake but system ASC was the major Na(+)-dependent transport system, particularly for glutamine. The specific hepatic system N was functional, but only partially mediated glutamine uptake. The study of Na(+)-independent arginine uptake demonstrated the presence of the cationic transport system Y+, reflecting the transformed nature of Hep G2 cells.


Purification and characterization of a sialidase from Bacteroides fragilis SBT3182. A sialidase from Bacteroides fragilis SBT3182 was purified 2,240-fold to apparent homogeneity by ammonium sulfate precipitation and sequential chromatographies on DEAE-Toyopearl 650M, Hydroxyapatite, MonoS and Superose6 columns. The N-terminal amino acid sequence of this sialidase, Ala-Asp-X-Ile-Phe-Val-Arg-Glu-Thr-Arg-Ile-Pro-, was determined. Substrate specificity of this enzyme using a variety of sialoglycoconjugates showed a 1.5- and 2.2-fold preference for sialyl alpha 2-8 linkages when compared with alpha 2-3 and alpha 2-6 bound sialic acids, respectively. The native sialidase had a molecular weight of 165kDa, as determined by Superose6 gel filtration chromatography and consisted of three subunits each of 55kDa by SDS-polyacrylamide gel electrophoresis. This enzyme had optimal activity at pH6.1 with colominic acid as substrate.


Comparison of cytotoxicity of the (-)- and (+)-enantiomer of 2',3'-dideoxy-3'-thiacytidine in normal human bone marrow progenitor cells. The effects of racemic cis-2',3'-dideoxy-3'-thiacytidine [(+/-)-BCH-189] and its two enantiomers on human myeloid and erythroid colony-forming cells were studied by clonogenic assays. The (+)-isomer was the most toxic with a median inhibitory concentration approximating 2 microM in both cell lineages. In contrast, concentrations of the (-)-isomer required for 50% inhibition of granulocyte macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) were 33.9 +/- 15.1 and 169.4 +/- 87.9 microM, respectively. The racemic BCH-189 was quite toxic to these cells, but to a lesser extent than observed with 3'-azido-3'-deoxythymidine and 3'-fluoro-3'-deoxythymidine (positive controls).


Effect of stilbene derivatives on gastric H+, K(+)-ATPase. The effect of naturally occurring hydroxystilbene, 3,3',4,5-tetrahydroxystilbene (piceatanol), and its derivatives on gastric H+, K(+)-ATPase was studied. Piceatanol inhibited H+, K(+)-ATPase in a dose-dependent manner. The 50% inhibition value was 4.3 x 10(-6) M. It was found from the kinetic study that the inhibition of the enzyme by piceatanol was competitive with respect to ATP and was noncompetitive with respect to K+. Piceatanol also effectively inhibited gastric acid secretion. However, methylation of phenolic hydroxy groups of piceatanol resulted in a complete loss of inhibition of the enzyme and acid secretion, suggesting the role of phenolic hydroxy groups in the inhibition. The study on hydroxystilbene derivatives also showed that phenolic hydroxy groups are important in the interaction with H+, K(+)-ATPase and that stilbenes with neighbouring hydroxy groups are the most effective inhibitors.


Woodfruticosin (woodfordin C), a new inhibitor of DNA topoisomerase II. Experimental antitumor activity. Woodfruticosin (woodfordin C) (WFC), a new inhibitor of DNA topoisomerase II (topo-II), was isolated from methanol extract of Woodfordia fruticosa Kurz (Lythraceae) and studied for in vitro and in vivo antitumor activities in comparison with Adriamycin (ADR) and etoposide (ETP), well known inhibitors of topo-II. The inhibitory activity against DNA topo-II shown by WFC was much stronger than that shown by ETP or ADR. WFC inhibited strongly intracellular DNA synthesis but not RNA and protein synthesis. On the other hand, WFC had a weaker growth inhibitory activity against various human tumor cells than ETP or ADR, but it showed remarkable activity against PC-1 cells and moderate activity against MKN45 and KB cells. Furthermore, WFC had in vivo growth inhibitory activity against s.c. inoculated colon38. These results indicate that the mechanism by which WFC exhibits antitumor activity may be through inhibition of topo-II.


Chlorpromazine increases the turnover of metabolically active phosphoinositides and elevates the steady-state level of phosphatidylinositol-4-phosphate in human platelets. Non-permeabilizing concentrations (< 40 microM) of chlorpromazine (CPZ) increase the radioactivity of phosphatidylinositol-4-phosphate (PIP) in platelets pre-labelled with [32P]Pi, but the biochemical mechanisms underlying this increase are poorly understood. Incubation of [32P]Pi-labelled, gel-filtered platelets with 25 microM CPZ for 10 min increased: (1) the mass of PIP from 315 to 476 nmol/10(11) platelets but not the total inositol phospholipid mass, (2) the specific phosphodiester radioactivities in phosphatidylinositol (PI), PIP and phosphatidylinositol-4,5-bisphosphate (PIP2) by 34, 63 and 37%, respectively, and (3) the specific phosphomonoester radioactivities in PIP and PIP2 by 53 and 10%, respectively. In control platelets (no CPZ) the specific radioactivity of the phosphodiester was the same in PI, PIP and PIP2, and the specific radioactivity in the phosphomonoester in PIP and PIP2 was 55% of that of the gamma-phosphoryl in ATP, measured as metabolically active, actin-bound ADP. These results suggest that 55% of each of PI, PIP and PIP2 constitutes a metabolic pool which is labelled by 32P in the platelets, while the remainder is in a metabolically inactive pool and not labelled. CPZ has two major effects: (1) CPZ interferes with the kinase and phosphohydrolase reactions that maintain the steady-state level of PIP in the metabolic phosphoinositide pool, resulting in a 92% increase in the PIP level of this pool, and (2) CPZ causes synthesis (45% in 10 min) of new phosphodiester in the metabolically active phosphoinositides by tentative stimulation of the turnover of the phosphoinositide cycle, de novo phosphoinositide synthesis and/or diacylglycerol formation through phospholipases C and D. The marked alteration by CPZ of phosphoinositide metabolism may be part of the mechanism by which this drug effects its psychotropic action.


