Comparison of the effect of the GABA uptake blockers, tiagabine and nipecotic acid, on inhibitory synaptic efficacy in hippocampal CA1 neurones. The action of the novel gamma-aminobutyric acid (GABA) uptake blocker, tiagabine, has been studied on isolated GABAergic fast inhibitory postsynaptic potentials (IPSP) and currents (IPSC) in rat hippocampal CA1 pyramidal cells in the slice preparation. Tiagabine (20-50 microM) had little effect on the peak amplitude of the IPSC, but caused a robust increase in the half-width (by 109 +/- 15%). These results contrasted with those obtained using the established uptake blocker, nipecotic acid (100 microM to 1 mM), which reduced the amplitude of the IPSC by 35 +/- 6% and caused only a modest prolongation of the recovery phase. These effects, which were poorly reversible, are probably explained by the fact that nipecotic acid is a substrate for the GABA-uptake carrier and can act as a false transmitter. Tiagabine is not transported by the GABA carrier and results with this substance demonstrate the role of uptake in determining the kinetics of activation of GABAA receptors. Tiagabine is proposed as the blocker of choice for the GABA uptake system.


Effects of enflurane on the voltage-gated membrane currents of bovine adrenal chromaffin cells. The effects of the volatile anesthetic enflurane on voltage-gated ionic currents of bovine adrenal chromaffin cells were studied using the patch clamp technique. Bath application of 3.5% (1.7 mM) enflurane decreased the outward Ca(2+)-dependent K+ current (IK(Ca)) 'hump' by 88 +/- 6% (mean +/- S.E.M., n = 5 cells) and the peak inward Ca2+ current by 60 +/- 3% (n = 5), whereas the Ca(2+)-independent K+ current fell by only 34 +/- 3% (n = 5) and peak inward Na+ current was unchanged. Exposure of excised patch 'BK' Ca(2+)-dependent K+ channels to 3.5% enflurane revealed that the anesthetic directly suppressed the channel probability of opening by 68 +/- 10% (n = 4) with no effect on open state conductance. The differential sensitivity of depolarizing and hyperpolarizing current pathways may contribute to the biphasic response, excitation and depression, observed in certain neuronal systems in response to this inhalational anesthetic.


[125I]-labelled iodomelatonin binding sites in the duck bursa of Fabricius: binding characteristics and diurnal variation. Melatonin receptors in membrane preparations of the duck bursa of Fabricius were studied by using [125I]-labelled iodomelatonin as the radioligand. Specific binding of [125I]-labelled iodomelatonin in the membrane preparations of bursa was stable, saturable, reversible and of high affinity. Scatchard analysis of the specific binding revealed an equilibrium dissociation constant (Kd) of 48.5 +/- 7.4 pmol/l and a total number of binding sites (Bmax) of 1.38 +/- 0.12 fmol/mg protein at mid-light. A diurnal study showed that the Bmax of [125I]-labelled iodomelatonin binding sites at mid-light was 94.4% higher (P < 0.05) than that at mid-dark. There was no significant difference in the mid-light and mid-dark Kd values. The Kd value determined by kinetic analysis was 42.0 +/- 9.3 pmol/l at mid-light. The pharmacological characteristics indicated that [125I]-labelled iodomelatonin binding sites are highly specific for melatonin. Our results suggest that the bursa is a target organ of melatonin action.


Tumor necrosis factor alters synaptic transmission in rat hippocampal slices. The effects of human recombinant tumor necrosis factor (TNF-alpha) on the synaptic transmission were studied in rat hippocampal slices by using extracellular field potential recordings. Population spikes and/or excitatory postsynaptic potentials were extracellularly recorded in hippocampus CA1 region from stratum pyramidale and stratum radiatum, respectively, and synaptic transmission was examined in the Schaffer collateral/commissural-CA1 pathway. Basal neurotransmission slightly and promptly increased in slices acutely exposed to TNF-alpha (1-100 nM). Examination of the long-term potentiation (LTP) revealed that a brief treatment with the cytokine did not influence LTP, while a long-lasting application of TNF-alpha (50 min or more) inhibited LTP in a dose-dependent way in the range of 1-100 nM. A role for TNF-alpha as a peptide of immunological significance belonging to the family of brain neuromodulators is discussed.


Characterization of 2-[125I]iodomelatonin-binding sites in quail testes at mid-light and mid-dark. The binding and pharmacological characteristics of 2-[125I]iodomelatonin binding sites in testis membrane preparations of quails were examined. Scatchard analyses yielded an equilibrium dissociation constant (Kd) of 46.6 +/- 8.6 pmol/l and maximum binding capacity (Bmax) of 2.77 +/- 0.20 fmol/mg protein for the gonadal 2-[125I]iodomelatonin binding sites. Except for melatonin, 6-chloromelatonin, 2-iodomelatonin and N-acetylserotonin, all compounds tested elicited no significant inhibition of radioligand binding. Significant diurnal variations were detected in serum melatonin levels of 24-week-old quails while no diurnal difference was detected in the affinities or densities of the gonadal 2-[125I]iodomelatonin binding sites in quails. Results of the present study suggest possible direct gonadal action by pineal melatonin in birds.


Hyperexcitability of hippocampal CA1 region in brain slices after GABA withdrawal. The interruption of GABA infusion in the cerebral cortex and in the hippocampus produces electrographic seizures in rats. Here, we have used the hippocampal slice preparation to induce a 'GABA withdrawal syndrome (GWS)'. With the stimulation parameters used (0.2 Hz, 200 microseconds), activation of the Schaffer afferents produced one population spike in the CA1 subfield, while multiple population spikes were observed in the slices previously incubated in GABA. Also, we recorded an increase in the amplitude of the population spike when compared to its control value. Paired pulse test showed absence of recurrent inhibition in these slices. These results suggest a dysfunction in GABAergic neurotransmission.


Effects of intrathecal capsaicin and an NK-1 antagonist, CP,96-345, on the thermal hyperalgesia observed following unilateral constriction of the sciatic nerve in the rat. We evaluated the effect of intrathecal (i.t.) capsaicin (CAP) and the NK-1 selective non-peptidic antagonist, CP,96-345, on the thermal hyperalgesia ordinarily observed after unilateral partial ligation of the sciatic nerve in rats. CAP was injected i.t. 2 days after constriction injury. Seven days after partial ligation, the levels of substance P (sP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) were the same in the left and right dorsal horns of the lesioned rats which were injected with vehicle (VEH). CAP (75 micrograms/15 microliters of 20% 2-hydroxypropyl-beta-cyclodextrin) resulted in an equal reduction (40-50%) in the dorsal horn levels of sP and CGRP, but not VIP. After 7 days, i.t. CAP increased the paw withdrawal latency (PWL) of the non-injured hind paw. In contrast, there was no change in the PWL of the injured paw when compared to that of VEH-treated animals. Thus, CAP did not abolish the hyperalgesic state. We concluded that the thermal hyperalgesia after sciatic nerve constriction injury is not mediated by CAP-sensitive C fibers. CP,96-345 given i.t. at a dose which is physiologically active (400 micrograms) had little effect on the thermal response latency of either the normal or hyperesthetic paw. This provides further evidence that neither the normal pain response nor hyperalgesic state is dependent upon a dorsal horn action of sP.


Unusual causes of obstructive jaundice in children: diagnosis on CT. Three cases are presented with unusual causes of obstructive jaundice diagnosed on abdominal CT in children under the age of 15 years. All the cases were initially examined by ultrasound which was inconclusive. CT studies were subsequently performed were diagnostic. These cases included hepatocellular carcinoma, a common bile duct web and duodenal hematoma. The first two cases were surgically confirmed, while the third case was proved by clinical follow-up.


Malignant endobronchial lesions of adolescence. Rare primary endobronchial malignancies were diagnosed in four adolescents: atypical bronchial carcinoid, mucoepidermoid carcinoma, bronchogenic (squamous cell) carcinoma, and non-Hodgkin's lymphoma. Metastatic disease was evident in each case. Three of the four patients died within one year of diagnosis. This paper describes each of these entities, illustrates their plain film and CT abnormalities (bronchial "cut-off", hilar mass, local congestive edema, lobar collapse, hyperinflation), and discusses the utility of CT in their diagnosis.


Nephroblastomatosis: the whole spectrum of abnormalities in one case. A case of nephroblastomatosis is described in a previously healthy child with a palpable mass in the right upper quadrant as an incidental finding. This case of nephroblastomatosis demonstrates the whole spectrum of abnormalities from local regression to formation of true Wilms' tumors bilaterally over a 4-year period. The evolution of these changes provides a graphic and dramatic example of this interesting entity, indicating its potential malignant association with Wilms' tumor.


A rapid quantitative bioassay for evaluating the effects of ligands upon receptors that modulate cAMP levels in a melanophore cell line. A new method for rapidly evaluating the effects of drugs on receptors that regulate intracellular cAMP in a cell line derived from Xenopus laevis melanophores has been developed. Melanophores were plated into sterile 96 well microtiter plates, and 3 days later the cells were treated with melatonin for 30 min to induce melanosome aggregation. Subsequent exposure to MSH or adrenergic agonists caused dose dependent pigment dispersion that peaked within 30 min. The cumulative pigment displacement from cells could be quantitated by using a microplate reader to measure changes in transmittance of light through the wells. The acquired data enabled detailed and reproducible dose response curves and time course analyses to be generated. In addition, the assay followed for the rapid characterization of the effects of antagonists upon the beta adrenergic receptor (beta AR). The assay has the potential to test the effects of ligands upon any receptor capable of mediating pigment translocation in the melanophore cell line.


Detection of human papillomavirus (HPV) DNA type 6/11 in a conjunctival papilloma by in situ hybridization with biotinylated probes. Four cases of conjunctival papilloma in two different patients were examined by in situ hybridization for HPV DNA type 6/11, 16/18 and 31/33/51. Formalin-fixed and paraffin-embedded tissues were hybridized by biotinylated probes. One tumor and one of its recurrences showed nuclear positivity for HPV 6/11 in the superficial cells of the epithelium. The results suggest that HPV type 6/11 may be etiologic agent of conjunctival papillomas. The benign behavior of these neoplasms may be related to the etiologic role of this type of HPV.


[Oxidative stress in 29 HIV seropositive patients. Two-year results of a double-blind trial of diethyldithiocarbamate versus placebo]. Among 29 seropositive subjects who had participated in the HIV 87 therapeutic trial (Mérieux laboratories), the oxidative stress was evaluated at 24 months in 16 treated with diethyldithiocarbamate (dithiocarb) and in 13 who had received the placebo. No significant difference was found between these two groups, whereas the existence of an oxidative stress has been confirmed in seropositive subjects compared with controls.


