Growth hormone stimulates granulation tissue formation and insulin-like growth factor-I gene expression in wound chambers in the rat.

It has been reported that GH stimulates fibroblast growth and wound healing. In the present study we measured the effect of locally applied GH on insulin-like growth factor (IGF-I) mRNA concentrations and granulation tissue formation in wound cylinders, implanted s.c. Four stainless-steel wiremesh cylinders were implanted s.c. in the back of male rats (280 g). Each cylinder was then injected every day with either 0.014 or 0.14 U human GH, or vehicle only. Ingrown granulation tissue and wound fluid was obtained on day 17 after implantation. The wet weight of granulation tissue was determined and concentrations of IGF-I mRNA in the tissue were measured by solution hybridization/RNAase protection assay. Similar assays were used to measure the levels of IGF-I receptor mRNA and GH receptor mRNA, while the IGF-I concentration in wound fluid and serum was determined by radioimmunoassay (RIA) after acid-ethanol extraction. The concentrations of IGF-I mRNA in ingrown granulation tissue as well as the wet weight of this tissue were significantly higher in the GH-treated cylinders. There was no significant effect of GH on IGF-I receptor mRNA and GH receptor mRNA levels. Consistent with the results of previous studies, wound fluid IGF-I levels were lower than serum IGF-I levels, but no significant difference was found between the GH-treated cylinders and the control cylinders. The results of the present study show that GH stimulates granulation tissue formation and increases the concentration of IGF-I mRNA in the ingrown granulation tissue.


Immunolocalization of mineralocorticoid receptor in human kidney, pancreas, salivary, mammary and sweat glands: a light and electron microscopic immunohistochemical study.

Using a polyclonal antibody against a synthetic fusion protein corresponding to 167 amino acids of the N-terminal region of the human renal mineralocorticoid receptor (MR), an immunohistochemical study was performed to investigate the intraglandular and intracellular localization of the receptor in human kidney, salivary gland, pancreas, mammary gland and sweat gland both at the light and electron microscopic levels. In the kidney, immunoreactivity was observed in distal convoluted tubules, branches of Henle's loop, and collecting tubules in the renal cortex, and papillary and Henle's loops' ducts in the renal medulla. No significant differences in the distribution of immunoreactivity were observed using different fixatives (10% neutral formalin, 100% methanol, 4% paraformaldehyde, PLP (periodate-lysine-2% paraformaldehyde) solution and Zamboni solution) and processing methods (paraffin embedding and frozen sectioning). Immunoreactivity in the kidney was observed in both the cytoplasm and nucleus, with cytoplasmic staining predominant, regardless of the methods of tissue preparation. Immunoelectron microscopy, employing a pre-embedding method, demonstrated the presence of immunoreaction precipitates in nuclei, endoplasmic reticulum, perinuclear cisternae, free cytoplasm and cell membranes. In nuclei, immunoreactivity was observed in euchromatin but not in heterochromatin, which is consistent with an association of MR with specific DNA regulatory elements located in transcriptionally active euchromatin. In other organs, MR was expressed in cells of the excretory ductal system where mineralocorticoids are known to play a role in electrolyte homeostasis.


Clostridium botulinum toxins: a general review of involvement in disease, structure, mode of action and preparation for clinical use.

The neurotoxins produced by Clostridium botulinum are the most potent acute toxins known and are the causative agents of the neuroparalytic disease botulism. The toxins act primarily at peripheral cholinergic synapses by blocking the evoked release of the neurotransmitter acetylcholine. There are seven distinct serotypes of toxin. All are polypeptides of Mr about 150 kDa that have similar structure and pharmacological action. In their most active forms the toxins exist as dichain molecules in which a heavy (H) chain is linked by disulphide bonding to a light (L) chain. The H chain is believed to be associated with the highly specific and avid binding of toxin to the motor nerve end plates and also with the process of internalisation of the toxin. The toxic activity appears to be associated with the L chain which blockades the calcium-mediated release of acetylcholine, probably by interfering at the molecular level with the mechanisms whereby neurotransmitter-containing vesicles merge with the plasmalemma. The type A toxin is now used therapeutically to treat a variety of conditions involving involuntary muscle spasm. The therapeutic toxin is a neurotoxin-haemagglutinin complex isolated from cultures of C. botulinum. A controlled manufacturing process has been developed for the therapeutic toxin which is specially formulated to give a freeze-dried product having good stability.


Fluctuation of serum phenytoin concentrations during autologous bone marrow transplant for primary central nervous system tumors.

We reviewed our experience for adult patients receiving oral anticonvulsant therapy during high-dose chemotherapy and autologous bone marrow re-infusion for primary malignant tumors of the central nervous system. Nineteen patients received either iv carmustine (BCNU) 900-1050 mg/m2 and 6120 cGy cranial irradiation (N = 10), iv carmustine 900-1050 mg/m2 and iv cisplatin 200 mg/m2 (N = 8), or iv carmustine 600 mg/m2, iv cisplatin 200 mg/m2, and iv etoposide 2400 mg/m2 (N = 1). Anticonvulsant therapy consisted of phenytoin alone (N = 8), phenobarbital alone (N = 4), carbamazepine alone (N = 2), phenytoin and carbamazepine (N = 2), carbamazepine and phenobarbital (N = 1), and no anticonvulsant therapy (N = 2). Serum anticonvulsant concentrations were monitored frequently and doses adjusted to keep values in the therapeutic range. While phenobarbital and carbamazepine doses remained relatively stable, all patients required increased doses of phenytoin anticonvulsant therapy after beginning chemotherapy (mean onset 3.7 days after initiation of chemotherapy). The increase in phenytoin dose ranged from 50% to 300% above baseline (mean 134%). By the time of discharge from the hospital (approximately 3-4 weeks after the start of chemotherapy) anticonvulsant dose was decreased to near pre-therapy levels. These swings coincided with the initiation of dexamethasone therapy for antiemetic effect and were more pronounced in patients also receiving cisplatin therapy. Due to close monitoring of serum phenytoin concentrations, no instances of toxicity due to excessive drug concentration, or seizures due to subtherapeutic doses, were noted in patients with primary CNS malignancies. Serum phenytoin concentrations fluctuate markedly during high-dose chemotherapy and must be analyzed frequently during the course of therapy.


Iron absorption from bread in humans: inhibiting effects of cereal fiber, phytate and inositol phosphates with different numbers of phosphate groups.

Iron absorption was measured from five kinds of bread made from various types of flour and fermented in different ways in order to obtain a wide variation in the content of fiber, phytate (inositol hexaphosphate) and its degradation products, inorganic phosphate and inositol phosphates with fewer numbers of phosphate groups (inositol pentaphosphate through monophosphate). Each experiment had 9-10 subjects and, in each subject, iron absorption was measured from control rolls made from low extraction wheat flour and one kind of test roll using two different radioiron tracers: 55Fe and 59Fe. The inhibition of iron absorption was closely related to the content of phytate-phosphorous as determined using the AOAC method, and to the sum of the tri- through hexaphosphate groups as determined using the HPLC method. As an example, prolonged fermentation of whole-rye bread reduced total inositol phosphates to the same amount as in the control rolls and increased fractional iron absorption to the same high level, in spite of a fiber content five times as great. The results strongly suggest that the inhibitory effect of bran on iron absorption is due to its content of phytate and other inositol phosphates present after fermentation, rather than to its content of fiber or other constituents. Thus, effective fermentation will increase the bioavailability of iron in whole-meal bread.


Bile acid metabolism in rats fed two levels of corn oil and brans of oat, rye and barley and sugar beet fiber.

High concentrations of fecal bile acids are associated with a higher incidence of colon cancer. Dietary changes that alter bile acid metabolism are therefore of interest. Here, we report the effect of feeding diets containing four fiber sources and two fat levels for 7 wk on bile acid excretion and small intestinal bile acids (an index of pool size) in rats. The fiber sources were oat bran, rye bran, barley bran and sugar beet fiber. Fiber-containing diets were 8% dietary fiber and contained either 5 or 20% corn oil. All fiber sources caused significantly greater fecal output compared with the fiber-free basal diet. All fiber sources also resulted in significantly (P less than 0.05) lower fecal bile acid concentration compared with the fiber-free basal diet. Only rye bran resulted in significantly (P less than 0.05) higher total fecal bile acid excretion. Oat bran resulted in a slightly but significantly (P less than 0.05) higher quantity of small intestine bile acids compared with the other diets. Dietary fat level had no significant effect on fecal bile acid concentration or excretion or quantity of small intestinal bile acids. We conclude that all four fiber sources tested resulted in lower fecal bile acid concentration, by effectively causing greater fecal mass. Changes in dietary fat level as corn oil had no effect on fecal bile acids.


Cholesterol-lowering in hamsters fed rice bran at various levels, defatted rice bran and rice bran oil.

This study was conducted to determine the relative cholesterol-lowering effects of several levels of full-fat rice bran in hamsters. In addition, the separate effects of defatted rice bran and/or crude rice bran oil were investigated at levels equivalent to those present in 43.7% full-fat rice bran. Diets containing 10.9, 21.8, 32.8 or 43.7% full-fat rice bran, 35% defatted rice bran and/or 9% rice bran oil were fed to 4-wk-old male hamsters. All diets contained 10% total dietary fiber, 9% fat and 3.2% nitrogen; hypercholesterolemic diets contained 0.3% cholesterol; two diets were cholesterol-free, i.e., 10% cellulose and 43.7% full-fat rice bran. After 21 d, plasma and liver cholesterol, plasma triglycerides and liver weights were significantly greater in hamsters fed the 10% cellulose diet with 0.3% cholesterol compared with those fed the cholesterol-free cellulose diet. In animals fed cholesterol-free diets, plasma cholesterol values were significantly lower in those fed the 43.7% full-fat rice bran diet than in those fed the cellulose diet. In animals fed cholesterol-containing diets, plasma and liver cholesterol were significantly lower in animals fed the 43.7% full-fat rice bran diet than in those fed the cellulose diet. Plasma cholesterol reductions were significantly correlated to the level of rice bran in the diet. In cholesterol-fed hamsters, total liver cholesterol content was significantly lower in those fed the defatted rice bran diet with rice bran oil compared with those fed the cellulose diet. Full-fat rice bran was the only treatment that significantly lowered both plasma and liver cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)


Intestinal calcium transport and calcium extrusion processes at the basolateral membrane.