Effect of delta-9-tetrahydrocannabinol and theophylline on hepatic microsomal drug metabolizing enzymes. Theophylline (Th) under in vitro conditions stimulated the activities of rat liver microsomal aniline hydroxylase, N-demethylase and O-demethylase, while delta-9-tetrahydrocannabinol (delta-9-THC) inhibited the activities of these hepatic microsomal drug metabolizing enzymes under similar conditions. delta-9-THC-induced inhibition of hepatic microsomal drug metabolizing enzymes was significantly reduced in the presence of Th. Analysis of Lineweaver-Burk plots showed that Th-induced stimulation of hepatic microsomal drug metabolizing enzymes occurs due to an increase in substrate affinity (1/Km) and of Vmax. delta-9-THC-induced inhibition of N-demethylase and O-demethylase is probably due to competition of the drug with the substrates for a common intermediate in the microsomal electron transport chain. Non-competitive and mixed-type inhibition caused by delta-9-THC on aniline hydroxylation appears to be associated with a non-specific action of delta-9-THC. Blocking of delta-9-THC-induced inhibition or reduction of Th-induced stimulation of hepatic drug metabolizing enzymes with Th or delta-9-THC was due to an increase or decrease in either Vmax, substrate affinity (1/Km) or both with respect to the corresponding Km and Vmax observed with delta-9-THC or Th alone.


Oxidative metabolism of amsacrine by the neutrophil enzyme myeloperoxidase. Oxidative metabolism of the anti-cancer drug amsacrine 4'-(9-acridinylamino) methane-sulphan-m-anisidide has been suggested to account for its cytotoxicity. However, enzymes capable of oxidizing it in non-hepatic tissue have yet to be identified. A potential candidate, that may be relevant to the metabolism of amsacrine in blood and its action in myeloid leukaemias and myelosuppression, is the haem enzyme myeloperoxidase. We have found that the purified human enzyme oxidizes amsacrine to its quinone diimine, either directly or through the production of hypochlorous acid. In comparison, the 4-methyl-5-methylcarboxamide derivative of amsacrine, CI-921 9-[[2-methoxy-4[(methylsulphonyl)-amino]phenyl]amino)-N, 5-dimethyl-4-acridine carboxamide, reacted poorly with myeloperoxidase, although it was oxidized by hypochlorous acid. Detailed studies of the mechanism by which myeloperoxidase oxidizes amsacrine revealed that the semiquinone imine free radical is a likely intermediate in this reaction. Oxidation of amsacrine analogues indicated that factors other than their reduction potential determine how readily they are metabolized by myeloperoxidase. Both amsacrine and CI-921 inhibited production of hypochlorous acid by myeloperoxidase. CI-921 acted by trapping the enzyme as the inactive redox intermediate compound II. Amsacrine inhibited by a different mechanism that may involve conversion of myeloperoxidase to compound III, which is also unable to oxidize Cl-. The susceptibility of amsacrine to oxidation by myeloperoxidase indicates that this reaction may contribute to the cytotoxicity of amsacrine toward neutrophils, monocytes and their precursors.


Inhibition of rat liver microsomal lipid peroxidation by boldine. The alkaloid boldine, found in the leaves and bark of boldo, was an effective inhibitor of rat liver microsomal lipid peroxidation under a variety of conditions. The following systems all displayed a similar sensitivity to boldine: non-enzymatic peroxidation initiated by ferrous ammonium sulfate; iron-dependent peroxidation produced by ferric-ATP with either NADPH or NADH as cofactor; organic hydroperoxide-catalyzed peroxidation; and carbon tetrachloride plus NADPH-dependent peroxidation. Boldine inhibited the excess oxygen uptake associated with microsomal lipid peroxidation. Thus, boldine was effective in inhibiting iron-dependent and iron-independent microsomal lipid peroxidation, with 50% inhibition occurring at a concentration of about 0.015 mM. Boldine did not appear to react efficiently with superoxide radical or hydrogen peroxide, but was effective in competing for hydroxyl radicals with chemical scavengers. Concentrations of boldine which produced nearly total inhibition of lipid peroxidation had no effect on microsomal mixed-function oxidase activity nor did boldine appear to direct electrons from NADPH-cytochrome P450 reductase away from cytochrome P450. Boldine completely protected microsomal mixed-function oxidase activity against inactivation produced by lipid peroxidation. The effectiveness of boldine as an anti-oxidant under various conditions, and its low toxicity, suggest that this alkaloid may be an attractive agent for further evaluation as a clinically useful anti-oxidant.


Interaction of the malonyldialdehyde molecule with membranes. A differential scanning calorimetry, 1H-, 31P-NMR and ESR study. The membrane interactions of malonyldialdehyde (MDA), natural product of polyunsaturated fatty acids peroxidation were investigated by differential scanning calorimetry, and ESR or NMR spectroscopy. This component is located in the superficial part of the bilayer, where it increases the local fluidity. High concentrations of MDA induce major membrane damage. Similar consequences of MDA-membrane interactions were observed on erythrocyte ghosts.


Protective effect of liposome-associated alpha-tocopherol against paraquat-induced acute lung toxicity. The present study was undertaken to investigate whether alpha-tocopherol, entrapped in liposomes and delivered directly to the lung, could protect against paraquat-induced lung damage in the rat. Plain liposomes (composed of dipalmitoylphosphatidylcholine, DPPC) or DPPC/alpha-tocopherol liposomes were administered intratracheally to animals 24 hr prior to an intraperitoneal injection of paraquat (20 mg/kg); rats were killed 24 or 48 hr after paraquat treatment. Results of this study showed that lungs of animals treated with paraquat were damaged extensively as evidenced by an increase in lung weight and a significant reduction in lung angiotensin-converting enzyme (ACE) activity and cytochrome P450 concentration. Furthermore, paraquat treatment resulted in a significant decrease in reduced glutathione (GSH) concentrations and a marked elevation in microsomal lipid peroxidation levels as measured by the formation of diene conjugates. Pretreatment of rats with DPPC liposomes alone did not alter significantly the paraquat-induced changes of all parameters examined. On the other hand, pretreatment of rats with DPPC/alpha-tocopherol liposomes 24 hr prior to paraquat challenge resulted in a significant increase in pulmonary alpha-tocopherol concentrations and antagonized paraquat-induced changes in lipid peroxidation, GSH/GSSG ratio, lung ACE activity and cytochrome P450 concentrations. Results of this study suggested that alpha-tocopherol, delivered directly to the lung in a liposomal formulation 24 hr prior to paraquat administration, confers protection against paraquat-induced lung damage.


