Can somatosensory evoked potential monitoring predict energy dynamics during controlled hypotension?

Somatosensory evoked potentials and redox (reduction/oxidation ratio) of cytochrome a,a3 were studied simultaneously before, during, and after controlled hypotension used for arteriovenous malformation resection. These studies were also conducted before and after ED-IC (external-internal carotid) bypass procedures for treatment of patients with transient ischaemic attacks. The former served as an acute model and the latter as a chronic model of low blood flow (ischaemia). The use of non-invasive reflection spectrophotometry (for redox studies) in conjunction with somatosensory evoked potentials (SSEP) has demonstrated that metabolic and electrophysiological changes parallel each other during "controlled" hypotension. The authors conclude that an analysis of SSEP changes are valuable in the study of metabolic and functional states of the brain during controlled hypotension.


DNA synthesis and intracellular calcium elevation in porcine cerebral arterial smooth muscle cells by cerebrospinal fluid from patients with subarachnoid haemorrhage.

To understand the molecular mechanism of the pathogenesis of cerebral vasospasm following subarachnoid haemorrhage, we analysed the effect of cerebrospinal fluid from patients with subarachnoid haemorrhage on DNA synthesis and cytosolic-free calcium elevation in cultured porcine cerebral smooth muscle cells. Cerebrospinal fluid from patients on day 2 after subarachnoid haemorrhage induced transient elevation in cytosolic-free calcium levels. In contrast, the maximal elevation of cytosolic-free calcium levels induced by cerebrospinal fluid from control patients (without subarachnoid haemorrhage) was significantly lower than that induced by cerebrospinal fluid from patients with subarachnoid haemorrhage. In cultured porcine cerebral arterial smooth muscle cells, cerebrospinal fluid from patients with subarachnoid haemorrhage promoted levels of [3H]-thymidine incorporation (DNA synthesis) more than 2.5-fold higher than that promoted by cerebrospinal fluid from control patients without subarachnoid haemorrhage. However, in cultured aortic smooth muscle cells, there was no significant difference in [3H]-thymidine incorporation between cerebrospinal fluid from patients with subarachnoid haemorrhage and that by control cerebrospinal fluid. From these results in cerebral arterial smooth muscle cells, cerebrospinal fluid from patients following subarachnoid haemorrhage may play not only constrictive functions, evidenced by cytosolic-free calcium elevations, but also proliferative functions, demonstrated by promotion of [3H]-thymidine incorporation. The relevance of these factors to vasospasm will be discussed.


Correction of ischaemic brain acidosis with SQ29,548/1-benzylimidazole.

Thromboxane A2 (TXA2) is a proaggregatory vasoconstrictor that is synthesized and released during reperfusion of ischaemic brain. We administered a TXA2 receptor antagonist, SQ29,548, and a thromboxane A synthase inhibitor, 1-benzylimidazole (1-BI), to rats subjected to 30 min of reversible forebrain ischaemia. Cerebral thromboxane B2 (TXB2), the stable metabolite of TXA2, measured after 60 min of reperfusion was 0.37 +/- 0.08 ng/mg brain protein in animals treated with SQ29,548/1-BI compared with 1.20 +/- 0.16 in ischaemic controls (p < 0.05). Cerebral pH determined by 31P magnetic resonance spectroscopy was higher in treated animals, 7.06 +/- 0.04, than in ischaemic controls, 6.5 +/- 0.01, after 20 min of reperfusion (p < or = 0.01). The significant elevation of cerebral pH in treated animals persisted at 30 (7.17 +/- 0.05 vs. 6.5 +/- 0.01; p < or = 0.01), 35 (7.17 +/- 0.05 vs. 6.44 +/- 0.04; p < or = 0.01), and 40 min of reperfusion (7.06 +/- 0.06 vs. 6.37 +/- 0.01; p < or = 0.05). We conclude that SQ29,548/1-BI reduces thromboxane levels and promotes resolution of tissue acidosis in ischaemic brain. The combination of a TXA2 receptor antagonist with a thromboxane A synthase inhibitor deserves further study as a potential treatment for acute cerebral infarction.


Cholinergic deafferentation prevents delayed neuronal death of the hippocampal CA1 pyramidal neurons after transient forebrain ischaemia.