The intestinal absorption of calcium has been proposed to occur by the transcellular transfer of Ca2+ through the enterocyte proper and between the cells of the intestinal epithelium, i.e., the paracellular path. Attention in this report is given to the transcellular models of Ca2+ absorption and, more specifically, the Ca2+ extrusion events occurring at the basolateral membrane. These extrusion processes include the operation of an ATP-dependent Ca2+ pump and a Na+/Ca2+ exchanger, as well as exocytosis as the terminal event in a proposed vesicular transport mechanism. Evidence for the presence of an ATP-dependent Ca2+ pump at the basolateral membrane is documented and illustrated with biochemical and immunological data from studies on the avian intestinal basolateral membrane. As shown immunohistochemically, the Ca2+ pump was primarily localized on the enterocyte basolateral membrane. The ATP-dependency and vitamin D enhancement of Ca2+ uptake by isolated basolateral membrane vesicles are shown. Western blot analysis of intestinal mucosa, by using a monoclonal antibody produced against the erythrocyte Ca2+ pump, indicated that the number of pump units is increased by 1,25-dihydroxycholecalciferol. The possible involvement of calbindin-D28K as a direct stimulator of the Ca2+ pump is discussed, and the quantitative relationship between Ca2+ transport rates and Ca2+ pumping activity has been estimated. Information related to the basolateral membrane Na+/Ca2+ exchanger and the vesicular transport model of Ca2+ absorption is also briefly reviewed.


A comparative study of the efficacy and morbidity of five techniques for ridge augmentation of the mandible.

Severely atrophic mandibles were augmented in 254 patients using five different surgical procedures, followed by total lowering of the floor of the mouth, vestibuloplasty, and skin graft. Patients were evaluated on a long-term basis to compare efficacy of treatment subjectively by recording patient complaints and by objectively assessing ridge form clinically and radiographically. Patients, in general, were pleased with the short- and long-term results of all five procedures. The ideal ridge form, comma-shaped, was obtained at a much higher rate with the three procedures using hydroxylapatite (HA) alone or in combination with collagen (HA-PFC). Ridge height loss both anteriorly and posteriorly was more severe in the two visor groups with bone graft or with HA than in those with only the alloplast. Labial sensory alterations more frequently occurred in the visor bone graft group, and wound dehiscence and lack of skin graft take were seen most often in the HA-PFC groups. Despite great differences in the quality of ridges obtained and in the number of complications between the visor groups and the alloplast groups, general patient satisfaction with any of the five procedures could be explained by the severity of the original complaints and because muscle interference has been eliminated in all cases by lowering of the floor of the mouth.


Central salivary gland tumors of the maxilla and mandible: a clinicopathologic study of 11 cases with an analysis of the literature.

Centrally occurring salivary gland neoplasms of the jaws are rare. The clinical and histologic features of 11 cases including mucoepidermoid carcinoma, adenoid cystic carcinoma, and adenocarcinoma were reviewed. The patients ranged in age from 10 to 67 years, with a mean age of 45 years. Males and females were almost equally affected. Ten cases were intramandibular and 1 case was of maxillary origin. Eight of 11 tumors either were histologically associated with an odontogenic cyst, or there was some recent history of exodontia in the tumor area. A review of the pertinent literature yielded 127 previously reported centrally occurring primary salivary gland tumors arising within the maxilla and mandible. An analysis of these cases, their clinical and histologic characteristics, as well as a discussion regarding their probable histogenesis and new suggestions for clinical staging, has been included.


Osseous response to implanted natural bone mineral and synthetic hydroxylapatite ceramic in the repair of experimental skull bone defects.

The purpose of this study was to assess and compare the osseous responses to implanted particles of resorbable anorganic xenograft bone mineral and non-resorbable dense synthetic hydroxylapatite of two different granule sizes. Four trephine calvarial defects were produced in each of 13 adult rabbits. The experimental materials were subsequently implanted in three defects, leaving the fourth defect for control purposes. Six animals were killed 4 weeks after surgery and seven at 14 weeks. The tissue responses were assessed by contact radiography, light microscopy, and histometry. The biocompatibility of the implants was confirmed. All defects healed uneventfully, although the resorbable hydroxylapatite seemed to promote initial bone regeneration. The importance to orthognathic surgery of early and effective healing of bone gaps, as well as of the advantage of implant resorbability to bone remodeling, are discussed.


The effect of therapeutic radiation on canine alveolar ridges augmented with hydroxylapatite.

The purpose of this investigation was to evaluate the effect of radiation on hydroxylapatite (HA) implanted subperiosteally for alveolar ridge augmentation in dogs. All bicuspids and molars were extracted from 16 dogs. After 6 weeks, nonporous HA granules were implanted subperiosteally on the alveolar ridge. Following 4 months of healing, 12 dogs (experimental group) underwent therapeutic radiation therapy (Co60, 4,000 rad [40 Gy]) to the head and neck region. Four dogs were not irradiated and served as controls. Four animals (three experimental and one control) were killed at 5,6,7, and 8 months after HA augmentation. Light microscopic evaluation showed that approximately 25% of HA granules were encased by bone while the others were surrounded by fibrous connective tissue. Dissolution of the HA was observed. Microparticles of HA were phagocytized as part of a granulomatous inflammatory reaction. This reaction decreased significantly as time elapsed after implantation. Osteoclastic activity was seen at the junction of HA and periosteum and as part of bone remodeling. Dissolution of the HA granules and the granulomatous inflammatory reaction were not significantly increased by therapeutic radiation. The radiation did not cause development of dehiscence or osteonecrosis.


Detection of hepatitis C virus (HCV) RNA in paraffin embedded tissue sections of human liver of non-A, non-B hepatitis patients by in situ hybridization.

Localization of hepatitis C virus (HCV) RNA was investigated by non-radioactive in situ hybridization in human liver specimens of chronic non-A, non-B (NANB) hepatitis patients who were seropositive for antibodies to HCV (anti-HCV). For in situ hybridization, T-T dimerized synthetic oligodeoxynucleotide probes were used and DNAs hybridized in situ were detected immunohistochemically using specific antibodies against T-T dimer. The data demonstrates that HCV-RNA was localized in the cytoplasm of hepatocytes in human liver biopsies obtained from the patients with chronic NANB hepatitis seropositive for anti-HCV.


Synthesis and structure-activity relationships of 7-diazabicycloalkylquinolones, including danofloxacin, a new quinolone antibacterial agent for veterinary medicine.

A series of novel 6-fluoro-7-diazabicycloalkylquinolonecarboxylic acids substituted with various C8 (H, F, Cl, N) and N1 (ethyl, cyclopropyl, vinyl, 2-fluoroethyl, 4-fluorophenyl, 2,4-difluorophenyl) substituents, as well as, 9-fluoro-10-diazabicycloalkylpyridobenzoxazinecarboxylic acids, were prepared and evaluated for antibacterial activity against a range of important veterinary pathogenic bacteria. The diazabicycloalkyl side chains investigated at the 7-position (benzoxazine 10-position) include (1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptane (2), (1S,4S)-2,5-diazabicyclo[2.2.1]heptane (3), (1R,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptane (4), 8-methyl-3,8-diazabicyclo[3.2.1]octane (5), 9-methyl-3,9-diazabicyclo[4.2.1]nonane (6), 1,4-diazabicyclo[3.2.2]nonane (7), 1,4-diazabicyclo[3.3.1]nonane (8), and 9-methyl-3,9-diazabicyclo[3.3.1]nonane (9). Among these side chains, in vitro potency was not highly variable; other properties therefore proved more critical to the selection of possible development candidates. However, the relative potencies observed for several of these compounds in mouse, swine, and cattle infection models correlated well with those seen in vitro. A combination of the N1 cyclopropyl group and the C7 (1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]hept-2-yl appendage conferred the best overall antibacterial, physiochemical, and pharmacodynamic properties. Hence, danofloxacin (Advocin, 2c) (originally CP-76,136, 1-cyclopropyl-6-fluoro-7-[(1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1] hept-2-yl]-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid) was selected as a candidate for development as a therapeutic antibacterial agent for veterinary medicine.


3,4-Dihydroquinolin-2(1H)-ones as combined inhibitors of thromboxane A2 synthase and cAMP phosphodiesterase.

A series of 1H-imidazol-1-yl- and 3-pyridyl-substituted 3,4-dihydroquinolin-2(1H)-ones was designed and synthesized as combined inhibitors of thromboxane (TXA2) synthase and cAMP phosphodiesterase (PDE) in human blood platelets. A number of structures, e.g. 4b, 7a, 7e, 13a, and 21-25, were superior to dazoxiben 26 as inhibitors of TXA2 synthase in in vitro ADP-induced aggregation experiments with human blood platelets. The TXA2 synthase inhibitory activity was confirmed by measurement of the prostanoid metabolites derived from 14C-labeled arachidonic acid. Three compounds (7a, 7e, and 25) demonstrated in vitro inhibition of human platelet cAMP PDE at micromolar concentrations in conjunction with their TXA2 synthase inhibitory activity. Synergistic enhancement of antiaggregatory and antithrombotic actions was expected when simultaneous stimulation of adenylate cyclase (through increased PGI2 production) and inhibition of platelet cAMP PDE were possible from the same compound. Ex vivo and in vivo experiments were conducted in rats and mice, respectively, to evaluate the effects of compounds 7e and 23 on platelet aggregation and thrombotic events within these animals. Compound 7e, which has a comparable level of TXA2 synthase (IC50 1.2 microM) and human platelet cAMP PDE (IC50 6.4 microM) inhibitory activities, was found to be orally bioavailable with a long duration of action and offered effective protection against mortality in a collagen-epinephrine-induced pulmonary thromboembolism model in mice. Significant blood pressure and heart rate effects were observed for several compounds, e.g. 7e, 9e, 13a, 13d, 18, 20, 21, and 23, when dosed orally in conscious spontaneously hypertensive rats.


Systemic analgesic activity and delta-opioid selectivity in [2,6-dimethyl-Tyr1,D-Pen2,D-Pen5]enkephalin.