Structural characterization of rabbit brain ubiquitin. Ubiquitin has been isolated and purified from rabbit brain using gel permeation and reverse-phase high-performance liquid chromatography. The 76-residue protein exhibits one difference towards a murine form, is identical to other characterized vertebrate ubiquitins, and confirms an extensive conservation of the ubiquitin structure. No positional microheterogeneities were detectable between two sub-forms.


Differential PGE2 and cAMP production by allogeneic and syngeneic pregnant mice uteri. Prostaglandin E2 (PGE2) and cAMP production in allogeneic and syngeneic pregnant mice uteri, were measured in relation to the ratio of plasma estrogen/progesterone levels. PGE2 generation by allopregnant uteri varied with the days of pregnancy. The increment of the prostanoid coincided with the increase in plasma estrogen concentration, whereas the decrement of its production was in parallel with the increment of plasma progesterone. The syngeneic pregnant uterus was unable to increase the release of PGE2 above basal values during the whole pregnancy. The rise of PGE2 production by the allogeneic pregnant uterus was correlated with an increase in cAMP levels. It is proposed that the pregnant mouse uterus increases its ability to release PGE2 in response to an ovarian steroids.


Effects of eicosapentaenoic acid, gamma-linolenic acid and prostaglandin E1 on three human colon carcinoma cell lines. Several studies have demonstrated that certain essential fatty acids present a specific cytotoxicity for tumor cells. However, no investigation of this type has been performed on human colon cancer cells to date. This study investigated the effect of gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and prostaglandin (PG) E1 on the proliferation and metabolism of three human colon cancer cell lines: HT 29, HRT 18, and CACO 2. GLA, EPA and PGE1 all inhibited the proliferation of the three cell lines, but with a decreasing gradient of sensitivity: HRT 18 > HT 29 > CACO 2, and with different IC50 values. PGE1 was markedly less effective than the other two. GLA and EPA increased lipid peroxidation and membrane fluidity in a dose-dependent manner. The presence of indomethacin did not modify the effects of GLA and EPA. In addition, PGE1 had little effect on membrane fluidity and lipid peroxidation. The antitumoral effect thus does not appear to be mediated by PGE1. Addition of vitamin E decreased the effects of GLA and EPA, which supports the hypothesis of direct action by these fatty acids. In conclusion, while EPA and GLA have an antitumoral effect in vitro, their effect on primary cultures of normal human colon cells must be investigated to determine whether this effect is specific to tumoral cells, as has been observed for other cell types.


Endothelin-1 stimulates phospholipid hydrolysis and prostaglandin F2 alpha production in primary human decidua cell cultures. The regulation of prostaglandin (PG) production by the uterine decidua may be an important mechanism controlling the onset and maintenance of human parturition. The present in vitro study has evaluated the potential for endothelin-1 (ET-1) to activate cell signalling and PGE2 alpha production in human primary decidua cell cultures. ET-1 stimulated a dose-dependent increase in inositol phospholipid hydrolysis and PG precursor release as evidenced by respective increases in [3H] inositol monophosphate accumulation and [14C] arachidonate release from radiolabelled decidua cells. PGF2 alpha production was increased in some but not all cell preparations in response to ET-1 alone. Pretreatment of decidua cells with interleukin-1 beta (IL-1 beta) enhanced PGF2 alpha production but not arachidonate release in response to ET-1. These in vitro observations support a possible role for ET-1 in the regulation of decidual PG production during parturition.


Magic angle spinning NMR studies on the metarhodopsin II intermediate of bovine rhodopsin: evidence for an unprotonated Schiff base. Magic angle spinning (MAS)13C-NMR spectra of the metarhodopsin II intermediate have been obtained using bovine rhodopsin regenerated with retinal 13C-labeled at the C-13 and C-15 positions to investigate the protonation state of the retinal Schiff base linkage. The 13C-labeled rhodopsin was reconstituted into 1,2-dipalmitoleoylphosphatidylcholine bilayers to increase the amount of meta II trapped at low temperature. Both the 13C-15 (159.2 ppm) and 13C-13 (144.0 ppm) isotropic chemical shifts are characteristic of an unprotonated Schiff base, while the 13C-15 shift is significantly different from that of retinal (191 ppm) or a tetrahedral carbinolamine group (70-90 ppm) previously proposed as an intermediate in the hydrolysis of the Schiff base at the meta II stage. This rules out the possibility that meta II non-covalently binds retinal or is a carbinolamine intermediate and provides convincing evidence that Schiff base deprotonation occurs in the meta I-meta II transition, an event that is likely to be important in triggering the activation of transducin.


The two consecutive M substates in the photocycle of bacteriorhodopsin are affected specifically by the D85N and D96N residue replacements. The photocycle of the proton pump bacteriorhodopsin contains two consecutive intermediates in which the retinal Schiff base is unprotonated; the reaction between these states, termed M1 and M2, was suggested to be the switch in the proton transport which reorients the Schiff base from D85 on the extracellular side to D96 on the cytoplasmic side (Váró and Lanyi, Biochemistry 30, 5016-5022, 1991). At pH 10 the absorption maxima of both M1 and M2 could be determined in the recombinant D96N protein. We find that M1 absorbs at 411 nm as do M1 and M2 in wild-type bacteriorhodopsin, but M2 absorbs at 404 nm. Thus, in M2 but not M1 the unprotonated Schiff base is affected by the D96N residue replacement. The connectivity of the Schiff base to D96 in the detected M2 state, but not in M1, is thereby established. On the other hand, the photostationary state which develops during illumination of D85N bacteriorhodopsin contains an M state corresponding to M1 with an absorption maximum shifted to 400 nm, suggesting that this species in turn is affected by D85. These results are consistent with the suggestion that M1 and M2 are pre-switch and post-switch states, respectively.


Time-resolved Fourier transform infrared spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: Asp-96 reprotonates during O formation; Asp-85 and Asp-212 deprotonate during O decay. The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.


Photolysis of rhodopsin results in deprotonation of its retinal Schiff's base prior to formation of metarhodopsin II. Absorption changes following photolysis of bovine rhodopsin in mildly sonicated membrane suspensions are monitored at 25 degrees C. Difference spectra collected at 17 times between 1 microsecond and 75 ms following excitation are analyzed globally using singular value decomposition and non-linear least-squares fitting techniques. The results are not consistent with the simple scheme: Lumirhodopsin-->Metarhodopsin I<-->Metarhodopsin II, but indicate that an intermediate with a deprotonated Schiff's base is formed nearly simultaneously with metarhodopsin I upon the decay of Lumirhodopsin.


Light-sensitivity modulating protein in frog rods. Cyclic GMP is the second messenger in the phototransduction mechanism in rod photoreceptors. Light-induced activation of cGMP phosphodiesterase (PDE), the hydrolyzing enzyme of cGMP, reduces cytoplasmic cGMP concentration to close the cGMP-activated channel and thereby causes a hyperpolarizing light response. Ca2+ concentration decreases during light-adaptation and this decrease is thought to be at least one of the underlying mechanisms of light-adaptation. Our previous electrophysiological work suggested that PDE in frog rod photoreceptors is regulated by this Ca2+ concentration decrease. In the present work, we isolated a protein that binds to disk membranes at high Ca2+ concentrations. In the presence of this protein (a 26 kDa protein), PDE light sensitivity becomes high at high Ca2+ concentrations. The effect was observed at physiological ranges of Ca2+ concentrations. Thus we could explain high light-sensitivity of photoreceptors under the dark-adapted condition. According to its function, we termed the 26 kDa protein 'sensitivity-modulating protein' or 'S-modulin'. During the purification we noticed that there are additional mechanisms present that may contribute to light-adaptation in frog rod photoreceptors.


Absorption and photochemistry of sensory rhodopsin--I: pH effects. Pyranine (8-hydroxyl-1,3,6-pyrene-trisulfonate) was used as a pH-probe to test whether there is a light-induced proton release to the bulk phase during the photochemical reaction cycle of sensory rhodopsin-I (SR-I). We conclude that the retinylidene Schiff-base proton is retained by SR-I-containing envelope vesicles during the SR-I photocycle under the conditions described here. Bacteriorhodopsin containing vesicles were used as a control to show that light-induced proton release can be observed under identical data acquisition parameters as those used for SR-I-containing vesicles. In addition, the effects of extravesicular pH on the absorption maximum (lambda max) and the SR-I photocycle were studied. SR-I properties are insensitive to pH in the range approximately 3 to approximately 8 with lambda max remaining at 587 nm. The lambda max shifts to 565 nm below pH 3.0 and to 552 nm at pH 10.8 with an apparent pKa of 8.5. Flash-induced absorbance changes of SR-I are described under neutral, alkaline and acidic conditions. The neutral, alkaline and acid SR-I forms each undergo similar photoreactions producing long-lived (> 500 ms decay half-time) blue-shifted intermediates. The UV/near-UV absorption of the photoproducts from neutral and alkaline SR-I indicate a deprotonated Schiff base, whereas acid SR-I produces a species with lambda max > 460 nm indicative of a protonated Schiff base.


The role of standard-dose etoposide in the management of non-small cell lung cancer. Etoposide remains an integral component of therapy for non-small cell lung cancer. Its single-agent activity, and, hence, its activity in combination therapy, need to be reassessed in light of several reports of increased activity at higher doses. Although no effective means of overcoming resistance to etoposide appear to exist, topoisomerase II levels may predict sensitivity to treatment. Biologic response modifiers appear to add little or nothing to standard etoposide chemotherapy for non-small cell lung cancer. Continuous low-dose etoposide infusions do not appear to exhibit the same degree of activity as has been observed with prolonged oral dosing. The availability of effective means of reducing hematologic and emetic side effects of chemotherapy may permit rational trials of more intensive therapy.


Developments in therapy for extensive-disease small cell lung cancer. Developments in the treatment of extensive small cell lung cancer have proven effective in palliative management but have not yielded improvements in the cure rate for this disease. Strategies involving dose escalation of chemotherapy, including de novo and late intensification dose escalation and dose escalation with growth factor support, have had mixed results in this patient population. Alternating chemotherapy, especially with regimens containing etoposide, has achieved better results overall but has not had a significant impact on survival. Current treatment goals should therefore be focused on short-term palliation of symptoms, at least until more effective drugs are developed. Exploration of resistance modulation strategies and biologic approaches, as well as determining the true value of protracted etoposide treatment, may play a role in future treatment endeavors.


Treatment of limited-disease small cell lung cancer. Refinements in the use of chemotherapy, both alone and in combination with chest irradiation, have prolonged the survival of patients with limited-disease small cell lung cancer in the last two decades. Based on reviews of trials of patients with both limited and extensive disease, these improvements appear to transcend adjustments made in the staging of limited disease. Alternation of chemotherapy regimens according to the Goldie-Coldman hypothesis has not proven advantageous in this setting. Etoposide combined with cisplatin has been effective as a salvage regimen for previously treated small cell lung cancer, and is particularly useful in first-line treatment approaches when given concurrently with chest irradiation. Although prophylactic cranial irradiation has reduced central nervous system recurrence in complete responders, its role remains controversial due to an unclear effect on survival and associated long-term toxicities. Our present understanding of the biology of limited-disease small cell lung cancer should lead to further refinements in treatment.