The relation between CA1 neurons, fimbria-fornix and cholinergic neurons of the basal forebrain was examined with the aid of Acetylcholine esterase (AChE) staining, Woelcke's staining and immunohistochemistry of Choline-acetyl transferase (ChAT). The transected side of the hippocampus was poorly stained by AChE two weeks after the transection, when the ipsilateral medial septum ChAT-positive neurons were reduced, but showed good recovery with AChE six weeks later. Nerve growth factor (NGF) was added at a dose of 10 micrograms/100 microliters immediately after the aspiration, and after that once per week with cisternal puncture. As a result, ipsilateral medial septum ChAT-positive neurons were preserved, but cross innervation with relation to hypertrophy of the cholinergic neurons was not detectable even six weeks after the transection. Furthermore, delayed CA1 neuronal death on the transected side of the hippocampus following occlusion of four vessels for 30 minutes was not detectable two weeks after the operation, although neuronal density was reduced after six weeks. The density of neurons on the transected side of the hippocampus in the CA1 subfield with treated NGF had not decreased significantly six weeks later. Therefore, we suspect that the input from cholinergic fibres must be transported to the hippocampal pyramidal neurons responding to NGF, and it was confirmed that cholinergic deafferentation prevents the delayed neuronal death of CA1 pyramidal neurons during transient ischaemia.


Cranio-nasal median splitting for radical resection of craniopharyngioma.

A new surgical approach for radical resection of craniopharyngioma is presented. This approach (cranio-nasal median splitting) involves craniotomy in the centre of the frontal bone, removal of the median portion of the supraorbital bar that incorporates the nasal bone, and detachment of the medial canthal ligaments. The frontal lobes, the cribriform plates, the planum sphenoidale, and the upper nasal cavities are split in the midline. The extraventricular surface of the hypothalamus, the pituitary stalk, and the posterior portion of the Willis' arterial ring are well visualized through the midline infrachiasmatic route. The intraventricular surface of the hypothalamus is also visible in the same operative field through the lamina terminalis and/or the anterior portion of the corpus callosum. This excellent visualization is quite helpful for minimizing operative injury to the hypothalamus and the pituitary stalk whichever surface of the third ventricular floor the tumour is situated upon. Three cases of craniopharyngioma operated upon by this approach are presented. Discussions are focused not only on the indication, but on the advantages and disadvantages of this approach. The surgical techniques for reconstruction of the cranial base are also described, together with some precautions that should be taken to prevent possible postoperative complications.


Changes of cerebral haemodynamics during rebleeding subsequent to subarachnoid haemorrhage with vasospasm.

A 40-year old patient with spontaneous subarachnoid haemorrhage and vasospasm in whom an acute rebleeding occurred during transcranial Doppler ultrasonography monitoring is reported. The Doppler recordings of the middle cerebral artery displayed an extreme modification in their configuration in the form of an orthograde systolic flow component with zero diastolic flow or an oscillating flow immediately after occurrence of rebleeding. Reoccurrence of a diastolic flow component in still markedly raised cerebrovascular resistance index could be demonstrated a short time later. Rebleeding in subarachnoid haemorrhage with preexisting vasospasm leads to a severe temporary disturbance of cerebral perfusion. The vasospasm is likely to lead to a delayed normalization of perfusion and thus to prolonged ischaemia.


Modulation of cholecystokinin (CCK) gene-expression in a human neuroblastoma cell line: effects of serum on enhanced CCK and c-fos mRNA expression.

The effect of fetal-calf serum (FCS) and Forskolin (FKN) on cholecystokinin (CCK) and proto-oncogene c-fos mRNA expression in the human neuroblastoma cell line SK-N-MC, cultured in serum free medium was studied by Northern blot analysis and nuclear run-off transcription analysis. Addition of FCS or FKN gradually increased the basal CCK mRNA level approximately four to six-fold after 2-4 h. In contrast, a strong and transient increase of the c-fos mRNA-level was observed, approximately forty to fifty-fold after 50-60 min over unstimulated cells. Nuclear run-off transcription analysis indicates that c-fos mRNA is constitutively expressed and transcription may be further stimulated by FCS and FKN. Moreover, in SK-N-MC nuclei, transcription of the c-fos gene clearly precedes stimulated CCK-mRNA expression. This suggests that FOS, which is known to form a AP-1 heterodimer transcription complex with the proto-oncogen product, Jun, may bind to the tentative AP-1 binding site, found within the human CCK promoter and thereby modulates basal and enhanced CCK-mRNA expression in SK-N-MC cells.


Pre-synaptic dopaminergic control mechanisms for CCK-8 like immunoreactivity in the rat medial frontal cortex.