The cyclic peptide [2,6-dimethyl-Tyr1,D-Pen2,D-Pen5]enkephalin (2) was synthesized by solid-phase techniques and contains the optically pure unnatural amino acid 2,6-dimethyltyrosine (DMT) as a replacement for the Tyr1 residue of [D-Pen2,D-Pen5]enkephalin (DPDPE, 1). This structural modification resulted in a 10-fold increase in the potency of 2 at the delta opioid receptor and a 35-fold increase in potency at the mu receptor while substantial delta receptor selectivity was maintained. In addition, 2 was 86-fold more effective than 1 at inhibiting electrically stimulated contractions of the mouse vas deferens. In the hot plate test, 2 was 7-fold more potent than 1 after intracerebroventricular administration in the mouse. While 1 was inactive following systemic administration of doses as high as 30 mg/kg, subcutaneous administration of 2 significantly inhibited writhing with an ED50 of 2.6 mg/kg. These results demonstrate that the potency and systemic activity of DPDPE are significantly increased by replacement of Tyr1 with DMT.


Benzazepinone calcium channel blockers. 4. Structure-activity overview and intracellular binding site.

We have synthesized a series of benzazepinones (2) in order to determine the structure-activity relationships (SAR) for calcium channel blockers related to diltiazem. A prerequisite for calcium channel blocking activity in vitro and in vivo is the presence of two pharmacophores: a 4'-aryl methyl ether and a basic substituent appended to N1 with a pKa in the physiological range. When these constraints are satisfied, a wide variety of substitution is tolerated at C6, C7, and C3. The presence of an electron-withdrawing group at C6 appears to enhance potency in vitro and in vivo. For such benzazepinones, activity is primarily dependent upon lipophilicity, as measured by log P. We believe these compounds must partition into the cell membrane in order to access their receptor. The quaternary methiodide 15k was used to demonstrate that the binding site for benzazepinones is on the intracellular face of the membrane. This work represents the first comprehensive SAR of diltiazem-like calcium channel blockers.


Depolarization exposes the voltage sensor of the sodium channels to the extracellular region.

Two domains of Na channels were mapped with site-specific antibodies raised in rabbit against synthetic peptides corresponding to a part of the voltage sensor of internal repeat 1-C1+ (amino acids 210-223) and to a region designated dipole (amino acids 1690-1699) of eel electroplax sodium channels. The antibodies bind to their respective domains in both purified and membrane-bound channels and immunoprecipitate the channels from eel electroplax and rat brain synaptosomes. Anti-C1+ depresses the action potential of rat sciatic nerve in a concentration-dependent way. It binds to the external side of rat brain synaptosomal vesicle, and its binding is potentiated by depolarization. Anti-dipole binds to the inner side of the vesicle, and the binding is inhibited by depolarization.


Regulation of principal cell pH by Na/H exchange in rabbit cortical collecting tubule.

Changes in intracellular pH (pHi) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pHi. Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Nai) would alter pHi in a predictable way. Maneuvers designed to alter Nai were without significant effects within a 10-min time frame. Specifically, addition of 100 microM ouabain to increase Nai or exposure of the tubules to 10(-5) M amiloride to decrease luminal Na entry and reduce Nai did not have an effect on pHi. In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pHi. These results suggest that Na/H exchange is a major regulator of pHi in principal cells. However, regulation of Na transport by changes in pHi in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.


Evidence that plasma membrane electrical potential is required for vesicular stomatitis virus infection of MDCK cells: a study using fluorescence measurements through polycarbonate supports.

We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (delta psi pm) and infectivity of vesicular stomatitis virus (VSV). Complete substitution of K+ for extracellular Na+ blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells. When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K+ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K+ applied apically had no effect on VSV infection. This morphological specificity correlates with a large decrease in delta psi pm of MDCK cells when high K+ buffer is perfused across the basal-lateral surface. Depolarization of the plasma membrane by 130 mM basal K+ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF. Depolarization also causes a transient increase of cytosol Ca2+ from 70 to 300 nM as reported by the dye Fura-2. Neither increase could explain the block of VSV infectivity by plasma membrane depolarization. One alternative hypothesis is that delta psi pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli.


N-methyl-N-nitrosourea-induced mutations in human cells. Effects of the transcriptional activity of the target gene.

In this study we addressed the question as to whether the mutagenesis by methylating agents is affected by the transcriptional activity of the damaged gene. An Epstein-Barr virus (EBV)-derived shuttle vector system was developed where the genetic target for mutation analysis, the bacterial gpt gene, is under the control of an eukaryotic inducible promoter in plasmid pF1-EBV and lacks the eukaryotic promoter in plasmid pF2-EBV. Two human cell lines that episomically maintain these shuttle vectors were established. In clone 6NT cells, which contain pF1-EBV plasmid, the gpt gene is actively transcribed and the transcription rate is regulated by zinc ions. In clone 3 cells, which harbor pF2-EBV plasmid, the gpt gene is not transcribed. Following treatment of both cell lines with the potent alkylating carcinogen N-methyl-N-nitrosourea (MNU), G.C to A.T transitions were the major mutagenic event, consistent with the miscoding potential of O6-methylguanine. The mutations were predominantly generated in the non-transcribed DNA strand of the active gpt gene. The same strand-bias was observed when the gpt gene was transcriptionally inactive, indicating that MNU-induced strand-specific formation of mutations is not due to transcription. Our data identify as major determinants of this phenomenon the sequence-specificity of MNU mutagenesis and the conformational properties of the target protein. Differences in mutation distribution were observed between the transcriptionally active and inactive gpt gene. This finding suggests that the organization of active genes in chromatin might modulate DNA alkylation and/or DNA repair.


Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins.

Two crystal forms (P6(3) and R3) of human annexin V have been crystallographically refined at 2.3 A and 2.0 A resolution to R-values of 0.184 and 0.174, respectively, applying very tight stereochemical restraints with deviations from ideal geometry of 0.01 A and 2 degrees. The three independent molecules (2 in P6(3), 1 in R3) are similar, with deviations in C alpha positions of 0.6 A. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five alpha-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the molecule opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholipase A2 calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the molecule is formed by a channel of polar charged residues, which also harbors a chain of ordered water molecules conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. We suggest that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport.


Determination of the nuclear magnetic resonance solution structure of the DNA-binding domain (residues 1 to 69) of the 434 repressor and comparison with the X-ray crystal structure.

The DNA-binding domain of the phage 434 repressor consisting of N-terminal residues 1 to 69 (434 repressor(1-69)), was expressed in Escherichia coli with natural isotope abundance, uniform 15N-labeling and biosynthetically directed fractional 13C-labeling in extent of about 10%. With these protein preparations the three-dimensional structure was determined in solution. The techniques used were nuclear magnetic resonance (n.m.r.) spectroscopy for the collection of conformational constraints, calculation of the protein structure from the n.m.r. data with the program DIANA and structure refinements by restrained energy minimization with a modified version of the program AMBER. A group of 20 conformers characterizes a well-defined structure for residues 1 to 63, with an average of 0-6 A for the root-mean-square deviations (RMSD) calculated for the backbone atoms of the individual conformers relative to the mean co-ordinates. The spatial structure of C-terminal residues 64 to 69 is not defined by the n.m.r. data. The molecular architecture of the 434 repressor(1-69) in solution includes five alpha-helices extending from residues 2 to 13, 17 to 24, 28 to 35, 45 to 52 and 56 to 60, which enclose a well-defined hydrophobic core. The n.m.r. structure is closely similar to the reported crystal structure of the 434 repressor(1-69), with an RMSD value of 1.1 A for the backbone atoms of residues 1 to 63. Small differences between the two structures in regions of the first helix and the loop between helices 3 and 4 were analyzed relative to possible correlations with protein-protein contacts in the crystal lattice and the different milieus of pH and ionic strength in the crystals and n.m.r. samples. Further systematic comparisons of local conformational features indicated that there are correlations between amino acid types, local precision of the structure determination by both techniques and local differences between the structures in the crystals and in solution. Overall, hydrophobic residues are most precisely characterized and agree most closely in the two environments.


Proton beams in radiation therapy.

The rationale for study of proton radiation therapy is that, for some anatomic sites and tumors, the treatment volume is smaller; i.e., there is less irradiation of nontarget tissue while the target is included in three dimensions at each treatment session. As a result, the dose to the target can be raised. The consequence is that the tumor control probability improves and the frequency and severity of treatment-related morbidity decrease. These results come about from the physical fact that the proton range in tissue is finite; in comparison, absorption of photons is an exponential function and, hence, some dose is received for the full-beam path through the body. Accordingly, the dose deep to the target for proton treatments can be zero for each beam path. This situation provides a virtually certain means of improving the treatment outcome for selected categories of patients. Experience to date with proton radiation therapy has been quite limited. As of June 1991, the total number of proton radiation-treated patients was 11,763 from the various centers. Of that number, approximately 46% and 32% have been treated for small benign intracranial lesions (principally pituitary adenomas and arteriovenous malformations) and for tumors of the eye, respectively. Thus, only some 2500 patients have been treated for all other tumor types. The results from three centers and approximately 2800 patients with uveal melanoma are that the local control rate was 96% (for failures in-field, marginal, and in other parts of the eye). The local control results for chondrosarcomas and chordomas of the skull base are 91% and 65%, respectively. These percentages compare with some 35% achieved with conventional treatment. Experience with arteriovenous malformations indicates that control of bleeding and disappearance of the lesion are comparable to those achieved by other procedures. The developments from the proton therapy programs have contributed greatly to radiation treatment planning, e.g., the first three-dimensional treatment planning system put into regular clinical use (uveal melanoma), beam's eye view, digital-reconstructed radiograph, dose-volume histograms, and definitions of the uncertainty in dose around any defined point. The potential for clinical gains is high. In May 1991, the Proton Radiation Oncology Group was formed to design, supervise, and coordinate clinical trials and to assist in data analysis. The efficacy of proton radiation therapy will be compared with that of photon therapy of the very highest technology.


RNA expression of the WT1 gene in Wilms' tumors in relation to histology.