The role of etoposide therapy in germ cell cancer. In the last decade, etoposide has become a standard component of first-line combination chemotherapy in germ cell cancer. The replacement of vinblastine by etoposide in primary treatment came after the therapeutic efficacy of etoposide-containing regimens was determined to be equivalent or superior to vinblastine, with diminished short-term side effects. Subsequent investigations in the salvage setting have demonstrated that high-dose carboplatin and etoposide regimens coupled with autologous bone marrow transplantation can cure some patients with highly resistant disease, and that novel scheduling of etoposide using daily, oral, low-dose therapy can provide effective palliation in some patients. Although these therapeutic successes have been clouded by the demonstration of secondary acute leukemias in rare patients who have received high-dose etoposide, the challenge of the 1990s will be to capitalize on the clinical leads of dose and scheduling as well as to continue to document the long-term safety of these agents.


Catalysis by site-specific recombinases. Site-specific recombination reactions bring about controlled rearrangements of DNA molecules by cutting the DNA at precise points and rejoining the ends to new partners. The recombinases that catalyse these reactions can be grouped into two families by amino acid sequence homology. We describe our current understanding of how these proteins catalyse recombination, and show how the catalytic mechanisms of the two families differ.


The unusual organization and processing of genomic DNA in hypotrichous ciliates. Hypotrichs are a large group of ciliate species that cut, splice, reorder and eliminate DNA sequences to an extraordinary extent during their sexual life cycle. Such DNA processing occurs when a ciliate converts a copy of its germ-line nucleus into a somatic nucleus after cell mating.


DNA inversions in phages and bacteria.

DNA inversions in phages and bacteria. In certain phages and bacteria, there is a recombination system that specifically promotes the inversion of a DNA fragment. These inversion events appear to act as genetic switches allowing the alternate expression of different sets of genes which in general code for surface proteins. The mechanism of inversion in one class of inversion systems (Gin/Hin) has been studied in detail. It involves the formation of a highly specific nucleoprotein complex in which not only the two recombination sites and the DNA invertase participate but also a recombinational enhancer to which the DNA-bending protein Fis is bound.


[The effect of a supplement of microbial phytase on zinc availability]. A study of 35 (5 x 7) male, individually housed, albino rats (initial average weight = 50 g) was undertaken to examine the effect of an addition of microbial phytase to a diet containing phytate on the availability of zinc. The rats were fed a semisynthetic diet based of egg white and cornstarch over a 3-week period. All diets were supplemented with 20 mg Zn/kg. Group I (control) was fed the basal diet free of phytic acid (PA) and phytase. By replacing cornstarch by Na-phytate (0.5% in group II and 1.0% group III), molar phytate: Zn ratios of 25 and 50:1 were obtained, respectively. In groups IV (0.5% PA) and V (1.0% PA) 1000 U of microbial phytase were added. A molar phytate:Zn ratio of 25 (group II) and 50:1 (group III) resulted in a dose-dependent depression of growth and feed efficiency ratio. These negative effects of the addition of PA could be completely counteracted by the supplementation of 1,000 U of phytase in group IV and partially so in group V. Similarly, the apparent absorption and retention of Zn, Zn-concentration in femur and testes and different Zn-status-parameters in plasma (Zn-concentration, percent unsaturated plasma-Zn binding capacity, activity of alkaline phosphatase) were improved by adding 1,000 U microbial phytase/kg diet. The present study shows that an addition of microbial phytase to phytate-rich diets considerably improves the availability of Zn in growing rats.


[Carbohydrate metabolism upon disruption of monoaminergic regulatory mechanisms, developing in the early post-resuscitation period]. Within the early period after clinical death of various duration, hyperglycemia developed in dogs as a result of increased secretion of catecholamines; the rate of hyperglycemia depended on the severity of postresuscitation impairments of the central nervous system. Content of blood glucose was increased simultaneously with elevation of insulin production. Within 1-2 hrs after administration of dihydroxyphenylalanine the excessive secretion of catecholamines altered adrenoreceptor system sensitivity in Langerhans islands with the subsequent decrease in insulin production. Content of cAMP in brain of resuscitated dogs as compared with controls was increased less drastically.


Louping ill virus envelope protein expressed by recombinant baculovirus and vaccinia virus fails to stimulate protective immunity. We have constructed recombinant baculoviruses and vaccinia viruses containing cloned DNA, encoding either the envelope protein alone or all of the structural proteins (core, membrane and envelope) of louping ill virus. Glycosylated viral envelope protein, presented both inside and on the surface of insect and mammalian cells, was expressed by all four recombinant viruses. Differences in antigenic presentation of the envelope protein were observed between the envelope protein and structural protein constructs as well as between the insect and mammalian cell expression systems. Despite the expression of epitopes known to elicit neutralizing and protective antibodies when present in authentic antigen, the recombinant envelope protein expressed by either vector failed to induce, in mice or rabbits, either neutralizing or protective antibodies against louping ill virus.


The genomes of simian varicella virus and varicella zoster virus are colinear. Simian varicella virus (SVV) causes an exanthematous disease in non-human primates which is clinically similar to varicella zoster virus (VZV) infection of humans. In this study, the genetic relatedness of SVV and VZV was confirmed and the location of SVV DNA sequences homologous to VZV restriction endonuclease (RE) fragments and viral genes was determined. VZV DNA RE fragments representing 98.3% of the VZV genome were 32P-labeled and hybridized to RE digested, immobilized SVV DNA. Homologous sequences were located throughout the viral DNAs in similar map positions, indicating a colinear relationship between the VZV and SVV genomes. 32P-labeled VZV glycoprotein (gp I, II, III, and IV) and gene 62 DNA probes also hybridized to SVV DNA in a colinear manner. The results suggest that the location of specific SVV genes may be predicted from the known map positions of homologous VZV genes. This study provides further support for SVV infection of non-human primates as a model for VZV infection of humans.


[Activity of antioxidant enzymes and peripheral blood lymphocyte chemiluminescence in herpes simplex virus infection of the eye]. Activation of lipid peroxidation in ophthalmic herpes may be estimated by an increase in the rate of chemoluminescence in peripheral lymphocytes within 2, 5-7 and 14 days after injection of herpes simplex virus into the rabbit retina. In the peripheral lymphocytes of patients with naturally developed herpetic keratitis the activity of glutathione peroxidase was decreased, while that of superoxide dismutase was increased 2-2.5-fold in the ophthalmic herpes as compared with the normal state.


Reptilian viruses: adenovirus-like agent isolated from royal python (Python regius). An adenovirus-like agent was isolated from a moribund royal python (Python regius). The DNA containing virus replicated in IgH2-cells at 30 degrees C forming eosinophilic intranuclear inclusion bodies. The virus proved to be stabile to treatment with chloroform, pH3 und pH 12 but it was labile to heat (56 degrees C). Infected IgH2 cells revealed symmetric hexagonal virus particles measuring 67-79 nm in the nucleus. The isolate shared characteristics with the viruses of the family Adenoviridae.


Pathogenicity and preliminary antigenic characterization of six infectious bursal disease virus strains isolated in France from acute outbreaks. Six isolates originating from acute outbreaks of infectious bursal disease recently reported in broiler and pullet flocks in France were studied with respect to their pathogenicity and their antigenic relatedness to the Faragher 52/70 reference strain. Although the mortality experimentally induced in susceptible chickens by the field strains was sometimes four times higher than that which followed the inoculation of the reference strain (16 to 48% versus 12%), neither mortality nor morbidity were observed in chickens previously vaccinated with a commercial live vaccine and then challenged under the same conditions. Agar gel precipitation tests demonstrated the existence of common antigens in the different strains, and high cross-neutralization indices measured in embryonated specific pathogen free eggs showed them all to belong to serotype I. These data are discussed with reference to previous European and North-American studies on the antigenic status of infectious bursal disease virus.


Progesterone increases basal 3',5'-cyclic adenosine monophosphate formation and down-regulates the agonist-induced inositol phosphates generation in human term placenta. Whether the placenta is a target tissue for estrogens and progesterone, and their putative mechanism of action, is still a controversial question in the literature. The effect of progesterone and estradiol on 3',5'-cyclic adenosine monophosphate (cAMP) and inositol phosphates generation in human term placenta was investigated. Placental explants were incubated in vitro for up to 48 h in the absence and in the presence of estradiol, progesterone or both steroids (0.1 mumol/l final concentration in all cases), and were stimulated with terbutaline, a beta-adrenergic agonist, (0.1 mmol/l) or angiotensin II (1 mumol/l). The cAMP content was measured by a competitive protein binding assay, and the generation of labelled inositol phosphates formation in explants prelabelled with 3H-myo-inositol was measured by anion exchange chromatography. Progesterone increased significantly basal cAMP concentrations in comparison with control or estradiol-treated tissues (169 +/- 13, 72 +/- 8, and 69 +/- 2 pmol/g wet wt tissue, mean +/- SEM, respectively). However, following terbutaline stimulation cAMP levels (mean +/- SEM) increased to similar values under all conditions (182 +/- 33, 197 +/- 36, and 237 +/- 17 pmol/g wet wt tissue for control, estradiol-, and progesterone-treated tissues, respectively). Angiotensin II stimulated inositol phosphates generation in placental explants by an average of fivefold, but this increase was significantly reduced in the presence of progesterone (5.2 +/- 0.7, 3.7 +/- 0.4, and 2.2 +/- 0.3 fold increase vs non-angiotensin-stimulated tissues, for control, estradiol-, and progesterone-treated placenta, mean- +/- SEM, respectively). These data suggest that progesterone modulates the formation of second messengers in human placenta at term.


Catecholamines stimulate testicular testosterone release of the immature golden hamster via interaction with alpha- and beta-adrenergic receptors. Several lines of evidence suggest that catecholamines are involved in the regulation of the development of the testis. We have therefore investigated the ability of testicular parenchyma (decapsulated pieces of testes) from 18 to 20-day-old golden hamsters to respond to catecholaminergic stimuli in vitro. Norepinephrine and epinephrine, as well as the beta-receptor agonist isoproterenol and the alpha-adrenoreceptor agonist phenylephrine were able to significantly stimulate testicular testosterone production. Dopamine and serotonin were not effective. The stimulatory action of norepinephrine on testosterone production was dependent on the concentration. In incubations of testes with human chorionic gonadotropin (hCG) and norepinephrine, no synergistic effects on testosterone release were observed. The stimulatory effect of norepinephrine could be partially blocked by incubation with beta-receptor antagonist propranolol, or with alpha-receptor antagonist prazosin, while a combination of propranolol and prazosin completely inhibited the norepinephrine-induced testosterone production. Moreover, isoproterenol and phenylephrine in combination stimulated testosterone more than either drug did alone. Measurements of concentrations of norepinephrine and epinephrine in testicular homogenates revealed higher values for these catecholamines than in the plasma, implying that catecholamine levels in the interstitial spaces of the testis might be in the range of concentrations effectively stimulating testosterone production in vitro. This suggests that in the immature testis of the golden hamster, catecholamines acting through both alpha- and beta-adrenergic receptors may be potent physiological stimulators of testosterone production.