Regulation of ATP-sensitive K+ channels by chronic glyburide and pinacidil administration. Treatment of rats with the K(ATP)+ channel antagonist sulfonylurea, glyburide (3 mg/kg/day, i.p., every 12 hr for 9 days), increased the Bmax value of [3H]glyburide binding to heart and whole brain total membranes by 30 and 24%, respectively. The ligand affinity was unaltered. Treatment with the K+ channel activator, pinacidil (20 mg/kg/day, i.p., every 12 hr for 9 days), did not alter the Bmax value for cardiac [3H]glyburide binding sites, but decreased the Bmax value in the brain by 21%. Chronic administration of hydralazine, which caused an acute reduction in systolic blood pressure equivalent to that of pinacidil, did not alter [3H]glyburide binding in either heart or brain. Treatment with glyburide, pinacidil or hydralazine did not alter L-type calcium channels, assessed by [3H]PN 200 110 binding, in cardiac and brain membranes or small size Ca(2+)-activated K+ channels in brain assessed by [125I]apamin binding. These studies show that the ATP-sensitive class of K+ channels can be regulated following chronic drug treatment in similar fashion to other receptor and channel systems.


GABAergic involvement in the acquisition of voluntary ethanol intake in laboratory rats. Treatment with the GABAA agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) or the GABAB agonist baclofen was shown to enhance the acquisition of voluntary ethanol consumption in laboratory rats. THIP administration resulted in an increased intake of absolute ethanol without an increase in total fluid intake. In contrast, baclofen administration, while increasing ethanol intake in a manner similar to that seen with THIP, also increased total fluid intake, indicating that its effects may not be specific to an interaction with ingested ethanol. The data obtained in the present study support the notion that GABAergic mechanisms, particularly those related to the GABAA receptor, may be involved in the acquisition of voluntary ethanol consumption in laboratory rats.


Use of head injury instruction cards in accident centres. This study reports on the value of head injury instruction cards as purveyors of information to patients. Patients over 10 years old attending the North Staffordshire Royal Infirmary Accident and Emergency Department with a mild head injury were invited to attend a special brain injury follow-up clinic, where they were asked to complete a questionnaire. Patients failing to attend this clinic were contacted and asked to complete the questionnaire. This revealed that they were no less severely affected by the injury as the attenders. They were asked why they had not attended and a significant number of patients had no recall of being given a head injury instruction card on which the information of the follow-up clinic was given. Memory loss was the most likely cause of this and is more common than is widely appreciated in patients attending accident and emergency departments.


Structural differences in active site-labeled thrombin complexes with hirudin isoinhibitors. Hirudin, a 65 amino acid polypeptide from the medicinal leech, is the most potent thrombin inhibitor known to date. Recently, recombinant forms have been reported, which are as effective as the isolated forms. The studies presented here demonstrate sensitive spectroscopic methods for distinguishing binding of two recombinant hirudins, HV1 and HV2-Lys 47, with active site-labeled human alpha-, epsilon- and zeta-thrombins. Specifically, fluorosulfonylphenyl nitroxide spin labels, dansyl fluoride and p-nitrophenylanthranilate, were employed as active site-directed covalent reporter groups. In general, the nitroxide immobilization was greater for spin-labeled thrombin complexes with HV2-Lys 47 vs. HV1. The two fluorophore moieties, dansyl and anthraniloyl, were also sensitive to differences in HV1 and HV2-Lys 47 binding, including interactions with loop 145-150 of the thrombin structure where the epsilon- and zeta-thrombin cleavages exist. Speculation over sequence differences between the two isoinhibitors centers on residues 24 and 47, both of which involve either a loss or gain of charge on the side chain.


Recombinant human glucagon: large-scale purification and biochemical characterization. Recombinant glucagon was expressed in Escherichia coli as a fusion protein including the glucagon sequence therein as previously reported [Ishizaki et al. (1992). Appl. Microbiol. Biotechnol. 36, 483-486]. We developed a large-scale method for the isolation and purification of recombinant glucagon. After cell disruption, the resultant pellets were solubilized with 2 M guanidine-HCl, to which Staphylococcus aureus V8 protease had been added, and were digested into intermediates composed of 53- and 60-residue peptides containing the glucagon moiety. After the digestion came to an end, the solution was desalted, and the remaining V8 protease was allowed to resume digestion of the intermediates into glucagon, followed by partial purification by S-Sepharose and Sephacryl S-100 chromatographies. The glucagon obtained was found to be not less than 99.5% pure by analytical HPLC. One liter of culture produced about 180 mg of pure glucagon. The amino acid composition and the sequence agreed well with the theoretical values. Radioreceptor assay gave an affinity constant similar to that of pancreatic glucagon, and similar activities in cAMP production and glycogenolysis were also observed. Thus, the recombinant glucagon was confirmed to be biochemically identical with pancreatic glucagon.


Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: a consequence of the formation of "native-like conformation". The influence of n-propanol on the overall alpha-helical conformation of beta-globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of alpha-helical conformation is induced into these proteins at pH 6.0 and 4 degrees C in the presence of relatively low concentrations of n-propanol. The induction of alpha-helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of alpha-globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The alpha-helical conformation induced into alpha- and beta-globin by n-propanol decreased as the temperature is raised from 4 to 24 degrees C. In contrast, the alpha-helical conformation of both alpha- and beta-chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced "alpha-helical conformation" of globins and the "alpha-helical conformation" of alpha- and beta-chains. A cross-correlation of the n-propanol induced increase in the fluorescence of beta-globin with the corresponding increase in the alpha-helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the alpha-helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a "native-like structure." The induction of alpha-helical conformation into these proteins in the presence of n-propanol and the consequent generation of "native-like conformation" is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an alpha-helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases.(ABSTRACT TRUNCATED AT 400 WORDS)


Identification of the ATP.Mg-dependent protein phosphatase activator (FA) as a synapsin I kinase that inhibits cross-linking of synapsin I with brain microtubules. The ATP.Mg-dependent protein phosphatase activating factor (FA) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factor FA has further been identified as a cAMP and Ca(2+)-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factor FA could phosphorylate synapsin I with a low Km value of about 2 microM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factor FA specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca2+/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factor FA could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factor FA as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission.


The motivational benefits of a dentifrice containing baking soda and hydrogen peroxide. Twenty-two family practice dentists, in a large metropolitan area, were recruited to act as independent examiners in a study to evaluate the compliance of their patients to accept a good oral hygiene regimen with the use of a fluoride dentifrice, containing hydrogen peroxide and baking soda, dispensed from a dual dispensing package. To evaluate compliance, the dentists attended an orientation seminar and were trained to assess gingival health using the CPITN periodontal probe. Each dentist evaluated the gingival health status of five to seven of his own patients, initially and after one and three months of product use following hygiene instruction and product assignment. One-hundred and thirty-one patients successfully completed the study. After one month of using the hydrogen peroxide/baking soda toothpaste, the mean reduction in bleeding sites was 53%; at three months the reduction was 62%. The hydrogen peroxide/baking soda dentifrice was well accepted by dentist and patient, and a discernible improvement in oral health of the patients was achieved when the product was used in a conscientious oral hygiene program.