On the basis of accumulating data, the recently isolated WT1 gene is a Wilms' tumor gene and a putative tumor suppressor gene. These findings include expression in developing fetal kidney, intragenic deletions in tumors, and germline mutations in predisposed individuals. Wilms' tumors, which exhibit a broad range of differentiation, are composed of three cell types: blastema, epithelium, and stroma. The purpose of this study was to investigate the relationship between WT1 gene expression and histologic composition in Wilms' tumors in an effort to elucidate how the WT1 gene functions in proliferation of these histologic components. We used Northern blot hybridization to study WT1 gene expression by messenger RNA (mRNA) accumulation in 20 tumors of varying histology and in adjacent uninvolved kidney tissue. In two patients, tumors were also compared before and after therapy. Tumors that were predominantly blastemal expressed high amounts of WT1 mRNA, whereas predominantly stromal tumors expressed either low or undetectable amounts. Blastemal tumors that were predominantly poorly differentiated expressed WT1 mRNA at higher levels than those that were more well differentiated. Although we expected that a putative tumor suppressor gene like WT1 would generally be expressed at lower levels in tumor than in normal kidney, this was true only in predominantly stromal cells. One of the two patients studied before and after therapy had a dramatic response to therapy accompanied by a decline in WT1 gene expression and disappearance of blastemal and epithelial elements. A correlation was observed between WT1 gene expression and histology of the tumors. Level of expression was inversely related to the degree of differentiation in blastemal tumors and in the patient with a dramatic response to therapy. These results, in conjunction with the observation that WT1 mRNA is abundant in normal fetal kidney, suggest that WT1 gene expression is related to kidney development, especially in differentiation of blastemal components. Further studies to search for alterations of the WT1 gene in tumors and to identify regulatory factors in gene expression will increase understanding of the role of this gene in normal development and tumorigenesis.


In vitro studies of the efficacy of reversed phase silica gel as a sorbent for hemo- and plasmaperfusion.

The clearance capacities of reversed phase silica gel, widely used in analytical chemistry, were studied in vitro. The plasma clearances of amitriptyline, quinidine, digoxin, digitoxin, methaqualone, phenobarbital and phenytoin were determined at typical toxic plasma levels. Between 88.8 and 99.5% of the drugs were eliminated from plasma by reversed phase silica gel, which compares favorably with amberlite XAD-4 and coated activated charcoal. The biocompatibility of reversed phase silica gel was also evaluated in vitro. Thrombocytopenia and leukopenia were noted while coagulation parameters, electrolytes, acid-base balance, glucose, urea, creatinine, uric acid, total protein, albumin, bilirubin, cholesterol and triglycerides were not significantly affected. Preliminary results in vitro support the high clearance capacity of reversed phase silica gel and suggest a biocompatibility similar to that of other sorbents currently in use. Further investigation of the clearance capacities and biocompatibility of reversed phase silica gel seems justified.


[Discovery and development of the proton pump inhibitor].

Gastric (H+/K+)-ATPase, the proton pump of the parietal cell, is responsible for the final step of acid secretion in the stomach. In 1981, picoprazole, a substituted benzimidazole, was found to inhibit (H+/K+)-ATPase. It was reported in 1983 that omeprazole has the most potent efficacy among the substituted benzimidazoles and today, omeprazole has been used for treatment of gastroduodenal disease. Recently, lansoprazole, similar to omeprazole in chemical structure, was developed in Japan, and several other compounds, such as pantoprazole, E-3810 and NC-1300-O-3, have also been reported to suppress acid secretion through inhibition of (H+/K+)-ATPase. In the present paper the background of the discovery of (H+/K+)-ATPase and development of proton pump inhibitors is reviewed.


[Clinical effect of proton pump inhibitors on reflux esophagitis].

The clinical efficacy of proton pump inhibitors (PPI, omeprazole 20 mg or lansoprazole 30 mg), once daily, after breakfast, was studied in patients with erosive/ulcerative reflux esophagitis. The following results were obtained. 1) Twenty-four hour esophageal pH monitoring was performed before treatment and on 7th day of PPI medications. Omeprazole reduced the percent time pH less than 4 from 29.1 to 1.2 and lansoprazole from 68.0 to 2.4. 2) The cumulative disappearance rate of overall symptom was 52% after 1 week and 62% after 2 weeks with omeprazole these were 66% and 91%, and with lansoprazole respectively 3) The endoscopic healing rate was 63% was after 2 weeks and 76% after 4 weeks with omeprazole medication, and 76% and 97% respectively with lansoprazole. These results indicate that PPI medication inhibits the acid reflux almost completely and is a more useful therapeutic agent for GERD than H2-antagonists.


[Recurrent peptic ulcer after gastric surgery].

Recurrent peptic ulcer after gastric surgery differs from duodenal ulcer in that they usually occur with low acid output, which is sufficient to cause ulceration in predisposed stomach or anastomosis. The proton pump inhibitor (PPI), significantly more potent and long-acting than H2-blocker, is expected to be more effective for postoperative recurrent ulcers. We evaluated the efficacy of omeprazole (OPZ) on recurrent ulcers in 12 patients following either gastrectomy or vagotomy. The healing rate after 4 week treatment with OPZ was 58% and increased to 100% after 8 week. Evaluation of quick symptom relief also supported the efficacy of OPZ treatment. These findings showed that OPZ treatment resulted in a more rapid healing of recurrent ulcers compared with H2-blocker. It remains to be clarified whether quit of treatment with PPI would induce high ulcer relapse rates, and whether maintenance therapy with PPI would be the only alternative therapy for surgical intervention.


[Proton pump inhibitor for maintenance therapy of peptic ulcer].

The strategy for peptic ulcer therapy has been changing with the clinical application of the gastric proton pump inhibitor (PPI). In Japan, Miyoshi et al and Takemoto et al reported an earlier reepithelialization of peptic ulcer with omeprazole (OME) or lansoprazole (LAN) than famotidine (FAM). Miyoshi et al also reported that there was no significant difference between OME and FAM in ulcer relapse rate during a one year follow-up period. Therefore, there were two problems. One is application of PPI for prevention of ulcer relapse, and the other is the more accurate diagnosis of ulcer healing. Application of PPI for maintenance therapy is not yet realized in Japan, but, Lauritsen et al had already reported on the efficacy and safety of OME, 20 mg, three days a week and 10 mg, daily in prevention of duodenal ulcer relapse. Reepithelialization (red scar) is already established as a starting point of maintenance therapy, and from Miyake's report, a white scar is believed a favorable (non relapsing) end point.


[Studies on the intracellular pharmacodynamic properties of proton pump inhibitors and the inhibitory mechanism of acid secretion].

In recent studies, proton pump inhibitors, such as omeprazole, were found to be transformed into sulfenamide derivatives in the acid space of isolated parietal cells. It is considered that these sulfenamide derivatives mainly inhibit H+, K(+)-ATPase activity. To clarify the inhibitory mechanism of proton pump inhibitors, we studied the effect on acid secretion of the isolated parietal cells. Proton pump inhibitors inhibited histamine-, carbachol- and gastrin-stimulated 14C-aminopyrine accumulation. Db-cAMP stimulation was also inhibited by these inhibitors. Consequently, it is believed that the origin of H+, K(+)-ATPase was located in the final stage of the acid production.


[Treatment of gastric ulcers with proton pump inhibitors--long-term treatment].

Pronounced inhibition of acid secretion appears to induce an intragastric environment suitable for N-nitrosamine formation, hyper-gastrinemia, ECL cell hyperplasia and carcinoid tumor formation. Development of gastric cancer, however, has not been obvious in clinical and experimental studies, but oncongenicity studies indicate an increased risk of gastric cancer with long-term use of proton pump inhibitors in subjects with hyperplastic or other, changes in the gastric mucosa. The findings suggest that proton pump inhibitors should be used only for short term treatment.


[Changes in the rat stomach after long-term administration of proton pump inhibitors (AG-1749 and E-3810)].

E-3810 and AG-1749, new proton pump inhibitors, were administrated in single doses or over a long period of time in order to evaluate their effect on the rat stomach. E-3810 inhibited gastric acid secretion for a shorter period of time than AG-1749 in both the single dose and long-term administration. While hypergastrinemia persisted with long-term administration of AG-1749, serum gastrin levels returned to normal upon cessation of treatment with E-3810. Both E-3810 and AG-1749 caused vacuolar formation of parietal cells and an increase in gastric acid secretion after cessation of long-term treatment, suggesting that these changes are common after treatment with proton pump inhibitors.


[Possible mechanisms for (H+ + K+)-ATPase inhibition by proton pump inhibitors, omeprazole, lansoprazole and SCH 28080].

Proton pump inhibitors are classified into two distinct types, and the mechanisms are reviewed. Substituted benzimidazole such as omeprazole and lansorrazole, inhibit (H+ + K+)-ATPase by reacting with SH groups of enzyme after the drugs are transformed into their active forms in the acidic environment of the intracellular canaliculi of parietal cells. This type of enzyme inhibition results in potent and long-lasting inhibition of gastric acid secretion. On the other hand, substituted imidazo pyridines, such as SCH 28080, inhibit (H+ + K+)-ATPase by competing with K+. This inhibition is reversible and the antisecretory effect is short-lived. Recent studies on DNA cloning and sequencing for (H+ + K+)-ATPase have led to a better understanding of enzyme structure and also the sites of action of the proton pump inhibitors.


[Protective effect of proton pump inhibitor on the gastric mucosa].

Although many authors reported that proton pump inhibitor showed mucosal protective activity, the precise mechanism is still unclear. In this study, we investigated the effect of proton pump inhibit on gastric mucosal hemodynamics, using a reflectance spectrophotometry system. The proton pump inhibitors had no effect on the gastric mucosal hemodynamics prior to the state of hemorrhagic shock state. However, during the hemorrhagic shock, proton pump inhibitors significantly inhibited the decrease in tissue oxygenation and significantly reduced ulcer formation. In conclusion, proton pump inhibitors maintained tissue oxygenation resulting in reduction of ulcer formation.


[Effect of proton pump inhibitor, in combination with epidermal growth factor, on the healing of chronic gastric ulcer in submandibular gland removed rats].

The effect of a proton pump inhibitor, omeprazole (OPZ), in combination with mouse epidermal growth factor (EGF), on the healing of chronic gastric ulcers induced by acetic acid in submandibular glands removed rats (SMR rat) was investigated. Four separate groups of SMR rats were orally administered 1 ml of distilled water (control), 100 mg/kg of OPZ alone (OPZ), 5 micrograms/kg of mouse EGF alone (EGF), or combination with (EGF + OPZ) twice daily. Two weeks later, ulcer areas of four groups were measured and expressed as the ulcer index (UI). The UI of the OPZ and EGF + OPZ group was significantly reduced as compared with that of the control. Furthermore, the UI of the EGF + OPZ group was significantly smaller than that of the EGF group. The present study indicates that the proton pump inhibitor promotes healing of chronic gastric ulcers in SMR rats and the effect of the proton pump inhibitor is enhanced by administration of mouse EGF.


[Characteristic features of proton pump inhibitors in the inhibition of gastric acid secretion: long-acting and complete inhibition].