Cytological maturity of zona fasciculata cells in the fetal sheep adrenal following ACTH infusion: an electron microscope study. Electron microscopy was used to assess the cytological maturity of the zona fasciculata cells in the adrenal cortex of fetal sheep at 105 days of gestation, following several ACTH infusion regimes. The aim of this study was to correlate the morphological appearance of the fetal adrenal zona fasciculata cells with the expression of the steroid hydroxylase genes and the fetal plasma cortisol concentrations in a parallel study. Immediately following infusion of ACTH for 24 or 72 h, the zona fasciculata cells at the cortico-medullary junction were more mature than those in the saline-infused controls. When ACTH infusions were withdrawn for 24-72 h prior to the termination of the experiment, the deep cortical cells appeared less mature than those in fetuses which had received ACTH right up until the time of tissue collection. Following ACTH administration, mitochondrial changes preceded changes in the smooth endoplasmic reticulum, and when ACTH was withdrawn, the smooth endoplasmic reticulum responded before the mitochondria. The study demonstrated a correlation between the cytological maturity of the deep zona fasciculata cells and the expression of the genes for the steroidogenic enzymes P-450(17)alpha and P-450scc in the 105-day fetal sheep adrenal following ACTH infusion.


Development of membrane currents in mammalian neuroventricular cells from the early neural tube stage in vitro: aspects of the neuronal lineage. Neuroepithelial cells from the murine brain anlage at the early neural tube stage (embryonic day 9 1/2, stage 15) were cultured. Their morphology and the development of membrane currents were studied during 2 weeks in culture. Immediately after dissociation the cells were equal in shape and no morphological or ultrastructural differences were evident. Patch-clamp measurements within the first 2 h, however, showed different membrane properties. Either sodium inward currents or potassium outward currents were observed in these undifferentiated cells, but no combined inward and outward currents were found. This would mean that some of the neuroventricular cells, but obviously not all of them, display neuronal membrane properties (sodium currents) in the immediate post neural plate stage. After 1 day, developing neurons could be identified morphologically by neurotubuli and process formation. The transformation of these cells into neurons was electrophysiologically characterized by an increasing sodium channel density and the expression of various kinds of potassium channels. After 4 days the vast majority of the neurons was electrically excitable, i.e. they could generate action potentials. The standard electrical profile at this time was characterized by a sodium inward current followed by a delayed potassium outward current. In the following days the complexity of membrane currents increased in some neurons by the emergence of transient potassium currents. After 2 weeks in culture different neuronal phenotypes could be identified.


The developing chick brain shows a dramatic increase in the omega-conotoxin binding sites around the hatching period. omega-Conotoxin CVIA, a 27 amino acid neuropeptide toxin believed to target voltage sensitive calcium channels (Cruz et al., 1987, Biochemistry 26 (3), 820-824) was bound to developing chick brain at embryonic day 9-post hatch day 10. A two-fold increase was observed in omega-Conotoxin binding sites around chick hatching, embryonic days 18-20. Depolarization induced 45Ca fluxes also increased around hatching. omega-Conotoxins block > or = 80% of these 45Ca fluxes throughout development. Competition binding did not detect large differences in the binding affinity of sites during development. We conclude from these data that the increases in omega-Conotoxin binding sites and 45Ca fluxes around chick hatching are related to one of the presynaptic mechanisms of neuronal maturation necessary for normal neuronal function and chick behavior after hatching.


Quantitative determination of sulfonated aliphatic and aromatic surfactants in sewage sludge by ion-pair/supercritical fluid extraction and derivatization gas chromatography/mass spectrometry. Secondary alkanesulfonate (SAS) and linear alkylbenzene-sulfonate (LAS) surfactants were quantitatively (> 90%) extracted from sewage sludges as their tetrabutylammonium ion pairs using 400 atm of supercritical CO2 for 5 min of static extraction followed by 10 min of dynamic extraction at 80 degrees C. Ion pairs of SAS and LAS quantitatively formed butyl esters in the injection port of the gas chromatograph and were determined by gas chromatography/mass spectrometry without class fractionation of the sewage sludge extracts. Concentrations of SAS and LAS in sludges from five different sewage treatment plants ranged from 0.27 to 0.80 g/kg of dry sewage sluge and from 3.83 to 7.51 g/kg, respectively. Good reproducibility was achieved with RSDs of typically 5% for replicate extractions and analyses. Homologue and isomer distributions of SAS in sewage sludge indicated an enrichment of the more hydrophobic components in sewage sludge during sewage treatment.


[Physiopathology and treatment of metabolic changes in transintestinal urinary diversions]. The use of intestinal segments in the urinary tract can cause metabolic changes that depend on the intestinal segment utilized. The severity of these changes basically depends on the area of the intestinal mucosa in contact with urine, the duration of exposure to urine and renal function. The length of time the intestinal mucosa is in contact with urine largely depends on the surgical technique employed. It is longer for the reservoirs, intestinal neobladders and ureterosigmoidostomies than for the intestinal conduits with cutaneous urinary diversion and therefore carry a higher incidence of metabolic changes. Jejunal urinary diversion causes metabolic acidosis with hypochloremia, hyponatremia, hyperpotassemia, azotemia and dehydration in at least 50% of the cases. Ileal and colonic urinary diversion can cause metabolic acidosis, although the incidence is significantly less. Acidosis presents with hyperchloremia, hyperammonemia, hypersulfatemia, increased osmolality and uremia with normal creatininemia and a tendency to develop hypocalcemia, hypophosphoremia and hypomagnesemia. Recent studies performed in our service show that acidosis is basically due to the secretion of sodium bicarbonate by the intestinal segment used in the urinary tract, which causes water-salt depletion that is compensated by secondary hyperaldosteronism. Mild chronic acidosis is neutralized via the respiratory system and by the bone buffers, which leads to bone remodelling manifested by the significant increase of serum alkaline phosphatase levels and increased calciuria. These calcium phosphate changes, although statistically significant, do not appear to be important since they were not accompanied by changes of serum PTH levels, 25 and 1-25-cholecalciferol. Nicotinic acid as inhibitor of cyclic AMP synthesis failed to correct metabolic acidosis in the patients with transileal diversion.


Bone metastasis in ovarian cancer.

Bone metastasis in ovarian cancer. Bone involvement in carcinoma of ovary occurs rarely. In a review of 103 patients over last 3 years we have seen 4 such patients (serous-2, mucinous-1, mixed germ cell tumour-1). Patients presented with severe localized bone pain, bony swelling and difficulty in walking. The common sites of involvement were vertebrae, pelvic bones and skull. Radiologically the osteolytic lesions were commonest. Bone lesions were associated with abdomino-pelvic disease in 3 patients. Cisplatinum based chemotherapy in addition to local radiation resulted in significant response in 2 patients; one complete and one partial response. The median survival was 7.5 months (range 6-39 months) after bone metastasis. We conclude bone involvement in cancer ovary is associated with poor prognosis.


Effects of glycerol acetonide on adrenergic neurotransmission in isolated rabbit heart. In the present investigation, the solvent glycerol acetonide (GA, CAS 100-79-8) was added (110 mg/min) to the Tyrode buffer perfusing an isolated rabbit heart preloaded with 14C-noradrenaline (NA). GA inhibits the neuronal uptake of NA but stimulates its spontaneous release. The latter effect was not ascribed to the stimulation of NA biosynthesis or to an inhibition of its catabolism. Moreover GA inhibits the evoked-release of the transmitter by tyramine and dimethyl phenyl piperazinium (DMPP). By stimulating the spontaneous release of NA, GA may induce both a depletion of myocardial stores and an important increase of the transmitter, inhibiting thereby the evoked-release by a negative feed-back mechanism on presynaptic alpha 2 adrenoceptors. Hence, the impact of GA on some steps of the cardiac adrenergic transmission may provide an adequate explanation for its observed hypotensive effect.


[Characteristics of the dosage of sodium hydrocarbonate for correction of metabolic acidosis in surgical patients]. It has been established that in conditions of intraoperative blood and plasma loss base deficiency is determined not only by hypocarbonatemia, but also by hypoproteinemia, hypophosphatemia and HCO3 metabolism disturbances caused by anemia. Correction of metabolic acidosis in such patients should include infusions of NaHCO3, protein preparations, blood, phosphates. Mellemgaard and Astrup's technique presupposes correction of the deficiency of all buffer bases only with NaHCO3, which dramatically increases its dosage. Thus, it is evident that the technique should be revised. The comparison of the results of metabolic acidosis correction using a conventional and adapted techniques (hydrocarbonate dose in mmol or ml of a 8.4% solution is 24-SB.body weight.0.2%) in statistically homogeneous groups has shown that differentiated "polybuffer" correction of metabolic acidosis with adapted NaHCO3 dose 1.7 times more frequently normalized acid-base balance parameters, reducing the risk of the onset of post-correction metabolic alkalosis to minimum.


Efficacy and safety of the novel Na+,K(+)-ATPase inhibitor 20R 14 beta-amino 3 beta-rhamnosyl 5 beta-pregnan 20 beta-ol in a dog model of heart failure. 20R 14 beta-amino 3 beta-rhamnosyl 5 beta-pregnan 20 beta-ol (LND 623, CAS 90520-42-6) was investigated and compared to digoxin in anesthetized dogs. The hemodynamic profiles showed: a) a pure positive inotropic action of LND-623; b) its potency was four-fold higher than that of digoxin and more marked in heart failure; c) its duration of action was maintained for at least 6 h. The onset and reversal of the inotropic effects of a single dose (3.3 nmol.kg-1.min-1) were faster with LND-623 than those of digoxin. This reversal is consistent with the faster dissociation profile observed at the level of the high affinity cardiac Na+,K(+)-ATPase receptor form. The advantage of LND-623 over digoxin resides in its larger therapeutic index (ratio of arrhythmogenic to inotropic responses) in anesthetized dogs with propranolol-induced heart failure. This index was 6 for LND-623 and 2 for digoxin.