Hippocampal electrical activity in the female rat: the estrous cycle, copulation, parturition, and pup retrieval. Hippocampal electrical activity was examined in female rats across the four phases of the estrous cycle, as well as during copulation, parturition, and pup retrieval. Possible hormonal effects on hippocampal activity were examined by recording daily during struggling and immobility throughout five estrous cycles. No differences in the characteristics of rhythmical slow activity (RSA), large-amplitude irregular activity (LIA), or their relation to behavior were found between the phases of the estrous cycle. Behaviors examined during copulation were lordosis, hopping, and ear wiggling. Lordosis and ear wiggling were both accompanied by irregular waves, with some low frequency RSA also present during lordosis. Hopping was accompanied by RSA which was greater in amplitude and frequency than RSA during walking. Parturition behaviors examined included body extensions, genital or pup licking, and 'body flattening'. Body extensions were found to be associated with irregular hippocampal activity, and appeared to be a Type 2 behavior. Interestingly, sudden suppressions of the hippocampal record lasting 1-5 s often preceded body extensions. Irregular hippocampal activity was present at all times during genital and pup licking. Body flattening was typically accompanied by very high amplitude irregular waves which, although the rats were awake, resembled a sleeping hippocampal pattern. Pup retrieval involved walking and was thus always accompanied by RSA.


GABAergic mediation of indirect transsynaptic control over basal and spatial memory testing-induced activation of septo-hippocampal cholinergic activity in mice. A neurochemical study of the transsynaptic interactions established between septal GABAergic interneurones and cholinergic septo-hippocampal neurones was conducted using mice. The effects of acute in vivo injections of either muscimol (20-500 ng/0.2 microliter), bicuculline (100 ng-1 micrograms/0.2 microliter) or saline vehicle (0.2 microliter) into the medial septum on septo-hippocampal cholinergic activity were evaluated using measures of hippocampal high affinity choline uptake at 30 min post-injection in two main groups of mice. The first (quiet control) remained in their home cages during the post-injection period whereas the second (active) were submitted, 10 min following injection to a 20-min period of spatial working memory testing in an 8-arm radial maze. Intraseptal injections of either muscimol or bicuculline produced significant (25-50%) inhibition of hippocampal cholinergic activity in quiet conditions (basal) as compared to intact or saline-injected mice. In the active groups, whereas memory testing induced significant cholinergic activation (+15-20%) in intact and saline injected mice at 30 s post-test no significant memory testing-induced activation was observed in either muscimol or bicuculline-injected mice at any dose. The role of septal GABAergic interneurones in the indirect transsynaptic control over the basal and activated states of septo-hippocampal cholinergic activity is discussed with respect to the concept that these complex neuronal interactions contribute to the physiological mechanisms involved in the modulation of working memory performance.


Reversal learning of the rabbit nictitating membrane response following kindling-induced potentiation within the hippocampal dentate gyrus. The following experiment examined the effects of kindled seizures on reversal learning and the effects of both kindling and classical conditioning of the rabbit nictitating membrane on granule cell responsivity to perforant path input. Kindling resulted in significant potentiation of the population spike, the excitatory postsynaptic potential (EPSP) and the magnitude of twin pulse inhibition. Following kindling, rabbits were trained in a discrimination-reversal paradigm with either a tone or light paired with a corneal airpuff. Kindling did not affect acquisition of the initial discriminative response but did retard the rate of reversal learning. Kindling-induced potentiation, within dentate excitatory and inhibitory circuits, persisted for the duration of training. Thus, these results do not distinguish between the contribution of kindling-induced potentiation within dentate excitatory and inhibitory circuits to discrimination-reversal training. Spikes evoked during tone presentations were of reduced amplitude compared to spikes evoked either between trials or during light trials. The EPSP was not affected by stimulus conditions. In control rabbits, the magnitude of both the spike and EPSP increased across training. Training-related potentiation, in kindled rabbits, could not be separated from kindling-induced potentiation. These results demonstrate that an LTP-like effect of both the population spike and EPSP occurs with discrimination-reversal training.


Septo-hippocampal and nBM-cortical cholinergic neurones exhibit differential time-courses of activation as a function of both type and duration of spatial memory testing in mice. We previously showed that the initial acquisition session of a spatial discrimination (mixed reference/working memory) test in an 8-arm radial maze induced differential activations in the ascending cholinergic septo-hippocampal and nBM-cortical pathways in mice. This data showed that the duration of post-test cholinergic activation was longer in the nBM-cortical pathway than in the septo-hippocampal projection. Moreover, the post-test durations but not the immediate post-test amplitudes of activation in each pathway decreased progressively as a function of repeated daily acquisition sessions. In the present study we have thus tested the hypotheses that the time-courses of post-test cholinergic activation in the septo-hippocampal and nBM-cortical pathways may vary both as a function of the type of memory used (working vs. reference) and according to the duration of repeated daily testing. Cholinergic activity in vivo in the hippocampus or frontal cortex of mice was quantified using measures of sodium-dependent high-affinity choline uptake at two different times (30 s and 15 min) following specific spatial working or reference memory testing in an 8-arm radial maze. The memory tests were administered daily over a 13-day period to attain high levels of performance in each type of task. In comparison to control groups both types of memory testing induced significant post-test cholinergic activations in each brain region on Day 15. However, cholinergic activity remained elevated in frontal cortex at 15 min post-test following reference memory testing, whereas significantly shorter durations of cortical and hippocampal cholinergic activation were observed following working memory testing using short (1 min) retention intervals. The possible significance of these differential modifications to the time-course of the post-test activations in these cholinergic pathways in working and reference memory processes and the putative transsynaptic mechanisms involved are discussed.


Facilitation and recovery of shuttle box avoidance behavior after frontal cortex lesions is induced by a contingent electrical stimulation in the ventral tegmental nucleus. A bilateral ablation of the frontal cortex was performed in rats before and after training in an active avoidance task in a shuttle box. Animals with this lesion showed an impairment in learning and in the reversal of the avoidance task. If the animals with the lesion were implanted with an electrode in the ventral tegmental nucleus and received an electrical stimulation in this area contingent to a correct response (avoidance or escape response) in the behavioral task, they did not show any impairment in the performance of the task. Furthermore, the effect of the stimulation persisted after it was retrieved. The present findings indicate that the motivational and cue properties of the electrical stimulation of the ventral tegmental nucleus may serve to facilitate learning and reversal in an avoidance task and to induce at the long term a recovery process in animals in which the frontal cortex has been ablated. Therefore, this method may be useful to study the adaptative changes which take place in the nervous system after recovery from brain damage occurs.