Proton pump inhibitor is a compound recently applied for the treatment of peptic ulcers for its strong action to inhibit the gastric acid secretion. It works through inhibition of H+, K(+)-ATPase, so called proton pump, on the luminal surface of secretory canaliculi in the parietal cells, showing remarkable characteristics in the inhibition of gastric acid secretion; e.g., the long-acting and complete inhibition. At neutral pH, the unionized form of this compound as a weak base is lipophilic, and passes through the cell membrane to accumulate as the ionized form in an acidic environment in the secretory canaliculi of parietal cells, where it is transformed to an active molecule which binds covalently to the active site of H+, K(+)-ATPase, forming a highly stable complex. The long-acting and complete inhibition of gastric acid secretion by this compound is derived from this physico-chemical nature. The above characteristics of the proton pump inhibitor have been confirmed with the basal, stimulated and nocturnal gastric acid secretion and the 24-hour intragastric pH of healthy volunteers by several investigators prior to its nation-wide clinical trial in Japan. On the other hand, the increased endocrine and exocrine secretion, such as pepsin secretion and gastrin release, and the increased turnover of gastrointestinal endocrine cells by this compound have been reported in animal models, probably due to its accumulation in the acidic environment.(ABSTRACT TRUNCATED AT 250 WORDS)


[Magnifying endoscopic observation on the effect of a proton pump inhibitor on the healing process of gastric ulcer].

The effect of a proton pump inhibitor on the healing process of gastric ulcer was investigated through magnifying endoscopic observation of the regenerated mucosa. The findings were the same obtained as those by stereoscopic microscopic observation of the effect of conventional drugs. Namely, a structureless red area consisting of one layer of regenerated epithelium was the first to procede forward the ulcer base, and a radially arranged roughly striated pattern of regenerated mucosa, in which gastric glands were formed. The regenerated mucosa changed from a striated to a granular pattern. In a shallow ulcer, the ulcer scar showed a fine gastric mucosal pattern similar to that of the surrounding area. The strong inhibition of gastric acid secretion by a proton pump inhibitor is believed to raise no problems in the healing process of peptic ulcer. The magnifying endoscopic observation also disclosed that the protruding granulation disappeared gradually and changed to concave regenerated epithelium, suggesting that the ulcer base protrusion, which has been considered as an adverse effect of strong acid secretion inhibitors, has no influence on the healing of the ulcer.


[Evaluation of chromo-endoscopy for diagnosis of histological repair of gastric ulcer].

The chromoendoscopic features of healed gastric ulcers were investigated by the methylene blue test. Histological repair of healed ulcers was graded as either Ul3 (incompletely healed) or Ul4 (completely healed). Endoscopically, the Ul4 scar was observed as a flat granular area without any depression, and sometimes associated with redness, the Ul3 scar was observed as a distinct depression in the surrounding normal mucosal surface, and usually associated with homogenous redness. Follow-up studies of patients with Ul4 and Ul3 lesions showed that recurrence was significantly rare in patients with the Ul4 lesion than in patients with the Ul3 lesions. These findings indicate that healing of gastric ulcers should be defined endoscopically as crater disappearance. At 8 weeks after treatment with omepral, most of the ulcers were healed, but judged a as Ul3 scar.


Bradykinin-induced biphasic response in the rat isolated stomach fundus: functional evidence for a novel bradykinin receptor.

The responses of the rat isolated stomach fundus to bradykinin (BK) and des-Arg9-BK (DA-BK) have been examined. In rat isolated stomach fundus pre-contracted with BaCl2 (0.5-1 mM), BK caused concentration-dependent biphasic responses characterized by relaxation followed by contraction. DA-BK also caused marked relaxations, but, unlike BK, induced only small contractions. Removal of the mucosal layer initially abolished the relaxant responses to BK and both responses to DA-BK without affecting BK-induced contractions, but repeated challenges with BK or DA-BK revealed a time-dependent reappearance of the relaxant response, suggesting "de novo" synthesis of BK receptors. Pretreatment of rat stomach fundus with tetrodotoxin (1 microM), atropine (1 microM), captopril (3 microM), prazosin (1 microM) or glibenclamide (1 microM) did not significantly modify the biphasic responses to BK (300 nM). The biphasic responses to DA-BK were antagonized selectively by the B1 receptor antagonist des-Arg9-[Leu 8]-BK (DAL-BK) (1 microM). In contrast, the biphasic responses to BK were unaffected by DAL-BK or by several selective peptide antagonists of B2 receptors including NPC 431 (Thi5,8, D-Phe7)-BK, NPC 349 (D-Arg Hyp3,Thi5,8,D-Phe7)-BK, NPC 567 (D-Arg-Hyp3,D-Phe7)-BK and NPC 361 (D-Phe7)-BK (3 to 10 microM). These results are consistent with the view that the biphasic responses of the rat isolated stomach fundus to BK appear to be mediated by a novel BK receptor which is insensitive to blockade by B1 and B2 selective BK receptor antagonists.


Effects of hirsutine, an antihypertensive indole alkaloid from Uncaria rhynchophylla, on intracellular calcium in rat thoracic aorta.

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla (MIQ.) Jackson, on cytosolic Ca2+ level ([Ca2+]cyt) were studied by using fura-2-Ca2+ fluorescence in smooth muscle of the isolated rat aorta. Noradrenaline and high K+ solution produced a sustained increase in [Ca2+]cyt. Application of hirsutine after the increases in [Ca2+]cyt induced by noradrenaline and high K+ notably decreased [Ca2+]cyt, suggesting that hirsutine inhibits Ca2+ influx mainly through a voltage-dependent Ca2+ channel. Furthermore, the effect of hirsutine on intracellular Ca2+ store was studied by using contractile responses to caffeine under the Ca(2+)-free nutrient condition in the rat aorta. When hirsutine was added at 30 microM before caffeine treatment, the agent slightly but significantly reduced the caffeine-induced contraction. When added during Ca2+ loading, hirsutine definitely augmented the contractile response to caffeine. These results suggest that hirsutine inhibits Ca2+ release from the Ca2+ store and increases Ca2+ uptake into the Ca2+ store, leading to a reduction of intracellular Ca2+ level. It is concluded that hirsutine reduces intracellular Ca2+ level through its effect on the Ca2+ store as well as through its effect on the voltage-dependent Ca2+ channel.


Effect of sex steroids on body temperature in postmenopausal women. Role of endogenous opioids.

The role of endogenous opioids on the thermoregulatory effect of sex steroids was investigated in six postmenopausal women before and during treatment with transdermal 17 B-estradiol (TTS 50; 50 mcg/day) with or without vaginal progesterone (P4; 100 mg twice daily). In all the different endocrine conditions, saline or the opioid antagonist naloxone (10 mg/hr. preceded by 10 mg iv bolus) were randomly infused for 4 hrs., on two consecutive days. Measurements of body temperature (BT) variations were performed by a thermistor probe placed in the rectum. BT did not significantly vary from baseline values during saline infusion, whereas it significantly decreased during the infusion of naloxone performed, either before treatment (p less than 0.01), during TTS 50 administration (p less than 0.01), or during TTS 50 + P4 (p less than 0.025). The naloxone induced decrease of BT was greater during TTS 50 administration than before treatment (p less than 0.025). The addition of P4 to TTS 50 administration increased baseline BT of 0.4 degrees C (p less than 0.01), and reduced the ability of naloxone to reduce BT (p less than 0.01 vs. TTS 50). The hyperthermic effect of P4 was not abolished by the infusion of naloxone. Our data show that in postmenopausal women the effect of endogenous opioid peptides on BT is enhanced by estradiol and reduced by progesterone. The hyperthermic effect of progesterone does not seem to be mediated by an increased endogenous opioid activity.


An insulin-like growth factor II-producing histiocytoma associated with hypoglycemia: analysis of the peptide, its gene expression, and glucose transporter isoforms.

An insulin-like growth factor II (IGF-II)-producing histiocytoma was detected in a patient presenting with the classical findings of tumor-related hypoglycemia (low serum insulin and IGF-I concentrations, glucose intolerance, and only modestly increased serum IGF-II levels). Acid-gel filtration of serum extracts showed a single peak of IGF-II immunoreactivity that emerged at the same site as the 125I-labeled human IGF-II standard. High-performance liquid chromatography (HPLC) analysis of the tumor IGF-II demonstrated that it had an identical retention time to that of recombinant human IGF-II. The tumor IGF-II content was extremely high, messenger RNA (mRNA) for IGF-II showed a 100-fold increase in expression compared with normal human liver tissue. Of special interest, a newly identified exon (hE1) was shown to be predominantly expressed in the tumor by Northern blot analysis using leader exon-specific rat IGF-II complementary DNA (cDNA) probes. Although the significance of this finding remains uncertain, this is the first evidence of a new transcription unit in the human IGF-II gene. In addition, immunoblotting showed that the levels of the glucose transporters, GLUT1 and GLUT4, in the tumor were low and undetectable, respectively. This finding makes it unlikely that increased glucose consumption by the tumor accounted for the hypoglycemia in this patient. This case report provides an interesting insight into the pathophysiology of tumor-induced hypoglycemia and new evidence of the abnormal regulation of IGF-II gene expression in human tumors.


Fructose 2,6-bisphosphate levels and modulation of glycolysis by histamine, cholecystokinin, and forskolin in isolated rabbit gastric glands.

In isolated rabbit gastric glands incubated in the presence of 1 mmol/L glucose, the content of fructose 2,6-bisphosphate (F-2,6-P2) was 5.7 +/- 0.5 pmol/mg dry weight. This value was progressively incremented by increasing glucose concentration in the incubation medium, and was almost doubled at 10 mmol/L glucose. Under these conditions, a close correlation could be established between the levels of F-2,6-P2 and the rate of L-lactate formation (r = .98; P less than .05). Both histamine (0.1 mmol/L) and cholecystokinin octapeptide (CCK-OCT; 0.1 mumol/L) increased L-lactate production, without significant changes in either F-2,6-P2 concentration or the amount of 6-phosphofructo-2-kinase in active form. In contrast, forskolin, which markedly increased the glandular content of cyclic adenosine monophosphate (cAMP), partially blocked glucose consumption and caused a significant reduction in both F-2,6-P2 levels and the proportion of 6-phosphofructo-2-kinase in active form. Furthermore, forskolin partially blocked the rate of glucose uptake by isolated gastric glands. Our results suggest a regulatory role of F-2,6-P2 in the control of the glycolytic flux in response to glucose, but not in its response to histamine or CCK-OCT.


Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.