Different pharmacokinetics, antithrombotic activity and bleeding effects of heparin and two new fragments administered in rat by subcutaneous route. Native heparin (CAS 9005-49-6) and its two new fragments, low molecular weight heparin (LMW-H, 5 kDa) and oligo-heparin (oligo-H, 2 kDa) obtained by radical degradation were characterized as to their physicochemical properties. Heparin fragments differ from unfractionated heparin only in molecular weight. The pharmacokinetics and some pharmacological effects, bleeding and antithrombotic activity, of the three different molecular weight heparins were investigated. The plasma concentrations were determined by an amidolytic method which measures inhibiting effect on factor Xa. The blood levels of each substance were derived from their in vitro calibration curves. The examination of the pharmacokinetics parameters allowed to evaluate the differences in the bioavailability, absorption rate and elimination mechanisms between the three different heparins. The bioavailability, the absorption rate and the distribution of the molecules of heparins in biological compartments depend on the molecular weight. LMW-H and oligo-H exhibit greater antithrombotic activity than unfractionated heparin when administered subcutaneously. The pharmacokinetic behaviour of oligo-H considerably differs from that of unfractionated heparin and LMW-H. This new drug is able to bind cells and plasma proteins differently from heparin and LMW-H. The capacity of oligo-H to bind smooth muscle cells and to interact with myosin is discussed in relation to the bleeding effect.


Relaxant effect of C-type natriuretic peptide on guinea-pig tracheal smooth muscle. The relaxant effects of the C-type natriuretic peptide (CNP) on guinea-pig tracheal smooth muscle were investigated. CNP (10(-8)-10(-5) mol/l) induced dose-dependent relaxant effects on the resting tone of tracheal smooth muscle with EC50 of 3.6 +/- 0.8 x 10(-6) mol/l (mean +/- SE). CNP caused a dose dependent increase in the tissue cyclic GMP level in tracheal smooth muscle (191 +/- 10 fmol/mg protein in control, 583 +/- 33 fmol/mg protein at 10(-7) mol/l, 688 +/- 56 fmol/mg protein at 10(-6) mol/l, 1091 +/- 167 fmol/mg protein at 10(-5) mol/l). Tissue cyclic GMP levels showed a peak at 1 min. Relaxation of the tracheal smooth muscle began at 1 min, reaching peak value at 5 min after the perfusion of CNP (10(-5) mol/l). Elevations in cyclic GMP preceded the relaxation of tracheal smooth muscle. These results suggest that CNP may be a novel potent relaxant in tracheal smooth muscle and that its relaxant effect may be mediated by cyclic GMP levels.


Survey on spontaneous peripheral neuropathy in aging mice. The incidence of peripheral neuropathy in CD-1 mice (caesarean-derived) obtained from long term studies over a 10 year period is reported. The survey included peripheral nerve samples of 15,223 males and 15,075 females. The incidence of peripheral neuropathy is 8% in untreated males and 12% in untreated females aged 80 to 104 weeks. The total incidence in all mice in the survey was higher in females than males and was age-related. The present survey also showed interstrain differences. The peripheral neuropathy in CD-1 strain was low in incidence compared to B6C3F1 (cross between C57 BL/C6NCLB and C3H/HENCRLB). The peripheral neuropathy was spontaneous in origin and was age-related.


Decreased polyphosphoinositide metabolism accompanies myelinated fiber loss in human peripheral neuropathies. The distribution of incorporated 32P in phospholipids of sural nerve biopsy samples from patients with several peripheral neuropathies was measured. Both the absolute amount and the proportion of isotope in polyphosphoinositides was decreased in nerves that displayed substantial (> 50%) depletion of myelinated fibers as compared to nerves that exhibited minimal depletion. The results suggest that diminished metabolism of these substances is an indicator of myelin loss, and are consistent with the conclusion that polyphosphoinositide turnover in human nerve is nearly entirely localized to the myelin sheath.


The presence of neuron-associated microtubule proteins in the human U-251 MG cell line. A comparative immunoblot and immunohistochemical study. U-251 MG, a permanent cell line derived from human glioblastoma multiforme with the capacity to maintain glial fibrillary acidic protein (GFAP) production over repeated in vitro passages, was evaluated for the expression of three neuron-associated proteins (Class III beta-tubulin, MAP2, and tau) in three different in vitro systems: as free-floating suspensions, on coverslips, and on a gelatin foam (Gelfoam) matrix. Cells grown under the three in vitro conditions were analyzed by immunoblotting techniques, whereas immunohistochemical analyses were performed on cells grown on Gelfoam. By immunohistochemistry, cells were positive for Class III beta-tubulin isotype, a neuron-associated beta-tubulin, for microtubule-associated protein 2 (MAP2), but not for tau. Immunoblotting studies confirmed the presence of Class III beta-tubulin in extracts of cells grown under the three in vitro conditions. MAP2 and tau were clearly evident only in cell extracts grown in Gelfoam cultures. GFAP expression was observed in all three in vitro conditions by immunoblotting and also in foam matrix cultures by immunohistochemistry. In matrix cultures, Class III beta-tubulin- and GFAP-positive cells were found immediately adjacent to each other, but coexpression of these proteins was not observed, and the cells were morphologically indistinguishable. Our findings confirm the heterogeneity of malignant gliomas in vitro, and the implications of these observations require further study.


Synthesis and binding assays of H3-receptor ligands. The preparation of a representative group of derivatives of the known H3-antagonist thioperamide is described. Binding affinity for histamine H3-receptors of thioperamide and its derivatives, which were obtained by substitution on the imidazole ring, was measured on rat brain cortex synaptosomes. Competitive binding assays were performed with two different labelled ligands, the physiological agonist [3H]histamine ([3H]HA) and the potent H3-agonist N alpha-[3H]methyl-histamine ([3]NAMHA). We observed a remarkable difference in Ki values obtained versus the two labelled ligands, both for thioperamide and its derivatives. In particular, 5-methylthioperamide showed a considerable selectivity for the system recognized by [3H]NAMHA, being about 100 times more potent versus this system than versus the system recognized by [3H]HA. On the basis of these observations, we suggest that it is necessary to consider this difference in evaluating the affinity of new compounds for the H3-receptors.


Identification of yeast cloned genes by genetic analysis. Gene cloning in yeast is usually carried out by complementation of recessive mutations. However, the fact that a DNA fragment is able to complement a mutation in a certain gene does not necessarily mean that it contains that gene. The identification of a cloned gene can involve the use of Molecular and/or Classical Genetics techniques. In this paper we describe the strategy to be followed in order to establish the identity of a cloned gene, by using genetic crosses and tetrad analysis. As a practical example of the use of this strategy, we describe the cloning of the THR1 gene which codes for the homoserine kinase in S. cerevisiae. This gene has been isolated from a yeast genomic library by complementation of a thr1 mutation. The complementing DNA fragment has been subcloned and integrated into the yeast genome. By genetic crosses and tetrad analysis it has been demonstrated that integration has occurred at the THR1 locus. Since in this organism integration takes place mainly by homologous recombination, it can be inferred that we have, in fact, cloned the THR1 gene. Biochemical analysis of the transformant that carries multiple copies of the cloned gene confirms this result. It shows that this strain presents a homoserine kinase activity about 60 times higher than that of the wild type.


Identification and evaluation of new primer sets for the detection of lentivirus proviral DNA. We have developed sets of degenerate oligonucleotides designed to detect pol gene sequences from any member of the lentivirus subfamily when used as primers in amplification techniques such as the polymerase chain reaction (PCR). This pan-lentivirus-specific primer set (PLSPS) consists of primers, LV1, LV2, and LV3, based on conserved regions common to lentiviruses only. Our protocol is based on primary amplification with LV1 and LV2 followed by secondary amplification with a nested primer set based on the YM/VDD motif found in all reverse transcriptases (or "DDMY," in the opposite direction), and LV3, a block of lentivirus homology nested just downstream of LV1. PLSPS-PCR analysis of DNA from cells infected with HIV-1, HIV-2, SIVmac239, BIV, visna, EIAV, CAEV, OPPV, or FIV resulted in the amplification of appropriately sized products. Sequence analysis of the LV1/2 products, cloned into pBluescript (pBS), indicated that at least 20% (most often, > 80%) contained the predicted lentivirus pol sequence. Greater than 95% of the LV3/DDMY products contained the expected lentiviral sequences. Using the PLSPS, lentivirus pol sequences could typically be detected at levels of one copy in 2 x 10(6) cells after secondary amplification. No specific lentiviral PCR products were detected in DNA from uninfected human or mouse monocytes, feline or bovine leukocytes, mouse, rat or human fibroblast cell lines, chicken embryo fibroblasts, Tahr lung cells, or cell lines infected with the following retroviruses which are not lentiviruses: Rous sarcoma virus, Moloney leukemia virus or Kirsten sarcoma virus, mouse mammary tumor virus, human T-cell lymphotropic virus I, and feline leukemia virus.(ABSTRACT TRUNCATED AT 250 WORDS)


Infection by human herpesvirus 6 (HHV-6) of human lymphoid T cells occurs through an endocytic pathway. We have analyzed by immunoelectron microscopy the early events of binding and internalization of human herpesvirus 6 (HHV-6, strain GS) on a susceptible T-lymphoblastoid cell line, HSB-2. The virions bound to the cell surface at 4 degrees C were tightly associated with the plasma membrane. Gold immunolabeling of the viral envelope proteins was strong and specific. Warming at 37 degrees C for different times showed viral internalization through smooth surfaced pits and vesicles. Fusion events of the virions with the cell plasma membrane were never observed. Gold immunolabeling performed in parallel experiments before or after viral internalization showed: (1) absence of viral envelope proteins on the cell plasma membranes at all times of internalization, again excluding fusion events; (2) entry of the virions with their envelopes. Treatment of the cells with chloroquine, a drug known to affect the endocytic pathway, led to an almost complete inhibition of viral infectivity, suggesting that the endocytosed virions are responsible for a successful infection. Comparable results were obtained using a second strain of HHV-6 (BA92), with biologic and molecular characteristics similar to the prototype strain Z29. The chloroquine inhibition was effective on two different T cell lines (HSB-2 and J-Jhan), as well as on phytohemagglutinin-stimulated peripheral blood mononuclear cells.


Increase in Na+/K+ pump numbers in vivo in healthy volunteers taking oral lithium carbonate and further upregulation in response to lithium in vitro. 1. We have measured [3H]-ouabain binding to lymphocyte membranes in eight healthy volunteers before and after they had taken lithium carbonate for 14 days in doses which maintained the serum lithium concentration in the range 0.5-1.0 mmol 1-1. 2. There was a statistically significant increase in the [3H]-ouabain binding capacity of the lymphocyte membranes (reflecting the number of Na+, K+-ATPase molecules) after 14 days of lithium administration in vivo. This suggests that a failure to increase pump numbers after similar exposure to lithium in vivo in patients with manic-depressive psychosis is a primary abnormality associated with the disease. 3. In vivo lithium administration did not alter the normal adaptive (upregulatory) response of lymphocyte Na+, K+-ATPase to standard pharmacological challenges, involving in vitro incubation for 3 days with lithium chloride (8 mmol 1-1) or sodium ethacrynate (1 mumol 1-1). 4. We have previously found that there is an impaired response of the Na+, K+-ATPase to these in vitro stimuli in patients with manic-depressive psychosis, and our present data suggest that this abnormality is attributable to the disease itself and not to in vivo lithium therapy. 5. The data also suggest that the increase in vivo Na+/K+ pump activity which we have previously described in healthy volunteers after 21 days of lithium administration is at least partly due to an increase in Na+/K+ pump numbers.