Evidence for the involvement of serotonin in the antinociception induced by electrical or chemical stimulation of the mesencephalic tectum. A great deal of evidence has shown that electrical stimulation or microinjections of GABAA blockers, such as bicuculline, into the midbrain tectum (MT) produce escape behavior, which has been associated to fear. This study was aimed to examine the characteristics of the analgesia that follows the escape behavior induced by electrical (freezing and escape thresholds) and chemical (bicuculline microinjections) stimulation of the midbrain tectum. Immediately after the expression of the aversive responses the rats were submitted to the tail-flick test. The obtained results show that analgesia always follows aversive responses integrated at the MT level regardless of the kind of stimulation applied. The antinociceptive effects induced by either electrical or chemical stimulation of the MT were not antagonized by central microinjections of naloxone. On the other hand, the non-specific serotonin antagonist methysergide microinjected into the MT was effective in antagonizing the analgesia induced by any of the aversive stimulations. Based on these results we suggest that serotonin, but not opioid mechanisms, may be involved in the integration of antinociceptive responses to stimulation of the midbrain tectum.


A case-control study of dietary factors in patients with lung cancer. A case-control study was designed to investigate association of dietary factors with the risk of lung cancer in Sichuan, China. The cases consisted of 135 patients with preinvasive lung cancer which had been confirmed with histopathology, fiber bronchoscope, CT and X-ray film in three provincial hospitals in the recent one year. Controls were healthy subjects who went to one of these hospitals for health check-up; patients with pulmonary diseases was excluded. Controls were matched to cases for sex and age with a ratio of 1:1. Nutrient intakes, the eating habit and other relevant factors were investigated. The data analyzed with the conditional logistic regression model indicated that dietary beta-carotene intakes had a significantly inverse association with the risk of lung cancer. Vitamin C had a less significantly inverse association with the risk. Association of protein, fat, energy, retinol intakes or diet-balance index with the risk was not significant. Association of tea, alcohol, garlic or mushroom, respectively, with the risk was also not observed. Consumption of more processed foods and deep-fried foods were found to be risk factors. Smoking and air pollution from coal burning stoves were also observed as independent risk factors of lung cancer in the present study. The mental stress incidence in the case was significantly higher than that in the control.


Studies on biochemical mechanism of neurotoxicity induced by acrylamide in rats. The effects of acrylamide on calmodulin (CaM), cAMP, cGMP, Ca2+, Mg(2+)-ATPase and 45Ca2+ uptake in nervous system were determined in Wistar rats (ip, 10 or 50 mg.kg-1 for 12 d). The results indicate that acrylamide caused an alteration in calcium homeostasis in rat brain by decreasing the Ca(2+)-sequestering capacity of microsomes, and this may occur due to an efflux of calcium secondary to a microsomal structure damage. The changes of CaM in nervous system coupled with potential alteration in the intracellular Ca2+ concentration could affect many CaM-dependent enzymes (i.e. Ca2+, Mg(2+)-ATPase) and cyclic AMP system, and CaM or cAMP is known to be "toxicological second messenger" that could initiate neurotoxicity, though more work is needed to elucidate the details of the mechanism.


Is peroperative smear cytology necessary for CT-guided stereotaxic biopsy? Neuropathology is a relatively scarce resource with uneven geographical distribution, and some surgeons rely on macroscopic appearances of tissue and await later paraffin section histology for the final diagnosis. The effect of the introduction of a peroperative cytology service has been assessed in a 5-year series of CT-directed stereotaxic biopsies of intracranial mass lesions using a low cost adapted pre-CT stereotaxic frame. The technique has been mastered by 28 different surgeons with varying degrees of neurosurgical expertise and 259 procedures have been performed in 245 patients. Benign lesions were detected in 24 (9.8%) patients, confirming the importance of making a histological diagnosis prior to initiating treatment. Permanent morbidity was 6.5% and mortality 3.3% and morbidity was balanced by an improvement in 19.6% of the patients following the procedure. For the first 142 patients no peroperative histological diagnosis was available and a diagnostic rate of 86.6% was achieved. For the last 103 patients the availability of peroperative smears improved the diagnostic rate to 94.2% and reduced the number of second biopsies needed for diagnosis from seven to zero. Our series demonstrates the need for peroperative cytology in CT stereotaxic biopsies, and confirms that a complete neuropathology service is a prerequisite for neurosurgical patient care.


Continuous impedance monitoring during CT-guided stereotactic surgery: relative value in cystic and solid lesions. Continuous monopolar impedance monitoring was performed during CT-guided stereotactic procedures involving 46 probe passages in 33 patients. Four readings were obtained during each passage to the lesion corresponding to the gray matter (A), subcortical white matter (B), perilesional tissue (C), and the target (D). The mean impedance of low-grade gliomas (442.7 +/- 96.1 SD ohm) was lower than that of high-grade gliomas (675 +/- 67.3 SD ohm) (p = 0.01). But because of considerable overlap of values in the 400-700 ohm range only impedances of less than 400 ohm and greater than 700 ohm are capable of predicting pathology, the former being associated with low-grade gliomas and the latter with high-grade gliomas. The mean change in impedance from the perilesional tissue to the target (C-D) was higher for cystic lesions compared with the solid lesions (127.5 +/- 131.5 SD versus 78.9 +/- 72.4 SD ohm) but was not statistically significant (p = 0.148). Impedance monitoring was found to be particularly useful in determining the entry into thick-walled cysts. It also helped elucidate the structures encountered in the probe track. Overall this procedure is a simple and useful adjunct, which can enhance the accuracy of selected CT-guided procedures without unduly increasing the length of the procedure or altering the morbidity.


Ventriculitis and hydrocephalus caused by Candida albicans successfully treated by antimycotic therapy and cerebrospinal fluid shunting. A unique case of Candida albicans ventriculitis and hydrocephalus in the absence of any evidence of systemic candidiasis or immunosuppression is reported. Initial treatment with CSF shunting and intravenous antimycotic therapy appeared to have eradicated the infection. Recurrence occurred 5 months after discharge and this was treated by intravenous and intrathecal antimycotic therapy in addition to removal of the shunt system, external ventricular drainage and then replacement of the shunt. A concomitant pyogenic brain abscess responded to burrhole aspiration and antibiotics. The role of mannan antigen monitoring is discussed.