Identification of women at unsuspected risk of primary infection with herpes simplex virus type 2 during pregnancy.

Primary infections with herpes simplex virus type 2 (HSV-2) acquired by women during pregnancy account for about half of the morbidity and mortality from HSV-2 among neonates. The other half results from reactivation of old infections. Better methods are needed to identify which women are at risk for primary HSV-2 infection. We prospectively studied HSV-2 infections among pregnant women who were patients in private obstetrical practices. Using an enzyme-linked immunosorbent assay that detects type-specific antibodies to HSV-2 glycoprotein G, we determined the prevalence at base line of HSV-2 infections among pregnant women and their husbands, the frequency of discordance for infection between partners, and the risk of seroconversion during pregnancy among the seronegative women whose husbands were seropositive. The seroprevalence of HSV-2 was 32 percent among the 277 women followed throughout their pregnancies and 25 percent among the 190 husbands studied. Two thirds of the HSV-2-seropositive women had no history of genital herpes. Of the 190 couples, 139 (73 percent) were serologically concordant for HSV-2 antibodies (57 percent being seronegative and 16 percent being seropositive), whereas 51 couples (27 percent) were discordant, despite having been sexually intimate for a mean of 6.1 years. Eighteen women who were seronegative for HSV-2 (9.5 percent) had seropositive partners, of whom 10 (56 percent) had no history of genital herpes. Thus, approximately 5 percent of these pregnant women had an unsuspected risk of contracting a primary HSV-2 infection. One of the 18 seronegative women with a seropositive husband seroconverted to HSV-2 during pregnancy; none of the other women seroconverted. In this study about 10 percent of pregnant women were at risk of contracting a primary HSV-2 infection from their HSV-2-seropositive husbands. In addition, about a third of these women were seropositive for HSV-2 and thus at risk for asymptomatic, reactivated infections. Serologic testing of couples can identify women who are at risk for primary or reactivated HSV-2 infections during pregnancy.


Parainfluenza virus respiratory infection after bone marrow transplantation.

Pneumonia complicates about half of all bone marrow transplantations, and in about a third of the cases no specific cause is identified. Although parainfluenza virus is a common cause of respiratory infection in normal children, its role in transplant recipients is unknown. We describe the incidence and clinical course of parainfluenza infection among the 1253 recipients of bone marrow transplants at our center from 1974 through 1990. We performed viral cultures on all such recipients who had manifestations of a viral infection or fever without apparent cause. Among the 1253 patients, we found 27 (2.2 percent) who had parainfluenza virus infection as demonstrated by culture (12 of 580 adults and 15 of 673 children). Eight of these patients had only upper respiratory tract involvement, all of whom had positive nasopharyngeal cultures. Of the remaining 19, 8 had symptoms of both upper and lower respiratory tract involvement, and 11 had only lower respiratory involvement, of whom only 6 had positive nasopharyngeal cultures. Four required bronchoalveolar lavage for diagnosis. A median of nine days elapsed from the onset of symptoms until the culture became positive, and overall only 33 of 118 cultures obtained were positive. Respiratory failure developed in 6 of the 19 patients with lower respiratory tract involvement, and all died. Parainfluenza virus is a cause of serious lower respiratory tract involvement in both adults and children who undergo bone marrow transplantation. Given the insensitivity of current culturing techniques, it may be underdiagnosed.


An ACTH4-9 analog enhances social attention in aging rats: a longitudinal study.

Effect of chronic treatment of the ACTH4-9 analog, Org2766, on social attention was studied longitudinally in aging rats. A 6-week treatment temporarily enhanced specific combinations of behaviors of two interacting animals. During a 6-month treatment social attention of treated animals remained at the same level, whereas control animals showed a gradual decrease. Two months after the treatment was ceased this effect on social attention was still present; moreover, the peptide-treated animals performed better in a spatial water maze task three months after the last injection. In addition, the nerve conduction velocity of the major caudal nerves and the sciatic nerve were measured; aged, peptide-treated animals preserved their conduction velocity. The present study shows beneficial long-lasting effects of the ACTH4-9 analog in aging rats on complex behavioral indices and on a physiological measure.


The effect of aging on stress responsiveness and central corticosteroid receptors in the brown Norway rat.

The present study examined the stress responsiveness of the hypothalamic-pituitary-adrenal axis in relation to the properties of corticosteroid receptors in the brain and pituitary of old (30 months) and young (3 months) male Brown Norway rats. Adrenocorticotropin hormone (ACTH) and corticosterone (B) were measured following exposure to novelty and to a conditioned emotional stimulus in blood samples sequentially obtained from chronically cannulated animals. Mineralocorticoid (MR) and glucocorticoid (GR) receptors were quantified by radioligand binding assay and in situ hybridization. The receptor binding constants were determined in tissue of rats that were adrenalectomized 24 hours previously, whereas gene expression was measured in the brain of intact animals. Aged Brown Norway rats showed a small but significant elevation in basal circulating ACTH level. The conditioned emotional stimulus, rather than the exposure to novelty, triggered a more than two-times higher ACTH response in the aged compared to the young rat. The termination of the stress-induced ACTH response seemed to proceed more efficiently in the aged rat. Basal and stress-induced total plasma B level did not differ in the young and old rats. The latter showed a 65% lower binding capacity of corticosteroid-binding globulin (CBG). Interestingly, in the aged rat the stress-induced rise in free circulating plasma B level was not elevated, but only prolonged. The hippocampus of aged rats displayed a decrease of maximally 44% in the apparent Bmax of MR, but no change in GR number. The Bmax of GR showed a 40% reduction in the hypothalamus and a 50% reduction in the anterior pituitary. GR affinity was considerably increased in the anterior pituitary, but was unchanged in the hippocampus and hypothalamus. Old age affected MR and GR gene expression differentially. GR mRNA was significantly reduced in cell field CA3 (-42%), CA4 (-41%) and the dentate gyrus (-26%) of the dorsal hippocampus, but did not change either in hippocampal cell field CA1 or in the hypothalamic paraventricular nucleus (PVN) of the old rat. There was no significant difference in MR mRNA between young and aged rats in the different cell fields of the hippocampus. The aged rat, therefore, is characterized by site- and receptor-specific changes in binding constants as well as by changes in receptor transcription and translation. The data demonstrate that in the old Brown Norway rats, a conditioned emotional stimulus results in enhanced pituitary ACTH release.(ABSTRACT TRUNCATED AT 400 WORDS)


Pharmacological characterization of regulation of phosphoinositide metabolism by recombinant 5-HT2 receptors of the rat.

Transfection of 5-HT2 receptor cDNA in 293 cells induced the expression of a protein binding domain, exhibiting the classical 5-HT2 receptor transduction mechanism. Both [3H]DOB and [3H]spiperone high affinity binding sites were present in membranes of sense but not of antisense, 5-HT2 receptor cDNA transfected cells. Addition of 1 microM 5-HT induced a time-dependent increase of phosphoinositide (PI) metabolism in sense but not in antisense, 5-HT2 receptor cDNA transfected cells. Graded concentrations of 5-HT and of different serotonergic agonists showed different potencies (DOI greater than 5-HT greater than quipazine greater than DOM greater than alpha-methyl-5-HT greater than 8-OH-DPAT greater than 2-methyl-5-HT greater than CGS-12066B) in stimulating turnover of PI in cells transfected with cDNA encoding for 5-HT2 receptors of the rat. The ability of different antagonists to inhibit 5-HT-stimulated turnover of PI bore a direct relationship with their potency to inhibit 5-HT2 receptor binding in cells transfected with 5-HT2 receptor cDNA (spiperone greater than ketanserin greater than ritanserin greater than mianserin greater than haloperidol). Preincubation of transfected 293 cells with pertussis toxin failed to modify either 5-HT- or DOI-induced activation of metabolism of PI. Pretreatment of transfected 293 cells with DOI (100 nM) for 2 hr or more, significantly reduced activation of turnover of PI elicited by graded doses of 5-HT. When the transfected 293 cells were exposed to DOI (100 nM) for 12 hr and the challenge was performed after a 2-hr wash-out period, the desensitization of the response to 5-HT was virtually abolished.(ABSTRACT TRUNCATED AT 250 WORDS)


The effect of systemic administration of baclofen on food intake in rats.

The effects of systemic administration of the GABAB agonist, baclofen was investigated on food intake in non-fasted rats. Baclofen (1.0, 2.0 and 4.0 mg/kg, s.c.) produced a dose-related increase in food intake in a free-feeding paradigm during the first 90 min after administration, with maximum increases occurring at a dose of 2 mg/kg (Experiment 1). Baclofen (0.5 and 1.0 mg/kg, s.c.) also increased food intake in the 40 min post-drug recording period in non-fasted rats, trained to make operant responses for food on a fixed-ratio schedule (Experiment 2). These results demonstrate that systemic administration of baclofen can stimulate ingestive behaviour in satiated rats and suggest a possible role for a GABAB receptor-mediated mechanism in the control of food intake.


Identification of normal and pathological aging in prospectively studied nondemented elderly humans.

Results of a standardized histochemical and immunocytochemical analysis of the brains of 14 nondemented elderly humans for whom prospective neurological and neuropsychological data had been collected for 3 to 8 years before death suggested that nondemented elderly humans fall into two pathological subgroups that are not clinically distinguishable. One was associated with moderate to marked cerebral amyloid deposition ("pathological aging"), while the other had either minimal or no amyloid deposition ("normal aging"). Neocortical and hippocampal neurofibrillary degeneration was either completely absent or of very limited degree in both subgroups. Both subgroups had ubiquitin-immunoreactive dystrophic neurites in the cerebral cortex and granular degeneration of myelin in white matter. These ubiquitin-immunoreactive structures seem to be a universal and invariant manifestation of brain aging, but the same cannot be said for amyloid deposition and neurofibrillary degeneration. Pathological aging might be preclinical Alzheimer's disease, but it currently cannot be distinguished from normal aging by even sensitive neuropsychological measures. These findings provide strong support for the hypothesis that cerebral amyloid deposition is not necessarily associated with clinically apparent cognitive dysfunction and that additional factors, such as neuronal or synaptic loss or widespread cytoskeletal aberrations, are necessary for dementia in AD.


Electrophysiological effects of phencyclidine and the sigma agonist ditolylguanidine in the cerebellum of the rat.