The effect of i.v. sumatriptan, a selective 5-HT1-receptor agonist on central haemodynamics and the coronary circulation. 1. Sumatriptan (GR43175) is a selective 5-HT1-receptor agonist effective in the acute treatment of migraine. Vasoactive properties in other vascular beds have been suggested by recent in vitro studies. 2. Its effects on coronary artery dimensions and central haemodynamics were assessed in 10 patients undergoing diagnostic coronary arteriography using digital subtraction angiography and invasive haemodynamic monitoring. 3. Following a 10 min i.v. infusion of sumatriptan to a total dose of 48 micrograms kg-1 there was a significant increase (P < 0.05) in systemic and pulmonary arterial pressures. There was a significant reduction in coronary artery diameter from 4.3 +/- 1.6 mm to 3.6 +/- 1.6 mm 12.9 +/- 6.9% (P < 0.001). There was no significant change in heart rate or ECG morphology. 4. Sumatriptan, a 5-HT1-receptor agonist, causes a vasopressor response in the systemic and pulmonary arterial circulations and coronary artery vasoconstriction; in this study there were no clinical sequelae.


Psoriasis treatment with vitamin D derivatives. The naturally occurring 1,25(OH)2 vitamin D3 and the two synthetic derivatives 1,24(OH)2 vitamin D3 and calcipotriol (Calcipotriol, INN, Calcipotriene, USAN) have a profound effect on keratinocyte proliferation and differentiation. Clinical trials have shown an effect of all three derivatives in psoriasis. The limiting factor for the use of 1,25(OH)2 vitamin D3 is the risk of hypercalcemia, which is 100 to 200 times less for calcipotriol and that may be less also for 1,24(OH)2 vitamin D3. Presently, calcipotriol in an ointment base has been marketed in a number of countries and represents an alternative to treatment with topical glucocorticoids and dithranol.


Regression mechanism of cyanamide-induced inclusion bodies in the rat: a useful experimental pattern to study the beta-glycogen metabolization of hepatocytes. Cyanamide, a drug used in alcohol aversion therapy, induces a distinctive liver cell lesion, both in human beings and rats. The lesion consists of cytoplasmic inclusion bodies which give a ground-glass appearance to the hepatocytes. In human beings the inclusion bodies do not persist but disappear some time after withdrawal of the drug. In order to confirm their disappearance and determine how they regress rats were treated with cyanamide (32 mg/kg) for 6 months before partial lobectomy. At this time, inclusion bodies were observed. After a period without further treatment (5-19 weeks) the animals were killed and a marked decrease in the number of inclusion bodies was observed, paralleling the period of time without treatment. Inclusion bodies regress as a result of glycogen removal by enzymatic activity of the smooth endoplasmic reticulum which then undergoes hyperplasia, plus a process of autophagocytosis and necrosis of inclusion-body-bearing hepatocytes which are then phagocytosed by macrophages.


Detection of viral RNA in experimental coxsackievirus B3 myocarditis of mice using the polymerase chain reaction. The presence of the viral RNA in the myocardium in experimental coxsackievirus B3 myocarditis of mice was investigated using the polymerase chain reaction (PCR). Four-week-old C3H/He mice (n = 35) were inoculated with coxsackievirus B3 (Nancy strain, 10(5) plaque-forming units/mouse). We used a pair of primers, which encompass a part of the 5' end sequence of the coxsackievirus B3 genome and can also detect many enteroviral RNAs. We found that hearts were positive for the viral RNA from 2 to 21 days after virus inoculation by PCR, but negative after day 28 and in non-infected control mice (n = 5). The viral RNA were detected by PCR later than by culture. Thus, the detection of the viral RNA using enzymatic amplification is more rapid and easier and may be more useful for clinical diagnosis of viral myocarditis than conventional culture methods. However, virus persistence in the myocardium long after virus inoculation is unusual in this model.


Role of lymphocytes in silicosis: regulation of secretion of macrophage-derived mitogenic activity for fibroblasts. We investigated the role of pulmonary lymphocytes in regulating the secretion by alveolar macrophages (AM) of mitogenic activity for lung fibroblasts, in an experimental model of the initial stages of silicotic inflammation and fibrosis. Following intratracheal instillation of silica, pulmonary parenchymal lymphocytes produced a lymphokine(s) that caused modest stimulation of the secretion of mitogenic activity by normal AM. Co-culture of small numbers of lymphocytes from silica-injected animals with AM induced enhanced secretion of fibroblast growth factor activity which was comparable to the maximal response elicited by recombinant interferon-gamma. Lymphocytes from animals given non-fibrogenic titanium dioxide exhibited no such effects. The stimulatory effect of lymphocytes from silica-treated animals in co-culture with macrophages was abrogated when the cells were separated by a microporous membrane. Our findings demonstrate that lymphocytes participating in the response to pulmonary deposition of silica are able to induce the secretion of a growth factor(s) for fibroblasts by pulmonary macrophages, possibly via lymphokines expressed on the cell surface or secreted at sites of cell-to-cell contact.


Movement disorders: why don't those synapses fire? From mobility to disability; this is the plight of the patient with a movement disorder. Alterations in motor functioning may include gait, balance, coordination and tone as well as voluntary and involuntary movements. These changes need to be observed, assessed and monitored, then correlated with the neuropathology and neurochemistry. The nurse, by doing this, provides the physician with the crucial information for successful medical management. This presentation will identify the pathology and required nursing assessment for the more prevalent movement disorders and provide an overview of pharmaceutical management.


In vitro lipid metabolism, growth and metabolic hormone concentrations in hyperthyroid chickens. Indian River male broiler chickens growing from 7 to 28 d of age were fed on diets containing energy:protein values varying from 43 to 106 MJ/kg protein and containing 0 or 1 mg triiodothyronine (T3)/kg diet to study effects on growth, metabolic hormone concentrations and in vitro lipogenesis. In vitro lipid synthesis was determined in liver explants in the presence and absence of ouabain (Na+, K(+)-transporting ATPase (EC 3.6.1.37) inhibitor) to estimate the role of enzyme activity in explants synthesizing lipid. Growth and feed consumption increased (P < 0.01) when the energy:protein value decreased from 106 to 71 MJ/kg protein; however, both variables decreased as the value was further decreased from 53 to 43 MJ/kg protein. Triiodothyronine depressed (P < 0.01) growth, but not food intake. Large energy:protein diets (> 53 MJ/kg protein) and dietary T3 lowered (P < 0.01) plasma growth hormone. Large energy:protein diets (> 53 MJ/kg protein) increased (P < 0.01) lipogenesis, plasma growth hormone (GH) and decreased plasma insulin-like growth factor 1 (IGF-1). Also, T3 decreased plasma GH, IGF-1 in vitro lipogenesis. Ouabain inhibited a greater proportion of in vitro lipogenesis in those explants synthesizing fat at a high rate. Both dietary T3 and in vitro ouabain decrease lipogenesis, but, when combined, the effects are not cumulative.


Effects of guar gum and cellulose on glucose absorption, hormonal release and hepatic metabolism in the pig. Six Large White pigs (mean body-weight 59 (SE 1.7) kg) were surgically fitted with permanent catheters in the portal vein, the brachiocephalic artery and the right hepatic vein, as well as with electromagnetic flow probes around the portal vein and the hepatic artery, and allowed to recover. The non-anaesthetized animals were given a basal non-fibre diet (diet A) alone or together with 60 g guar gum/kg (diet B) or 150 g purified cellulose/kg (diet C) by substitution for mica. The diets were given for weekly periods and according to a replicated 3 x 3 Latin square design. On the last day of each such adaptation period, test meals of 800 g were given before blood sampling. Sampling was continued for 8 h. Guar gum strongly reduced glucose apparent absorption without changing the absorption and the hepatic uptake profiles. Production rates of insulin, gastric inhibitory polypeptide and insulin-like growth factor-1 (IGF-1) were lowest after guar gum ingestion. However, the reductions in peripheral blood insulin levels caused by guar gum were not associated with a change in hepatic insulin extraction. IGF-1 appeared to be strongly secreted by the gut, whereas the liver had a net uptake of the peptide. Ingestion of guar gum increased the hepatic extraction coefficient of gut-produced IGF-1. Guar gum ingestion appeared also to decrease glucagon secretion. Cellulose at the level consumed had very few effects on the variables considered. It is suggested that the modulation of intestinal mechanisms by guar gum was sufficient to mediate the metabolic effects described.


The effect of weaning diet on the subsequent colonic metabolism of dietary fibre in the adult rat. The effect of the weaning diet on the subsequent colonic metabolism of bran and pectin in the adult rat has been investigated. Feeding a fibre-reduced diet on its own or supplemented with bran (WB) and pectin (P) from weaning (fibre-reduced (weaning)) was compared with introducing the same diet to age-matched rats reared on a standard laboratory diet from weaning (fibre-reduced (6 weeks)). The effects of the diets on colonic metabolism were measured by wet and dry caecal contents and stool weights, caecal sac weight, and caecal and faecal short-chain fatty acids (SCFA). Final body-weights were greater for fibre-reduced (6 weeks) and fibre-reduced (6 weeks) + P groups, but not fibre-reduced (6 weeks) + WB, than those of the fibre-reduced (weaning) rats. Rats fed on fibre-reduced (6 weeks) diet had a higher total caecal SCFA content than fibre-reduced (weaning) control rats. Fibre-reduced (weaning) + P-fed rats had a threefold higher caecal concentration of both propionate and butyrate than the matched fibre-reduced (6 weeks) + P group. Fibre-reduced (weaning) + WB animals had a significantly higher butyrate caecal concentration compared with their matched fibre-reduced (6 weeks) + WB group. Fibre-reduced (weaning) + P-fed rats had a lower faecal output than the fibre-reduced (6 weeks) + P rats. There was no difference in faecal output in rats fed on either fibre-reduced (6 weeks) + WB or fibre-reduced (weaning) + WB. The faecal concentration of SCFA was in general higher in the rats fed on fibre-reduced (weaning) alone, + P, or + WB than in those fed on fibre-reduced (6 weeks) alone, + P or + WB. Faecal output of total and individual SCFA was increased on the fibre-reduced (weaned) + WB diet compared with fibre-reduced (6 weeks) + WB-fed animals. The diet at weaning may be important in determining the pathways of caecal bacterial metabolism in the adult rat. In studying the effect of a dietary fibre on caecal metabolism and faecal output, when the diet is changed appears to be important.