An analysis of the role of coenzyme Q in free radical generation and as an antioxidant. The vital role of coenzyme Q in mitochondrial electron transfer and its regulation, and in energy conservation, is well established. However, the role of coenzyme Q in free oxyradical formation and as an antioxidant remains controversial. Demonstration of the existence of the semiquinone form of coenzyme Q during electron transport, coupled with recent evidence that hydrogen peroxide (but not molecular oxygen) may act as an oxidant of the semiquinone, suggests that the highly reactive OH. radical may be formed from the semiquinone. On the other hand, data exist implicating the Fe-S species as the source of electron transfer chain, free radical production. Additional data exist suggesting instead that the unpaired electron of the coenzyme Q semiquinone most likely dismutases superoxide radicals. These concepts and those arising from observations at several levels of organization including subcellular systems, intact animals, and human subjects in the clinical setting, supporting the concept of reduced coenzyme Q as an antioxidant, will be presented. The results of recent studies on the interaction between the two-electron quinone reductase--DT diaphorase and coenzyme Q10 will be presented. The possibility that superoxide dismutase may interact with reduced coenzyme Q, in conjunction with DT diaphorase inhibiting its autoxidation, will be described. The regulation of cellular coenzyme Q concentrations during oxidative stress accompanying aerobic exercise, resulting in increased protection from free radical damage, will also be presented.


Enzymes that recognize dolichols participate in three glycosylation pathways and are required for protein secretion. We have explored the structure, function, and membrane topography of enzymes that recognize dolichols and participate in glycosylation pathways in the endoplasmic reticulum. Enzymes that interact with dolichols, including dolichyl phosphate mannose (Dol-P-Man) synthase and UDP-GlcNAc:Dol-P-transferase, revealed a conserved amino acid sequence in membrane-spanning regions. The consensus is Phe-Ile/Val-Xaa-Phe/Try-Xaa-Xaa-Ile-Pro-Phe-Xaa-Phe/Tyr, and we propose it is involved in dolichol recognition. We have used yeast mutants to demonstrate the role of dolichols in three glycosylation pathways. At its nonpermissive temperature, a Dol-P-Man synthase mutant (dpm1) was blocked in N-glycosylation, O-mannosylation, and glycosyl phosphoinositol membrane anchoring of protein, most likely because Dol-P-Man serves as mannosyl donor in all three pathways. The secretion mutant sec59 has a similar phenotype to dpm1, and the presence of a dolichol recognition sequence in the SEC59 protein gave a clue to its defect, which is in dolichol kinase. Comparison of yeast glycosylation mutant suggests that the ability to carry out N-glycosylation alone is sufficient to allow yeast to secrete glycoproteins and that an N-linked saccharide of a minimum size must be attached to proteins for cells to be able to secrete them and maintain a functional secretory pathway.


Dolichol and polyprenol kinase activities in microsomes from etiolated rye seedlings. Dolichol kinase activity in microsomes from etiolated rye seedlings had a pH optimum at 8 with a shoulder at pH 6.5. Triton X-100 (0.4%) was required for optimum activity. Exogenous divalent cations did not enhance activity, although Mg+2 was added routinely. Rye microsomes were found to contain dolichol and polyprenol in a ratio of 3 to 2. Rye, soybean embryo, and rat liver microsomes catalyzed the synthesis of 78, 52, and 516 nmol [14C]dolichyl phosphate/(mg microsomal protein.h) compared with 21, 22, and 49 nmol [3H]polyprenyl phosphate/(mg microsomal protein.h), respectively. It is clear that microsomes from plant systems can catalyze the phosphorylation of polyprenol better than rat liver when compared with their abilities to catalyze the phosphorylation of dolichol. It is not known whether one or more kinases is responsible for catalyzing the phosphorylation of these two closely related groups of compounds.


Dolichol and N-linked oligosaccharide synthesis in the rat testis: interaction between Sertoli and spermatogenic cells, evidence for paracrine effects. The relative contribution of the Sertoli cell and the pachytene spermatocyte to dolichol and N-linked oligosaccharide biosynthesis within the seminiferous tubule was investigated. Evidence is presented to show that the interaction between these two cell types affects dolichol and N-linked oligosaccharide biosynthesis. Analysis of the dolichol content of Sertoli cultures confirms earlier data suggesting that the Sertoli cell constitutes the major pool of dolichols within the seminiferous tubule. [14C]Acetate incorporation studies suggest that the Sertoli cell in culture synthesizes dolichol much more rapidly than does the isolated pachytene spermatocyte. This information, in addition to previous data in the literature, infers an interactive effect whereby the presence of the spermatogenic cell in the tubule stimulates dolichol synthesis in the Sertoli cell. The absence of normal Sertoli-spermatocyte interactions in in vitro incubations may also limit dolichol synthesis in the pachytene spermatocyte. The distribution of dolichol kinase between the Sertoli and the pachytene spermatocyte was also examined. The concentration of this enzyme in the Sertoli cell suggests the presence of an active salvage pathway within that cell. The correlation between the appearance of the pachytene spermatocyte and the previously described peak of dolichol kinase activity in the seminiferous tubules of the prepubertal animal implies cell-cell interactions. Radiolabelling studies of N-linked oligosaccharides were conducted using [3H]mannose and concanavalin A affinity chromatography to identify multiantennary, biantennary, and high-mannose oligosaccharide pools. An in vitro bicameral coculture system was used to demonstrate that pachytene spermatocytes stimulate incorporation of [3H]mannose into Sertoli cell oligosaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)


Cytochrome c and cytochrome c oxidase interactions: the effects of ionic strength and hydrostatic pressure studied with site-specific modifications of cytochrome c. Seven cytochromes c, in which individual lysines have been modified to the propylthiobimane derivatives, have been prepared. These derivatives were also converted to the porphyrin cytochromes c by treatment with HF. The properties of both types of modified proteins were studied in their reactions with cytochrome c oxidase. The results show that lysines 25, 27, 60, 72, and 87 do not contribute a full charge to the binding interaction with the oxidase. These five residues, with the exception of the lysine-60 derivative, on the front surface of the protein and contain the solvent-accessible edge of the heme prosthetic group. By contrast, lysines 8 and 13 at the top of the front surface do contribute a full charge to the binding interaction with the oxidase. The removal of the positive charge on any one lysine weakens the binding to cytochrome c oxidase by at least 1 kcal (1 cal = 4.1868 J). The presence of bimane at lysines 13 and 87 clearly forces the separation of the cytochrome c and oxidase, but this does not occur with the other complexes. The bimane-modified lysine-13 protein, and to a lesser extent that modified at lysine 8, show the interesting effect of enhanced complex formation with cytochrome c oxidase when subjected to pressure, possibly because of entrapment of water at the newly created interface of the complex. Our observations indicate that the two proteins of the cytochrome c - cytochrome oxidase complex have preferred, but not obligatory, spatial orientations and that interaction occurs without either protein losing significant portions of its hydration shell.