The electrophysiological actions of phencyclidine (PCP) and the sigma agonist 1,3-di(2tolyl)guanidine (DTG) were examined in the cerebellum of urethane-anesthetized rats. The object of the study was to determine if PCP and sigma agonists shared a common mechanism of action. The cerebellar Purkinje neuron was chosen because it has sigma receptors but not N-methyl-D-aspartate receptors, where PCP has additional effects. Both DTG and PCP decreased the spontaneous discharge rate of cerebellar Purkinje neurons after parenteral administration. When the drugs were applied locally to single Purkinje neurons, using pressure ejection through multibarrel micropipettes, both compounds decreased the spontaneous activity of the neurons with equal potency. Previous studies have shown that the actions of PCP in the cerebellum are dependent upon an interaction with noradrenergic terminals from the nucleus locus coeruleus. A similar finding was made in this study for DTG. Elimination of the noradrenergic input by lesion with the neurotoxin, 6-hydroxydopamine, diminished equally the effects of PCP and DTG. Treatment of the animals with haloperidol had similar effects. It is concluded that PCP and the sigma agonist DTG both act as indirect noradrenergic agonists in the cerebellum.


Inositol 1,4,5-trisphosphate receptor in developing and senescent rat cerebellum.

Numerous process associated with intracellular calcium homeostasis have previously been found to vary with age. To determine whether the binding sites for the calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (InsP3), also displays such variation, [3H]InsP3 binding was investigated in cerebellar or cerebral cortical membranes prepared from rats at different ages from birth up to 24 months of age. In the cerebellum, the InsP3 receptor density was very low during the first week after birth, increased markedly between days 8 and 28 and then reached an apparent plateau between 28 to 56 days of age. The InsP3 receptor binding affinity was comparable at different developmental stages. No age-related differences were found in InsP3 receptor density or affinity in the cerebral cortex of 3-, 6-, 9-, 12-, and 24-month-old rats. In the cerebellum, InsP3 receptor density but not affinity was significantly reduced in 24-month-old compared only to 3-month-old animals. Our data suggest that the changes in InsP3 receptor binding during early development might reflect the growth and maturation of neurons containing these receptors (i.e., Purkinje cells). Furthermore, the age-dependent reduction in InsP3 receptor density, together with the recent report of senescent changes in protein kinase C activity, indicate that disruption of phosphoinositide second messenger system may be of importance to the impairment of neuronal responsiveness and behavioral deficits observed with aging.


Receptor-mediated volume regulation in astrocytes in primary culture.

Changes in astroglial volume were studied after incubating primary cultures from the cerebral cortex of newborn rats for 5 hr in taurine, glutamate or gamma-aminobutyric acid (GABA), alone or together with monoamine receptor agonists. Control cell volume was 2.2 microliters/mg protein. In the presence of taurine or glutamate there was a small increase in cell volume, further augmented when the cells were incubated in isoproterenol plus taurine or phenylephrine, or isoproterenol plus glutamate. After incubation in 5-hydroxytryptamine (5-HT) plus taurine, on the other hand, the cell volume was not different from the control value. In the presence of GABA, alone or together with adrenoceptor agonists (alpha 1, alpha 2 or beta) or 5-HT, there were no significant changes in cell volume. The regulation of astroglial volume is complicated and affected by ion fluxes, free amino acids and metabolic events including changes in pH. The results are discussed in relation to late data on receptor regulation of active uptake of amino acids. Astroglial volume might be influenced by amino acid transport, partly under control of monoamine receptors.


Progressive decline in spatial learning and integrity of forebrain cholinergic neurons in rats during aging.

Rats distributed over five different age groups, 3, 12, 18, 24 and 30 months of age, were screened for their spatial learning and memory ability in the Morris water maze, and the degree of place navigational impairments was correlated with morphological changes in the four major forebrain cholinergic cell groups (medial septum, MS; vertical limb of the diagonal band of Broca, VDB; nucleus basalis magnocellularis, NBM; and striatum) using choline acetyltransferase (ChAT) and nerve growth factor receptor (NGFr) histochemistry. Impaired place navigation developed progressively with age, such that 8% of the 12-month-old rats, 45% of the 18-month-old, 53% of the 24-month-old, and over 90% of the 30-month-old rats were behaviorally impaired. Significant reductions in the number of ChAT/NGFr-positive cell bodies, amounting to between 19 and 45%, were observed in all four cell groups, and the remaining cells were reduced in size (6-24% reduction in cross-sectional area in the oldest age groups). Although the morphological changes were less severe and tended to develop later than the behavioral impairments, there was overall a significant correlation between water maze performance and ChAT/NGFr-positive cell counts, and to a lesser degree also cell size in all four cell groups. These changes were also highly correlated with age. The highest correlations were seen in MS, VDB and NBM, which are known to play a role in spatial memory performance in young rats. The results indicate that degenerative and/or atrophic changes in the forebrain cholinergic system and decline in spatial learning ability are parallel processes during aging. Although the magnitude of the morphological changes does not appear to be substantial enough, by itself, to explain the severe spatial learning impairments that develop in the oldest animals, the present data are consistent with the view that impaired function in the forebrain cholinergic system can contribute to age-dependent cognitive decline in rodents.


The effect of 3,3-dipyridylmethyl-1-phenyl-2-indolinone on the neuromuscular transmission in the rodent skeletal muscles.

The effects of a cognition enhancer, 3,3-dipyridylmethyl-1-phenyl-2-indolinone (DPMPI) (21.5-645 microM), on neuromuscular transmission were studied electrophysiologically on diaphragms of mouse and rat and the soleus muscle of rat. The drug DPMPI (21.5-645 microM) increased both direct and indirect twitch tension of mouse diaphragm. It also increased (a) the frequency of miniature endplate potentials and (b) the quantal content of endplate potential. However, DPMPI (64.5 microM) affected neither the amplitude of the directly elicited action potential of soleus muscle in the rat nor the magnitude of the resting membrane potential of mouse diaphragm, although DPMPI (215 microM) decreased the amplitude of the compound action potential of phrenic nerve. Based on these results, it is concluded that DPMPI had several effects on neuromuscular transmission, i.e. it (a) facilitated the transmitter releasing process of the motor nerve terminal, (b) decreased the conduction in the phrenic nerve and (c) increased the directly elicited twitch tension.


Characteristics and function of Ca(2+)- and inositol 1,4,5-trisphosphate-releasable stores of Ca2+ in neurons.

Molecular, biochemical and physiological evidence for the existence of releasable Ca2+ stores in neurons is strong. There are two separate molecules that function as release channels from those Ca2+ stores, the RyanR and InsP3R, and both have multiple regulatory sites for positive and negative control. Perhaps most intriguing is the biphasic, concentration-dependent action of cytosolic Ca2+ on both channels, first to stimulate release then, at higher concentration, to depress release. Whether the InsP3R and RyanR channels regulate Ca2+ release from different or identical functional compartments will need to be defined for each neuron type and perhaps even for each intracellular region within neurons since the evidence for functional separation of stores is mixed. The identification of Ca2+ storage and releasing capacity throughout all subcellular regions of neurons and the increasing evidence for a role for Ca2+ stores in neuronal plasticity suggests that the further characterization of the functional properties of Ca2+ stores will be an increasingly important and expanding area of interest in neurobiology.


Isolation of glutamate-binding proteins from rat and bovine brain synaptic membranes and immunochemical and immunocytochemical characterization.

We previously isolated two glutamate-binding proteins from rat brain synaptic membranes of 71,000 and 63,000 mol. wt [Chen et al. (1988) J. biol. Chem. 263, 417-426]. In the present study, the 71,000 and 63,000 mol. wt glutamate-binding proteins were purified from rat and bovine brain synaptic membranes by affinity chromatographic separation on an L-glutamate-derived Trisacryl matrix. The major protein component in the purified fractions was a 71,000 mol. wt protein as judged by sodium dodecyl sulfate gel electrophoresis. This fraction represented enrichment of bovine brain glutamate-binding proteins by a factor of 7000-8000 when compared with brain homogenates. The ligand binding characteristics of the proteins purified by this new procedure are very similar to those of the proteins purified by the procedures we described in previous studies. Polyclonal antibodies were raised in mice against the purified, native rat and bovine brain glutamate-binding proteins. These two sets of antibodies interacted specifically with the glutamate-binding proteins but not with any glutamate-metabolizing enzymes. Both sets of antibodies labeled a 71,000 mol. wt protein in Western blots of synaptic membranes obtained from either rat or bovine brain, an indication of homology between these proteins. Both sets of antibodies produced immunoprecipitation of approximately 70-75% of all glutamate-binding proteins from solubilized synaptic membrane proteins. These observations are an indication that the glutamate-binding proteins described above are the most common glutamate-binding entities in both bovine and rat brain synaptic membranes and that they can be easily purified in a one-step chromatographic procedure.


Contrasting patterns in the localization of glutamic acid decarboxylase and Ca2+/calmodulin protein kinase gene expression in the rat central nervous system.

The expression of the genes encoding the alpha subunit of type II calcium calmodulin-dependent protein kinase (CaM II kinase alpha) and the 67,000 mol. wt form of glutamic acid decarboxylase was examined throughout the rat central nervous system. In situ hybridization histochemistry, using cRNA probes, revealed a dense population of CaM II kinase alpha-expressing cells throughout the telencephalon and diencephalon. CaM II kinase alpha mRNA was also expressed in the midbrain, cerebellum and medulla oblongata, but at greatly reduced levels. No CaM II kinase alpha gene expression was detected in nuclei producing monoamines or acetylcholine. By contrast, the glutamic acid decarboxylase gene was moderately to highly expressed throughout the central nervous system. In several regions there was a complementarity in the distributions of cells expressing the glutamic acid decarboxylase or CaM II kinase alpha genes. Cells in certain nuclei such as the thalamic reticular nucleus or globus pallidus showed glutamic acid decarboxylase gene expression only; others such as the majority of the dorsal thalamic nuclei showed CaM II kinase alpha gene expression only. Several regions contained both glutamic acid decarboxylase and CaM II kinase alpha expressing cells. However, simultaneous immunostaining for both proteins revealed only two regions where CaM II kinase alpha and glutamic acid decarboxylase immunoreactivity were colocalized: the cerebellar Purkinje cells and the commissural nucleus of the stria terminalis. The results imply that CaM II kinase alpha is primarily expressed in non-GABAergic neurons. In several regions CaM II kinase alpha mRNA is concentrated in nuclei known to contain populations of neurons that use excitatory amino acid transmitters.