In order to study the control mechanism of cholecystokinin (CCK) contents of the rat brain mediated by pre-synaptic receptors in dopamine (DA) neurons, R(+) and S(-) compounds of 3-(3-hydroxyphenyl)-N-n-propylpiperidine (3-PPP), which are selective pre-synaptic dopaminergic agents, were administered in rats at low and high doses. CCK-8-like immunoreactivity (CCK-8 LI) was measured in the medial frontal cortex. In another experiment, a neurotoxin, N-methyl-D-aspartic acid (NMDA) was used to degenerate efferent CCK neurons and CCK interneurons of the medial frontal cortex, followed by an intraperitoneal administration of apomorphine hydrochloride (APO) to study the effect on CCK-8 LI via the pre-synaptic DA receptor. According to the results of these experiments, CCK-8 LI was increased in the medial frontal cortex in response to the stimulation of pre-synaptic DA receptor, suggesting a control of CCK-8 release, at least in part, by the pre-synaptic DA receptor.


Endogenous glutamate stimulates release of alpha-melanocyte-stimulating hormone from the rat hypothalamus.

Release of alpha-melanocyte-stimulating hormone (alpha-MSH) and glutamic acid was quantified from superfused slices of rat hypothalamus. Application of L-glutamic acid 10(-4) M failed to evoke release of alpha-MSH but, in the presence of 10(-4) M dihydrokainic acid (DHK) an inhibitor of glutamate uptake systems, caused significant stimulation of release. DHK caused gradual and sustained increases in both alpha-MSH and glutamate release. That in alpha-MSH was blocked by 10(-4) M DL-2-amino-5-phosphopentanoic acid, a competitive N-methyl-D-aspartic acid (NMDA)-type glutamate receptor antagonist. We conclude that hypothalamic glutamate is subject to rapid uptake through mechanisms blocked by DHK and that alpha-MSH release is stimulated by endogenous and exogenous glutamate through NMDA-type receptors.


A prospective study of the effect of the appropriateness of foot-shoe fit and training shoe type on the incidence of overuse injuries among infantry recruits.

In a prospective study of the effect of the appropriateness of foot-shoe fit and training shoe type on the incidence of overuse injuries among infantry recruits, it was found that three shoe widths for each shoe length size were necessary to adequately accommodate the recruit population's foot anatomy. Recruits compensated for the lack of available shoe widths by choosing larger shoe sizes. However, this compensation did not result in an increase in the incidence of overuse injuries. Recruits who trained in basketball shoes had a lower incidence of overuse injuries of the feet than recruits who trained in infantry boots (p = 0.0001), but the overall incidence of overuse injuries was the same in both groups.


Gonadotropin-releasing hormone (GnRH) cells of the terminal nerve as a model neuromodulator system.

Modulation of ionic channel properties by neurotransmitters and hormones is called neuromodulation and may be the basis for many long-lasting changes in animal behavior, e.g. changes in the arousal or motivational states. Gonadotropin-releasing hormone (GnRH), originally identified as a hypophysiotropic hormone, is now believed to act also as a neuromodulator. From studies of electrical activities and morphology of terminal nerve cells (major source of GnRH) of a fish brain, a general hypothesis regarding modulator neurons is proposed; modulator neurons have endogenous oscillatory activities which vary according to the animal's hormonal or environmental conditions. These modulator neurons, in turn, regulate neuronal excitabilities in a wide variety of brain regions simultaneously via multiple axonal branches.


Norepinephrine reverses N-methyl-D-aspartate-mediated inhibition of long-term potentiation in rat hippocampal slices.

Application of 1 microM N-methyl-D-aspartate (NMDA) either immediately before or following tetanic stimulation inhibits the induction of CA1 hippocampal long-term potentiation (LTP). We have examined the effect of trans-amino-1,3-cyclopentanedicarboxylic acid (ACPD), norepinephrine and acetylcholine on this NMDA-mediated LTP inhibition using extracellular recordings from in vitro rat hippocampal slices. When NMDA is accompanied by 100 microM ACPD or 10 microM norepinephrine, the block of LTP is overcome. The norepinephrine effect is mimicked by phenylephrine and methoxamine and is blocked by phentolamine and prazosin suggesting the involvement of alpha 1-adrenergic receptors.


Excitatory effect of adenosine on neurotransmission is due to increase of transmitter release in the hippocampal slices.