Metastasis-associated alterations in phospholipids and fatty acids of human prostatic adenocarcinoma cell lines. Metastatic variants of human prostatic adenocarcinoma cell lines (DU-145, LNCaP, and ND-1) were studied by using soft agar colony forming efficiency, nude mice tumorigenicity, in vitro invasion assay, and type IV collagenase assay. The DU-145 and ND-1 cell line showed higher metastatic potential than LNCaP. Lipids from DU-145, ND-1, and LNCaP cells were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. The major lipids were phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, fatty acids, and cholesterol. The sphingomyelin level was significantly higher in highly metastatic cells (DU-145 and ND-1) compared with the lower metastatic variant (LNCaP). The increase in the synthetic pathway and decrease in degradation pathway of sphingomyelin in microsomal fractions was sufficient to account for the measured increase in sphingomyelin in DU-145 cells compared with LNCaP cells. The major fatty acids of these lipids were palmitic (16:0), stearic (18:0), oelic (18:1), and arachidonic acid (20:4). The arachidonic acid level was significantly decreased in DU-145 and ND-1 compared with LNCaP cells. Electron microscopic studies showed no significant changes in the morphology of DU-145, ND-1, and LNCaP cells. The results of these investigations demonstrate for the first time that sphingomyelin and arachidonic acid contents are different in high and low metastatic variants of human prostatic adenocarcinoma cell lines.


Effect of diethyl pyrocarbonate modification on spectral and steady-state kinetic properties of bovine heart cytochrome oxidase. The histidine-specific reagent diethyl pyrocarbonate has been used to chemically modify bovine heart cytochrome oxidase. Thirty-two of sixty-seven histidine residues of cytochrome oxidase are accessible to modification by diethyl pyrocarbonate. Effects on the Soret and alpha bands of the heme spectrum indicate disturbance in the environment of one or both of the heme groups. However, diethyl pyrocarbonate modification does not alter the 830-nm absorbance band, suggesting that the environment of CuA is unchanged. Maximal modification of cytochrome oxidase by diethyl pyrocarbonate results in loss of 85-90% of the steay-state electron transfer activity, which can be reversed by hydroxylamine treatment. However, modification of the first 20 histidines does not alter either activity or the heme spectrum, but only when 32 residues have been modified are the activity and heme spectral changes complete. The steady-state kinetic profile of fully modified oxidase is monophasic; the phase corresponding to tight cytochrome c binding and low turnover is retained, whereas the high turnover phase is abolished. Proteoliposomes incorporated with modified oxidase have a 65% lower respiratory control ratio and 40% lower proton pumping stoichiometry than liposomes containing unmodified oxidase. These results are discussed in terms of a redox-linked proton pumping model for energy coupling via cytochrome oxidase.


Monomer and excimer fluorescence of horse plasma gelsolin labelled with N-(1-pyrenyl)iodoacetamide. Horse plasma gelsolin was labelled with the sulfhydryl-specific fluorescent reagent N-(1-pyrenyl)iodoacetamide. The level of incorporation of probe was 1.6 +/- 0.3 mol pyrene/mol gelsolin. The circular dichroism spectrum of pyrenyl-gelsolin and its ability to interact with muscle actin were not different from that found for unmodified gelsolin. The emission from pyrenyl-gelsolin was dominated by a broad emission band centred near 483 nm, characteristic of the presence of pyrene excimers. Analysis of excitation spectra for the monomer and excimer-type fluorescence suggested that ground-state interactions may occur between adjacent pyrenes in the gelsolin structure. In the case either of excimer formation or of ground-state pyrene-pyrene interactions in doubly labelled gelsolin molecules, the modified Cys residues must be in close proximity in the folded protein structure. Thermal denaturation of gelsolin could be monitored by observing the decrease in excimer emission that accompanied heating and unfolding of the tertiary structure. While heat treatment alone did not eliminate excimer fluorescence, digestion of gelsolin with chymotrypsin completely abolished such emission. Also, pyrenyl-gelsolin prepared and studied in 6 M guanidine-HCl exhibited fluorescence characteristic of pyrene monomers exclusively.


Sphingomyelin-metabolizing enzymes and protein kinase C activity in liver plasma membranes of rats fed with cholesterol-supplemented diet. The effect of cholesterol-supplemented diet on the activities of rat liver plasma membrane sphingomyelin-metabolizing enzymes and protein kinase C was studied. Protein kinase C, phosphatidylcholine:ceramide-phosphocholine transferase, and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase activities were found to increase continuously and almost in parallel during the experimental period on cholesterol diet (days 10, 20, and 30). Linear regression analysis showed a positive correlation between these activities with correlation coefficients r = 0.959 for protein kinase C and phosphatidylcholine:ceramide-phosphocholine transferase, and r = 0.998 for protein kinase C and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase. On the other hand, protein kinase C activation does not correspond to sphingomyelinase activity changes. These data suggest that protein kinase C activation observed in cholesterol-enriched plasma membranes is due to increased production of diacylglycerol and increased acylation of sphingosine to ceramide.


Phosphatidylcholine and phosphatidylinositol-specific phospholipases C of bull and rabbit spermatozoa. Acrosomal reaction is an essential prerequisite to fertilization. The changes in lipid composition of sperm membranes cause fusion of the plasma and outer acrosomal membranes that results in the exocytosis of acrosomal contents. We report that both bull and rabbit spermatozoa contain a phosphatidylcholine-specific phospholipase C (PC-PLC) that hydrolyzes L-alpha-dipalmitoyl-(choline-methyl-14C-153.0 Ci/mmol and a phosphatidylinositol-specific phospholipase C (PI-PLC) that hydrolyzes L-alpha-(Myo-Inositol-2-3H (N)-5.2 Ci mmol. PI-PLC from bull sperm acrosome has been purified 568 x fold with a specific activity 6.25 +/- 0.6 nmol/min/mg protein, km 0.004 mM, and Vmax 12 nmol/min/mg protein. Both enzymes had optimum at pH 7.5. The activity of PC-PLC remained unaffected by varying concentrations of Ca2+, whereas PI-PLC activity was significantly increased. The bulk of PI-PLC was found to be associated with inner acrosomal membrane of bull and rabbit sperm, while PC-PLC was found in the outer acrosomal membranes in the bull sperm and the plasma membrane of the rabbit sperm. Both enzymes are compartmentalized in sperm cell.