Preparation of a stable liquid material for calibration and quality control for lysosomal enzymes in plasma. Assay of enzymes of lysosomal origin in plasma, I. Several lysosomal enzymes present in human plasma (N-acetyl-beta-glucosaminidase, beta-glucuronidase, beta-galactosidase, alpha-galactosidase, alpha-L-fucosidase, alpha-mannosidase, beta-glucosidase) were maintained in a fully active state for at least 8 months by the addition of ethylene glycol (300 milligrams final concentration) to freshly prepared plasma and storage at -20 degrees C. Pools of human plasma from healthy humans, stabilized and stored as above, and containing a low, medium or high content of the above enzymes, were used to establish the analytical imprecision (within-run, day-to-day and total imprecision) of the fluorimetric assay. Ten replicates in ten different analytical series, covering a period of two months, were performed. The total imprecision (expressed as coefficient of variation) was in general lower than 10%; in a few cases, particularly plasma samples with a low enzyme content, the total imprecision was 18%. The isozymes A, B, I1, and I2 of N-acetyl-beta-glucosaminidase displayed the same stability upon storage as the unfractionated enzyme. It is concluded that pools of human plasma containing known amounts of lysosomal enzymes, stabilized by the addition of 300 micrograms ethylene glycol and stored at -20 degrees C, are suitable liquid materials for calibration and quality control for the assay of the same enzymes.


Characterization of gonadotropin-releasing hormone binding to pituitary receptors in the gilthead seabream (Sparus aurata). Receptors for GnRH in the pituitary of Sparus aurata were characterized using iodinated [D-Ala6-Pro9-NEt]-LHRH (GnRHa). Equilibrium binding of the ligand to the receptor was achieved after 1 h at 4 degrees C. Binding of the radioligand was a function of tissue concentration, with a linear correlation over the range of one-sixteenth to three-fourths pituitary per tube. Displacement experiments with salmon GnRH (sGnRH), GnRHa, as well as unrelated peptides demonstrated the specificity of the receptors. Binding was found to be saturable at ligand concentrations of 4 x 10(-9) M. Scatchard analysis of the saturation data suggested the presence of a single class of high-affinity sites (Ka = 0.567 +/- 0.136 x 10(9) M-1, Bmax = 1091 +/- 207 fmol/mg protein).


Stimulation of phospholipase A2 by xanthine oxidase in the rat corpus luteum. The ability of the superoxide radical (SOR) generated by xanthine oxidase to activate phospholipase A2 (PLA2) was examined in microsomes prepared from luteinized rat ovaries. Treatment of microsomes with xanthine oxidase resulted in a rapid burst in SOR formation followed by an increase in PLA2 activity. Stimulation of PLA2 activity was dose related and similar in microsomes prepared from control or prostaglandin F2 alpha (PGF2 alpha)-treated rats. Activation was inhibited by the antioxidants, vitamin E and nordihydroguaiaretic acid, and by superoxide dismutase and catalase, which metabolize SOR and H2O2 to remove reactive oxygen species from the cell. The stimulation of PLA2 activity by xanthine oxidase was dependent upon the addition of calcium ions, and it was highest in samples in which cytosol was added to membranes. These results indicate that the SOR and/or H2O2 may mediate PLA2 activation, which may be involved in the luteolytic process.


Rat ovarian angiotensin II receptors, renin, and angiotensin I-converting enzyme during pregnancy and the postpartum period. The ovarian renin-angiotensin system (RAS) has been studied extensively in the virgin cycling rat, but little information is available about this system in pregnant and postpartum rats. We show that renin and angiotensin I-converting enzyme (ACE)--the key enzymes involved in angiotensin II (Ang II) formation--and Ang II receptors, are present in pregnant and postpartum rat ovaries. From gestation Days 2-4 to 10-12, active ovarian renin ranged from 1.12 +/- 0.13 to 1.27 +/- 0.19 ng Ang I/h/mg and comprised between 68 and 86% of total (active+inactive) ovarian renin activity. Between Days 10-12 and Days 14-16 of pregnancy, ovarian active renin activity increased slightly, but inactive renin disappeared, suggesting its activation; the remaining active renin then decreased 62% by Days 18-20 (p < 0.05). On postpartum Day 2, both active and total ovarian renin activity exceeded that of Days 2-20 of pregnancy (p < 0.05); levels of both then declined sharply by postpartum Day 3 (p < 0.05). In pregnant rats, levels of ovarian Ang II receptors, identified by the specific binding of [125I]-[Sar1,Ile8]Ang II to ovarian membranes, were high between Days 2-4 and 10-12 of pregnancy, ranging from 12.8 +/- 1.7 to 15.7 +/- 3.4 fmol/mg, but steadily declined by 82% between gestation Days 10-12 and 18-20 (p < 0.05). Postpartum Ang II receptor levels on Days 2, 3, and 4 showed a gradual increase from low levels comparable to Days 18-20 of pregnancy. Ovarian ACE activity did not change throughout pregnancy or during the postpartum period.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of interferon on the steroidogenic functions and proliferation of immature porcine granulosa cells in culture. Recent studies suggest the relevance of several cytokines to the growth and differentiation of granulosa cells. In the present study, we investigated the effects of interferon (IFN) on the steroidogenic functions and proliferation of immature porcine granulosa cells. Human IFN-alpha inhibited FSH-induced progesterone secretion in a concentration-dependent manner. The effect of IFN-alpha was significant at a concentration as low as 10 pg/ml. Maximal inhibitory concentrations (10-50 ng/ml) of IFN-alpha reduced FSH-induced progesterone secretion by 70%. In contrast, estradiol secretion induced by FSH was significantly enhanced by relatively high concentrations (1-50 ng/ml) of IFN-alpha. IFN-alpha (0.1-10 ng/ml) reduced cAMP generation in response to FSH by as much as 80%, although its effect was not concentration-dependent. The proliferation of cultured granulosa cells was inhibited by IFN-alpha in a concentration-dependent manner. Human IFN-gamma did not affect granulosa cell functions. The stimulation of estradiol secretion and the inhibition of cell proliferation induced by IFN-alpha in cultured porcine granulosa cells in this study are in contrast with the effects of IL-1, which, as we reported previously, inhibited both progesterone and estradiol secretion and stimulated cell growth in these cell cultures. Such differences in the mode of action of cytokines may contribute to the regulation of granulosa cell functions under physiological or pathological conditions.


Oxytocin and bovine parturition: a steep rise in endometrial oxytocin receptors precedes onset of labor. Oxytocin receptors were measured in myometrium and intercaruncular endometrium of cows during pregnancy and parturition. Concentrations of estradiol-17 beta, estrone, and progesterone in peripheral blood were also measured. Receptor concentrations in the endometrium rose almost 200-fold from Day 20 to term (p < 0.0001, ANOVA), from 40 +/- 11 to 7300 +/- 1430 fmol/mg protein. Myometrial receptor concentrations increased 10-fold from 180 +/- 36 fmol/mg on Day 20 to 1850 +/- 360 fmol/mg protein at term (p < 0.0001, ANOVA). During labor, endometrial receptors (6600 +/- 1300 fmol/mg) remained at prelabor values, whereas myometrial receptor concentrations had decreased to 1190 +/- 316 fmol/mg (not significant) and declined further postpartum. Plasma concentrations of progesterone declined from 4-5 ng/ml to about 2 ng/ml between Days 250 and 282 and dropped to < 0.2 ng/ml shortly before delivery. Plasma concentrations of estrone and estradiol-17 beta were below 10-20 pg/ml until Day 230. Estrone concentrations were significantly (p < 0.05) increased by Day 250 and estradiol-17 beta by Day 270, and then both rose rapidly. During labor, plasma estrone was 1135 +/- 245 pg/ml and plasma estradiol-17 beta was 226 +/- 131 pg/ml. The molar ratio of estrone and estradiol-17 beta to progesterone rose from less than 0.01 to 4.4 during labor, and was correlated with oxytocin receptor concentrations in endometrium (r = 0.5160, p < 0.001), but not those in myometrium (r = 0.0122). The regulation of oxytocin receptors by ovarian hormones in the two tissues may therefore differ.(ABSTRACT TRUNCATED AT 250 WORDS)


Stimulation of in vitro steroidogenesis by pituitary hormones in a turtle (Trachemys scripta) within the temperature-sensitive period for sex determination. To investigate the possible involvement of pituitary hormones in the regulation of steroidogenesis during reptilian sexual differentiation, we tested the ability of gonadotropin (ovine FSH), adrenocorticotropin (porcine ACTH), and growth hormone (bovine GH) to stimulate in vitro steroidogenesis in embryonic adrenal-kidney-gonad complexes (AKGs) of a turtle, Trachemys scripta, during and after the temperature-sensitive period for sex determination (TSP). Radioimmunoassays were used to measure progesterone, testosterone, estradiol, and corticosterone in incubation media; additionally, immunoreactive ACTH was measured in plasma. Presumptive male and female AKGs were stimulated by both FSH and ACTH at each stage investigated. Secretion of progesterone and corticosterone was usually far greater than that of testosterone or estradiol in both basal and hormone-stimulated incubations. In general, AKGs from presumptive males secreted more progesterone and corticosterone than AKGs from presumptive females. Progesterone and estradiol secretions were stimulated by both FSH and ACTH, but testosterone secretion was stimulated only by ACTH. Corticosterone secretion was strongly stimulated by ACTH. GH failed to significantly stimulate steroid secretion. Plasma ACTH levels were significantly higher in males than in females, and both sexes had significantly higher plasma levels of ACTH after the TSP compared to during the TSP. Our data demonstrate that during the temperature-sensitive period AKGs are responsive to both gonadotropin and ACTH, and that there are significant sex differences in steroidogenesis, sensitivity to gonadotropin and ACTH, and plasma ACTH levels.