Adenosine has dose-dependent biphasic excitatory and inhibitory effects on neurotransmission in the hippocampus. The mechanism of the excitatory action is not known although that of the inhibitory action has been well analyzed. Here we report on the mechanism of excitatory action of adenosine, using hippocampal slices. Studies of intracellular recordings of CA3 pyramidal neurons showed that the amplitude of EPSP was dramatically enhanced by application of adenosine at low concentration (0.1 microM) without changing resting membrane potentials, membrane conductance or the threshold for spike generation by injecting current pulses. On the other hand, the presence of adenosine at a concentration of 0.1 microM during electrical stimulation to the slices increased 1.7 times the release of glutamate, an excitatory neurotransmitter in the hippocampus. These results indicate that the excitatory action of adenosine at low doses is due to the increase of transmitter release.


The involvement of muscarinic, beta-adrenergic and metabotropic glutamate receptors in long-term potentiation in the fimbria-CA3 pathway of the hippocampus.

Possible modulatory actions of endogenous neurotransmitters on long-term potentiation (LTP) were investigated in the fimbria-CA3 pathway of rat hippocampal slices. Bath application of atropine (10 microM), but neither timolol (10 microM) nor D,L-2-amino-3-phosphonopropionate (AP3, 100 microM), significantly attenuated LTP induced by 20 pulses of 50 Hz stimulation. When stronger stimulation (3 trains of 100 Hz, 100 pulses) was used for the induction of LTP, timolol significantly attenuated LTP, but atropine and AP3 did not. These results suggest that, under specified conditions, endogenous acetylcholine through muscarinic receptors, and noradrenaline through beta-adrenergic receptors may modulate the generation of LTP in the fimbria-CA3 pathway. Metabotropic glutamate receptors may be involved not in the generation of LTP but in low-frequency synaptic transmission, since 300-1,000 microM AP3 greatly reduced, or abolished synaptic transmission in this pathway.


Identification of a novel vertebrate homeobox gene expressed in haematopoietic cells.

This paper describes the characterisation of a novel chicken homeobox gene, Prh, whose encoded homeodomain sequence differs significantly from those of other factors which have been described. As expected, a portion of the encoded protein, containing the homeodomain, is capable of sequence-specific DNA-binding. Outside the homeodomain, Prh, possesses an N-terminal region extremely rich in proline residues and a C-terminal acidic portion, either of which may function as transcription regulatory domains. Since, among the chicken tissues tested, its transcription is restricted to haematopoietic cells, lung and liver, it may function in tissue-specific patterns of gene regulation. Human and murine Prh homologues have also been identified; so it is likely that such genes are a general feature of vertebrate genomes.


The regulation of the murine Hox-2.5 gene expression during cell differentiation.

The mouse Hox-2.5 gene containing a Drosophila Antennapedia-type homeobox sequence is expressed in a spatially and temporally restricted manner during embryogenesis. We found that the mouse embryonal carcinoma cell line P19 expresses Hox-2.5 during differentiation by the treatment with retinoic acid (RA). Expression of the Hox-2.5 gene was not detected in undifferentiated P19 cells, but detected 72 hours after treatment with RA. In order to analyze this inductive response, we first identified the Hox-2.5 transcription initiation site and a possible promoter region. Subsequently, we prepared constructs containing various Hox-2.5 DNA fragments fused to a firefly luciferase reporter gene and transfected these into undifferentiated or differentiating P19 cells. These studies have demonstrated that a region -279 to +15 with respect to the transcription initiation site has a differentiation-responsive promoter activity. Deletion analysis suggests that the sequences responsible for this induction are located in several distinct domains within the 294 bp promoter region. Two of the possible differentiation-responsive elements were identified by analysis of DNA-protein interactions, and in vivo competition assays lend support to the notion that these regions are involved in the differential expression of Hox-2.5 promoter activity.


Delta-type DNA polymerase characterized from Drosophila melanogaster embryos.

Genetic and biochemical evidence suggests there are at least three DNA polymerases required for replication in eukaryotic cells. However, Drosophila embryonic cells have a very short duration S phase which is regulated differently. To address the question of whether embryos utilize different DNA polymerases, we employed Mono Q anion exchange chromatography to resolve the DNA polymerase activities. Two types of DNA polymerase, DNA polymerase delta and DNA polymerase alpha, were distinguished by: 1. copurification of DNA primase or 3'-5'exonuclease activities; 2. immunoblot analysis with alpha-specific polyclonal antisera; 3. sensitivity to aphidicolin and BuPdGTP; and 4. processivity measurements with and without Proliferating Cell Nuclear Antigen. These observations suggest that Drosophila embryos, similar to nonembryonic cells, have both alpha- and delta-type DNA polymerases.


[An enzyme study of purine nucleotide biosynthesis in the plerocercoids of cestodes in the fam. Ligulidae].