Okadaic acid and p13suc1 modulate the reinitiation of meiosis in mouse oocytes. Short-term exposure to okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, induced resumption of meiosis, including metaphase spindle formation, in mouse oocytes treated with a phosphodiesterase inhibitor, while long incubations with OA arrested oocyte maturation at a step prior to spindle formation. To explore the basis for this difference, the overall patterns of protein synthesis and phosphorylation and the production of tissue-type plasminogen activator (tPA), the synthesis of which is induced after germinal vesicle breakdown (GVBD), were analyzed under various OA treatments. Short-term exposure to OA led to tPA production and did not greatly affect the maturation-associated changes in protein phosphorylation. By contrast, a long application of OA did not result in tPA production and induced more marked changes in protein phosphorylation. Microinjection into prophase oocytes of the product of the fission yeast gene p13suc1, known to inhibit p34cdc2 kinase activation and/or activity, prevented meiotic reinitiation. This effect was overcome by microinjection of OA, at concentrations higher than those required for induction of maturation in the absence of p13suc1. These observations suggest that inhibition of phosphatase 1 or 2A or both triggers meiotic resumption by acting at the same site or at a site proximal to the p13suc1-sensitive step of cdc2 kinase activation.


Automated analysis of rabbit sperm motility and the effect of chemicals on sperm motion parameters. Appropriate software settings and optimum procedures were determined for the measurement of the motion parameters of rabbit spermatozoa by the CellSoft (Cryo Resources Ltd., Montgomery, NY) computer-assisted digital image analysis system. The system was used to follow motion parameter changes occurring in spermatozoa incubated for 6 hr with or without exposure to chemicals. Mean amplitude of lateral head displacement (AALH) increased over the 6 hr period, while curvilinear velocity (Vc) first increased and then decreased. Values for linearity (Lin), or beat cross frequency (BCF), were unchanged. The majority of spermatozoa progressed linearly, with rapid rotation of the sperm head, but subpopulations of spermatozoa with different swimming patterns appeared after 1-3 hr of incubation. Percentage motile sperm and Vc were most sensitive to the action of the compounds (pyrogallol, hydroquinone, ammonium oxalate, triethyl phosphite, and pinocolyl alcohol), while BCF was least affected. The decline in percentage of motile sperm was dependent on duration of exposure and chemical concentration. Mean Vc of the sperm population decreased rapidly upon chemical exposure and remained at a low value until motility ceased. The initial decrease in Vc was dependent on the concentration of the added compound. Motion-based indices--motility concentration (MCI50), motility time (MTI50), and velocity (VI)--were defined and used as toxicological endpoints. The rank order of these indices, the end point of the neutral red in vitro assay for cytotoxicity, and LD50 values for the five compounds were the same, suggesting that chemical inhibition of sperm motility may be useful as a method for the in vitro assessment of chemical cytotoxicity.


Identification of adenylate cyclase-coupled histamine H2 receptors in guinea pig tracheal smooth muscle cells in culture and the effect of dexamethasone. Histamine acts on airway contractile elements through at least two different receptor subtypes: H1, which mediates Ca(2+)-dependent contraction, and H2, which stimulates cyclic adenosine monophosphate (cAMP) synthesis and possibly relaxation. The aim of this study was to determine the relative contribution of the different receptor subtypes to histamine-stimulated cAMP production by guinea pig tracheal smooth muscle (GPTSM) cells in primary culture. Histamine and N-alpha-methylhistamine induced concentration-dependent cAMP synthesis; these effects were entirely blocked by 10(-4) M cimetidine, an H2-receptor antagonist, whereas 10(-6) M thioperamide, a selective H3 blocker, was ineffective. The H3 agonist, R-(alpha)-methylhistamine, did not stimulate cAMP synthesis. Triprolidine, an H1 antagonist, did not modify histamine (10(-5) M)-stimulated cAMP synthesis. Histamine (10(-5) M) doubled [Ca2+]i in GPTSM. A 24-h pretreatment of GPTSM cells with 10(-6) M dexamethasone enhanced cAMP synthesized in response to 10(-5) M histamine and to 5 x 10(-6) M forskolin but did not significantly alter either the affinity or the binding capacity for [3H]-tiotidine, an H2-receptor antagonist. These results indicate that GPTSM cells in culture express H2 but not H3 receptors, which are linked to adenylate cyclase; their functional expression does not seem to be modulated by the concurrent activation of H1 receptors, whose presence in GPTSM is evidenced by a histamine-stimulated increase in [Ca2+]i. The most likely site of action of dexamethasone in enhancing histamine-stimulated cAMP synthesis is at the level of adenylate cyclase since the steroid had no effect on the H2 receptor itself.


Dual regulation by cAMP of beta-hexosaminidase-induced mitogenesis in bovine tracheal myocytes. beta-Hexosaminidases, potent mitogens in bovine tracheal myocytes (BTM), stimulate a rapid and transient increase in intracellular cyclic adenosine monophosphate (cAMP) accumulation. The objective of this study was to elucidate the contribution of cAMP in hexosaminidase-induced airway muscle proliferation. Rate of DNA synthesis was measured by 3H-thymidine incorporation in quiescent cells prepared by a low-serum treatment (0.4%) for 48 h after reaching confluency in microtiter wells. cAMP accumulation was measured in acetylated cell extracts in the presence of isobutyl methylxanthine (100 microM) by radioimmunoassay using 125I-cAMP as tracer. Exposure of quiescent cells to purified human placental hexosaminidase B (5 micrograms/ml, 50 nM) caused a significant transient increase in cAMP accumulation (49 to 107 fmol/micrograms protein, or a 20- to 70-fold increase from basal level). Maximum increase occurred at 15 min followed by a rapid decline in cAMP accumulation within 30 min after exposure to hexosaminidase. Similar results were obtained in cells treated with neoglycoprotein mannose bovine serum albumin (100 to 500 nM). The increase in cAMP accumulation was inhibited by mannan (mannose receptor blocker, 0.1 mg/ml), as well as phenylisopropyladenosine (PIA; A1 receptor agonist that inhibits adenylyl cyclase, 0.1 to 1.0 microM). The increase in 3H-thymidine incorporation induced by hexosaminidase B was also inhibited by mannan and PIA. Exposure to 8-(4-chlorophenylthio)-cAMP (cpt-cAMP; a cell-permeable analog of cAMP, 100 microM) or forskolin (a direct activator of catalytic subunit of adenylyl cyclase, 24 microM) up to 6 h enhanced 3H-thymidine incorporation. In contrast, a prolonged exposure (18 to 30 h) to these agents inhibited 3H-thymidine incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)