Receptors for prostaglandins F2 alpha and E2 in ovine corpora lutea during maternal recognition of pregnancy. Plasma membrane receptors for prostaglandins (PG) F2 alpha and E2 were quantified in ovine corpora lutea obtained from nonpregnant and pregnant ewes on Days 10, 13, and 15 post-estrus, and from additional ewes on Days 25 and 40 of pregnancy. Regardless of reproductive status or day post-estrus, concentrations of luteal receptors for PGF2 alpha were 7- to 10-fold greater than those for PGE2. In pregnant ewes the concentration of receptors for PGF2 alpha was highest on Day 10 (35.4 +/- 2.8 fmol/mg) and lowest on Day 25 (22.3 +/- 2.5 fmol/mg). A difference in the concentration of luteal receptors for PGF2 alpha between pregnant and nonpregnant ewes was apparent only on Day 15 post-estrus, at which time the concentration of receptors for PGF2 alpha was higher in pregnant ewes than in nonpregnant ewes (27.1 +/- 2.7 vs. 17.7 +/- 2.7 fmol/mg). Concentrations of receptors for PGE2 in pregnant ewes were similar (p > 0.05; 2.8 +/- 0.3 to 3.7 +/- 0.2 fmol/mg) between Days 13 and 40 but were higher (p < 0.05) than in corpora lutea obtained from nonpregnant ewes on Days 10 (5.0 +/- 0.4 vs. 4.1 +/- 0.2 fmol/mg) and 15 (3.7 +/- 0.2 vs. 2.0 +/- 0.4 fmol/mg) post-estrus. Although concentrations of receptors for both PGF2 alpha and PGE2 were lowest in corpora lutea obtained from nonpregnant ewes on Day 15, this was not due to luteal regression since the weights and concentrations of progesterone in corpora lutea on Day 15 were not lower than those for corpora lutea obtained on Days 10 and 13.(ABSTRACT TRUNCATED AT 250 WORDS)


Changes in mitochondrial and microsomal 3 beta-hydroxysteroid dehydrogenase activity in mouse ovary over the course of the estrous cycle. 3 beta-Hydroxysteroid dehydrogenase (HSD) is located in the endoplasmic reticulum and mitochondria. To determine whether the separate enzymes play different roles in steroidogenesis, the specific activity (SA) of both were measured at four different stages of the mouse estrous cycle. Microsomal HSD activity changed little throughout, averaging 8.7 +/- 0.7 nmol progesterone/min/mg protein. In contrast, mitochondrial HSD activity changed dramatically at diestrus, increasing to 14.4 nmol progesterone/min/mg protein. When measured at proestrus, estrus, and metestrus, mitochondrial HSD activity was 5.5, 7.4, and 4.5 nmol progesterone/min/mg protein, respectively. To ascertain whether the increase in mitochondrial HSD activity at diestrus could be due to a preferential induction of enzyme, its SA and the SA of a mitochondrial inner membrane enzyme, cytochrome C oxidase, were compared to the SA of a mitochondrial outer membrane enzyme, rotenone-insensitive NADH cytochrome C reductase. The SA of all three enzymes changed proportionally at diestrus, suggesting that the increase in mitochondrial HSD activity was not due to its preferential induction. Rather, we believe that the HSD activity in the mitochondrial fraction, as measured at the four stages of the estrous cycle, is a reflection of the combined contributions from an ever changing population of ovarian cells. Mitochondria from luteal cells have the highest HSD activity, and are very likely responsible for the major synthesis of progesterone during the luteal phase.


The development of staging data for use in the microsimulation of lung cancer. Lung cancer incidence rates by cell type and stage were required for a lung cancer microstimulation submodel developed at Statistics Canada. Lung cancer incidence rates not disaggregated by stage were calculated for different histological cell types using Canada's National Cancer Incidence Reporting System data. In the absence of national lung cancer staging information, staging data from the province of Alberta were collected and rates of occurrence were calculated for different stages of lung cancer at time of diagnosis. Imputation procedures were used to maximize the amount of usable staging data. The Alberta stage rates were combined with the Canadian incidence rates to obtain estimates needed by the microsimulation submodel of the annual probability of an individual of a particular sex and age being diagnosed with lung cancer of each cell type at each stage. This project, although highly specialized, illustrates the need for more extensive and standardized staging data for cancer in Canada.


C-fos expression in the rat brain after intraperitoneal injection of lithium chloride. The distribution of evoked expression of the proto-oncogene c-fos was immunohistochemically examined in the rat brain after intraperitoneal injection of isotonic LiCl, which is commonly used to induce internal malaise in the conditioned taste aversion paradigm. C-fos-like immunoreactive neurones (c-fos neurones) were most densely observed in the central amygdaloid nucleus, external lateral subnucleus of the parabrachial nucleus (PBN), posteromedial and commissural parts of the nucleus of the tractus solitarius (NTS) and area postrema (AP). Experiments including vagotomy, intravenous injection of LiCl and lesions of the area postrema suggest that NTS neurones are activated via both sides of the vagus nerves, while AP neurones, humorally as well as neurally via the vagal nerve with a right side predominance. The activated NTS and AP neurones project mainly to the external lateral subnucleus of the PBN and lightly to the central lateral subnucleus of the PBN. These results are discussed in terms of the role of LiCl in the formation of conditioned taste aversion.


The role of 5-HT1A and 5-HT1B receptors in spinal nociceptive transmission and in the modulation of NMDA induced behaviour. The effects on nociception of intrathecal (i.th.) administration of selective 5-HT1A and 5-HT1B agonists were studied in rats. Nociception was evaluated using the tail-flick test with adjustments for tail-skin temperature, the increasing temperature hot-plate test and the scoring of biting and scratching behaviour after i.th. N-methyl-D-aspartate (NMDA). Activation of the spinal 5-HT1A receptor induced an antinociceptive effect in the increasing temperature hot-plate test and produced a dose dependent decrease in NMDA-receptor mediated behaviour. No significant change in nociception measured by either of the nociceptive tests was found after administration of the 5-HT1B agonist. These results support the hypothesis that spinal 5-HT1A receptor activation has an antinociceptive effect, and indicate a possible interaction between the serotonergic and glutaminergic transmitter systems.


Long-term potentiation is lost in aged rats but preserved by calorie restriction. Recordings of synaptic population responses, post-tetanic potentiation (PTP) and long-term potentiation (LTP) were made from area CA1 in hippocampal slices from ad libitum-fed rats at about 2 and 24 months of age, and also in animals at about 24 months of age that had been restricted to 60% of the caloric intake of control animals since weaning. Both PTP and LTP were greatly reduced in the old ad-lib animals. Calorically-restricted rats at about 24 months of age showed hippocampal responses with initial peak amplitudes more like those of 2-month controls than the ad-lib animals at 24 months. These observations suggest that calorie restriction preserves nervous-system functions, including indicators of plasticity such as LTP, which are otherwise lost in aging.


D2O discriminates gamma aminobutyric acid A receptors with different lifetimes. Progress of gamma aminobutyric acidA receptor channel (GABAAR) mediated 36Cl-ion uptake by native membrane vesicles from the rat cerebral cortex has been examined using rapid kinetic techniques. Data on GABA responsive 36Cl-ion flux, measured in a series of buffered salt solutions made up of different H2O-D2O mixtures, were evaluated by fitting a model with the mechanisms of channel opening and desensitization of GABAAR. The overall isotope effect could be isolated into its component contributions from the GABAARs by their different active lifetimes. Ion flux mediated by the slower desensitizing GABAAR was completely inhibited by 20% D2O. Large and selective D2O effects on ion fluxes mediated by the kinetically distinguishable native GABAARs suggest distinct types of subunit combinations for these receptors.


Co-expression of NMDA and AMPA/kainate receptor mRNAs in cochlear neurones. The co-expression of NMDAR-1 and GluR1-4 mRNAs in spiral ganglions of rat and guinea-pig cochleas was checked using a non-radioactive in situ hybridization technique. NMDAR-1, GluR2 and GluR3 mRNAs were expressed in the large neurones (type I) of the ganglion which innervate inner hair cells (IHCs), a sensory cell type likely to use an excitatory amino acid as a neurotransmitter. The labelling was very intense with the GluR2 and GluR3 oligoprobes while it remained moderate with the NMDAR-1 oligoprobe. This is consistent with the predominant implication of AMPA/kainate receptors in the physiological and early pathophysiological aspects of the IHC neurotransmission. Spiral ganglion neurones did not express GluR1 and GluR4 mRNAs, but a glial expression of GluR4 mRNAs was observed.


Control of salivary phospholipid content and composition. The mediation of phospholipid secretion in rat sublingual salivary gland cells maintained in the presence of [3H]choline was investigated. The secretion of [3H]choline-containing phospholipids was enhanced by beta-adrenergic agonist, isoproterenol, to a greater extent than the cholinergic agonist carbachol. A 2.9-fold increase in phospholipid secretion occurred with isoproterenol, while carbachol evoked only about 1.3-fold increase. In contrast to carbachol, the enhanced phospholipid secretion due to isoproterenol was accompanied by an increase in cAMP concentration. The secretion of phospholipids was also stimulated by dibutyryl-cAMP and the protein kinase C activator, phorbol myristate acetate, but not by 4 alpha-phorbol 12, 13-didecanoate which does not activate protein kinase C. Furthermore, the effects of dibutyryl-cAMP and phorbol myristate acetate were additive. The phospholipids secreted in response to isoproterenol exhibited a 52% decrease in lysophosphatidylcholine, while those secreted in response to carbachol showed a 23% lower content of phosphatidylcholine, and were enriched in lysophosphatidylcholine (2.8-fold) and sphingomyelin (1.4-fold). The results suggest that salivary phospholipid secretion remains mainly under beta-adrenergic control, while the phospholipid makeup is under cholinergic regulation.


Activation of protein serine/threonine kinases p42, p63, and p87 in Rous sarcoma virus-transformed cells: signal transduction/transformation-dependent MBP kinases. We have used myelin basic protein immobilized in sodium dodecyl sulfate-polyacrylamide gels to identify protein kinases after gel electrophoresis, followed by protein kinase reactions. This technique has permitted us to detect three protein kinases in serum-deprived cells transformed by p60src. On induction of cellular transformation by a temperature-sensitive v-src, a p87 protein kinase is activated within 30 min and remains activated in fully transformed cells. The p63 protein kinase is not fully activated until 24 h but remains activated in transformed cells. The commonly studied p42MBPK is rapidly activated within 30 min, and its kinase activity decreases significantly by 24 h, when the p63 enzyme is fully activated. The p42MBPK, as well as the p63 and p87 enzymes, are stimulated by transforming p60c-src mutants but not normal c-src or nonmyristylated p60c-src. In addition, the kinase activity of p63 enzyme, but not of p42MBPK, can be induced in okadaic acid-treated chicken embryo fibroblasts, indicating that phosphatase 2A and/or phosphatase 1 may be involved in the regulation of its activity. Additional data indicate that either p42MBPK or p63 activity correlates with the stimulation of the protein kinase p90RSK. Thus, there may be two independent pathways leading to the activation of the RSK gene product.


Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm. We cloned the hamster cdc25C cDNA by using the human cdc25C cDNA as a probe and prepared an antibody to Escherichia coli-produced hamster cdc25C protein that is specific to the human cdc25C protein. The microinjected antibody inhibited a chromosome condensation induced by tsBN2 mutation, indicating that the cdc25C protein is required for an activation of p34cdc2 kinase caused by loss of RCC1 function. The hamster cdc25C protein located in the cytoplasm, prominently in a periphery of the nuclei of cells arrested with hydroxyurea, and seemed to move into the nuclei by loss of RCC1 function. Also, we found a molecular shift of the cdc25C protein in cells showing premature chromosome condensation (PCC), in addition to normal mitotic cells. This molecular-shift appeared depending on an activation of p34cdc2 kinase.