Dendritic localization of type II calcium calmodulin-dependent protein kinase mRNA in normal and reinnervated rat hippocampus.

In situ hybridization histochemistry has revealed a diffuse distribution of the alpha subunit of type II calcium calmodulin-dependent protein kinase (CaM II kinase alpha) mRNA in the neuropil of regions containing CaM II kinase alpha-expressing cells and has led some to propose that it may be expressed in dendrites. In order to determine if CaM II kinase alpha mRNA is expressed in dendrites and if the gene encoding CaM II kinase alpha is regulated in response to synaptic reinnervation, we examined its expression in the hippocampus of normal rats, of rats that had received a unilateral injection of kainic acid and of rats with a unilateral entorhinal cortex lesion. The relatively specific elimination of the CA3 pyramidal cells by kainate lesions precisely correlated with the loss of CaM II kinase alpha cRNA hybridization in the stratum radiatum as well as the stratum pyramidale. Following entorhinal cortex lesions, during the period of new synapse formation in the dentate gyrus, there was no detectable change in the level of CaM II kinase alpha gene expression. These data suggest that CaM II kinase alpha mRNA is expressed in the dendrites of hippocampal pyramidal cells and, therefore, is likely to be expressed in dendrites in other regions of the central nervous system exhibiting CaM II kinase alpha cRNA labeling in the neuropil. However, changes in expression were not found to accompany new synapse formation.


Cellular pattern of type-I insulin-like growth factor receptor gene expression during maturation of the rat brain: comparison with insulin-like growth factors I and II.

Insulin-like growth factors have a number of potent trophic effects on cultured neural tissue and most if not all of these effects appear to be mediated by the type-I insulin-like growth factor receptor. In order to establish the identity of cell types expressing this receptor in the rat central nervous system during development and maturity, we have used in situ hybridization to map sites of type-I insulin-like growth factor receptor mRNA synthesis in the developing and adult rat brain. In order to identify possible local sources of peptide ligands for this receptor, we have also mapped the sites of insulin-like growth factors I and II mRNA synthesis in parallel brain sections. From early development onward, there is a uniform and stable pattern of type-I insulin-like growth factor receptor gene expression in all neuroepithelial cell lineages, in which regional variations reflect primarily differences in cell density. In addition to this generalized pattern, during late postnatal development, high levels of type-I insulin-like growth factor receptor gene expression are found in specific sets of sensory and cerebellar projection neurons in conjunction with abundant insulin-like growth factor-I gene expression in these same neurons. While insulin-like growth factor-I expression is confined to the principal neurons in each system, receptor mRNA is also found in local interneurons. In the cerebral cortex and hippocampal formation, type-I insulin-like growth factor receptor mRNA and insulin-like growth factor-I are concentrated in different cell populations: receptor mRNA is abundant in pyramidal cells in Ammon's horn, in granule cells in the dentate gyrus, and in pyramidal cells in lamina VI of the cerebral cortex. Insulin-like growth factor-I mRNA is found in isolated medium- to large-sized cells which are rather irregularly distributed throughout the hippocampus and isocortex. In the hypothalamus, receptor mRNA is concentrated in the suprachiasmatic nucleus but is in low abundance elsewhere, including the median eminence, while insulin-like growth factor-I mRNA is not detected in this region at all. Type-I insulin-like growth factor receptor and insulin-like growth factor-II mRNAs are both abundant in choroid plexus, meninges and vascular sheaths from early development to maturity, but insulin-like growth factor-II mRNA is not detected in cells of neuroepithelial origin at any stage of development. This study provides evidence for two fundamentally different patterns of gene expression for the brain type-I insulin-like growth factor receptor.(ABSTRACT TRUNCATED AT 400 WORDS)


Primary receptor for inhibitory transmitters in lamprey spinal cord neurons.

The action of glycine and GABA on isolated lamprey spinal cord neurons was investigated by means of intracellular perfusion and concentration clamp techniques. These amino acids activated desensitizing chloride ionic conductances. The concentrations of agonists evoking half-maximum effects (ED50) were equal to 16 microM and 1.5 mM for glycine- and GABA-activated currents, respectively. Increase in the transmitter concentration led to a decrease in the time constant of desensitization. Current-voltage relationships of glycine- and GABA-activated currents were strongly dose-dependent. At low agonist concentrations the time constant of activation decreased with membrane hyperpolarization. Glycine- and GABA-activated currents exhibited complete cross-desensitization. The specific glycine antagonist, strychnine, suppressed both glycine- and GABA-activated currents to the same degree. Selective antagonists of GABA receptors, bicuculline and picrotoxin, produced equal blocking effects on glycine- as well as GABA-evoked responses. In the cells studied, taurine activated desensitizing ionic conductance. Responses evoked by taurine and glycine applications demonstrated complete cross-desensitization. Taurine-activated currents were sensitive to strychnine, bicuculline and picrotoxin. These results suggest the existence of one receptor-channel complex for the main inhibitory transmitters in lamprey spinal cord neurons. 3,4-Dioxy-L-beta-phenylalanine evoked desensitizing strychnine-sensitive ionic responses which exhibited cross-desensitization with glycine-activated currents.


Postischemic binding of [3H]phorbol 12,13-dibutyrate and [3H]inositol 1,4,5-trisphosphate in the gerbil brain: an autoradiographic study.

Postischemic alteration of second messenger systems was investigated in the Mongolian gerbil, utilizing [3H]phorbol 12,13-dibutyrate and [3H]inositol 1,4,5-trisphosphate receptor autoradiography. Transient ischemia was induced for 10 min, and animals were allowed to survive for various recirculation periods of up to one month. [3H]Phorbol 12,13-dibutyrate binding in selectively vulnerable areas showed no significant change 1-24 h after ischemia except for a transient decline in a few regions. Thereafter, the binding in most of the selectively vulnerable areas showed significant alteration 48 h or seven days after ischemia. Interestingly, dentate molecular layer which was resistant to ischemia showed a significant elevation in the number of [3H]phorbol 12,13-dibutyrate binding sites. One month after ischemia, [3H]phorbol 12,13-dibutyrate binding showed significant reduction only in the striatum and the hippocampal CA1 sector where severe neuronal damage was seen morphologically. A significant elevation in the number of [3H]phorbol 12,13-dibutyrate binding sites was still seen in the dentate molecular layer one month after ischemia. In contrast, [3H]inositol 1,4,5-trisphosphate binding showed significant reduction in the selectively vulnerable regions 1-24 h after ischemia. Thereafter, [3H]inositol 1,4,5-trisphosphate binding in most of the selectively vulnerable areas markedly decreased up to one month after ischemia. In the dentate molecular layer, [3H]inositol 1,4,5-trisphosphate binding also showed significant reduction during recirculation except for a slight recovery 48 h and seven days after ischemia. One month after ischemia, the binding in all regions showed significant reduction. These results suggest that postischemic alteration of two second messenger (protein kinase C and inositol 1,4,5-trisphosphate) binding sites was produced with different processes in selectively vulnerable areas.(ABSTRACT TRUNCATED AT 250 WORDS)


Adenosine-dependent enhancement by methylxanthines of excitatory synaptic transmission in hippocampus of rats.

Whole-cell patch-clamp was used to investigate synaptic transmission in hippocampal slices. Excitatory post-synaptic currents (EPSCs) were facilitated by low (less than or equal to 1 microM) adenosine (Ado) concentrations, while high concentrations had well-known inhibitory effects on the EPSC. When added on the background of preapplied Ado, methylxanthines caused a large potentiation of EPSCs. At saturation, the enhanced EPSC could exceed the control almost by an order of magnitude. Pertussis toxin strongly impaired the ability of Ado to block EPSCs but did not augment the facilitatory effect. The two components of the EPSC mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors were facilitated simultaneously and in equal proportions.


HS-142-1, a novel non-peptide ANP antagonist, blocks the cyclic GMP production elicited by natriuretic peptides in PC12 and NG108-15 cells.

HS-142-1 is a novel non-peptide antagonist for atrial natriuretic peptide (ANP) receptor. The effect of HS-142-1 on the cyclic GMP production elicited by natriuretic peptides in neuronal cell lines, PC12 and NG108-15 was examined. Natriuretic peptides such as ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) enhanced cyclic GMP production in a dose-dependent manner. HS-142-1 inhibited cyclic GMP accumulation elicited by natriuretic peptides in a dose-dependent fashion in both cells. The results suggest that HS-142-1 will be an important tool for identification and understanding of the mechanisms by which natriuretic peptides act in nervous systems.


Chronic elevation of cAMP levels induces changes in the adenylate cyclase system, opiate receptor sensitivity and levels of Gs-mRNA in cultured neurons.

Neurons in primary culture were treated (5 days) with the adenylate cyclase stimulator 10(-5) M forskolin. The basal adenylate cyclase was decreased by 57%. The acute stimulatory effect of forskolin was down-regulated by 48%. The inhibitory effects of the 3 opiate receptor agonists (mu, delta and kappa) were partly abolished. The abundance of mRNA encoding the stimulatory G-protein (Gs) was decreased prominently. The data indicate that the cAMP system in neuronal cells in primary culture is under dynamic regulation, possibly including altered Gs-protein gene expression. Furthermore, long-term forskolin treatment might induce increased proliferation in susceptible neural blast cells.


Fast and slow twitch skeletal muscle fibres differ in their distribution of Na channels near the endplate.

Sodium channel distributions were measured in fast and slow twitch rodent skeletal muscle fibres using the loose patch voltage clamp technique. Large differences were found between these fibre types with respect to Na channel density in the perijunctional region. Fast twitch fibres exhibited a large increase in Na channel density near the endplate, while slow twitch fibres did not.


Alteration of pituitary-adrenal responses to adrenalectomy by the immunological targeting of CRF neurons.

We previously demonstrated that cellular toxins added to a cytotoxic IgG2a monoclonal antibody to corticotropin releasing factor (CRF-MAb) may specifically penetrate some hypothalamic CRF neurons, after central injection near the paraventricular nuclei. We attempt here to evaluate the consequential effects on the CRF neurons functioning. Such a toxic mix, 4 weeks after its central injection, caused a marked reduction (66%) of the chronic adrenocorticotropic hormone (ACTH) release in response to a bilateral adrenalectomy (7th day). This change was accompanied by a reduction in the CRF concentration (43%) measured in the median eminence. We concluded that specific internalization of toxins, by the way of CRF-MAb, leads to a long-term dysregulation of the CRF synthesis and/or neuronal transport.