By means of spectrophotometric method there was determined the activity of three enzymes of biosynthesis of purine nucleotides: amino imidazole ribonucleotide-carboxylase (AIR-carboxylase, EC 4.1.1.21), an enzyme of biosynthesis of purine nucleotides de novo in plerocercoids of Schistocephalus pungitii and Digramma interrupta; inosine monophosphate-dehydrogenase (IMPh-dehydrogenase, EC 1.2.1.14), an enzyme of salvage path, and adenylosuccinate lyase (EC 4.3.2.2), an enzyme taking part both in biosynthesis de novo and salvage in plerocercoids of Schistocephalus pungitii. The activity of AIR-carboxylase was not determined. Specific activities of adenylosuccinate lyase and IMPh-dehydrogenase amount to (1.3 +/- 0.3) x 10(-3) and (1.2 +/- 0.4) x 10(-3) mumole/min.mg protein, respectively. The activity of the three enzymes was determined in the liver of ten-spined stickleback, a host of S. pungitii plerocercoids. The question of metabolic dependence of Ligulidae plerocercoids on hosts to provide for purine bases is discussed.


Purification and characterization of NF-R1 that regulates the expression of the human multidrug resistance (MDR1) gene.

We have purified a protein, NF-R1, that specifically binds to two unrelated motifs--the ATTCAGTCA motif and the GC-box motif--on the MDR1 proximal promoter. Purified NF-R1 has been confirmed by southwestern blotting to be a 110-kDa protein. Methylation interference analysis revealed that the nucleotides were in close contact with purified NF-R1 on the ATTCAGTCA and GC-box motifs. The nucleotides were required for the binding of NF-R1, as seen by competition gel mobility shift assay using point mutated oligonucleotides. In a CAT expression assay using the corresponding point-mutated MDR1 promoter fused to a CAT gene, binding inhibition of NF-R1 to the promoter resulted in 2- to 3-fold increases of CAT activity, as compared to the intact promoter in Adriamycin-resistant K562 cells. Thus NF-R1 has a relation to the negative regulation of the MDR1 gene transcription.


Ondansetron to prevent emesis following N-acetylcysteine for acetaminophen intoxication.

We present a 17-year-old girl who developed persistent vomiting following acetaminophen overdose. Because of the amount of drug ingested (300 mg/kg acetaminophen) and the four-hour postingestion level (256 micrograms/ml), administration of N-acetylcysteine (NAC) was indicated. Emesis occurred immediately following the first three doses of NAC despite administering the drug by continuous nasogastric drip over one hour. Prior to the next attempt, ondansetron (0.15 mg/kg) was administered intravenously as an antiemetic. Thirty minutes following ondansetron, NAC was tolerated without further emesis. Although several antiemetics may have prevented further emesis, we chose ondansetron since, as a serotonin antagonist, it does not cause extrapyramidal side effects or sedation. In patients with potentially toxic drug ingestions, these side effects may be confused with or mask the adverse effects of the ingested drug, thereby interfering with the ongoing evaluation of the patient. Although not previously administered for this indication, ondansetron has several advantages over other antiemetic agents in the setting of an acute drug ingestion.


Medical therapy in Crohn's disease.

Traditionally, sulfasalazine and corticosteroids have been used to treat Crohn's disease. However, therapy has been expanded to include other drugs, and several newer agents show clinical promise. The authors discuss the therapeutic options now available and describe results of several drug trials. Despite the apparent benefits of newer therapies, caution must be used in choosing appropriate treatment.


Medical treatment after myocardial infarction. Results of studies using various methods.

Patients who have had myocardial infarction and are at risk for continued problems may benefit from several treatment methods (table 1). Beta-adrenergic blockers reduce subsequent cardiovascular morbidity and mortality through their effect on the myocardial supply-demand balance. Administration of angiotensin-converting enzyme inhibitors reduces morbidity and mortality rates in myocardial infarction survivors who have diminished left ventricular ejection fraction. Aspirin and other anticoagulants are beneficial through their antiplatelet effects. Cardiac rehabilitation methods and aggressive management of lipids levels are also useful.


Beta-adrenergic responsiveness of the gastrointestinal tract in diabetic rats.

Recently, decreased gastrointestinal beta-adrenergic responses in experimental diabetes have been demonstrated. Gastrointestinal responses to beta-adrenoceptor agonists are impaired in both insulin-dependent and non-insulin-dependent diabetic rat. Insulin treatment improves the impaired gastrointestinal beta-adrenergic responsiveness of diabetic rats. The improvement seen with insulin treatment on beta-adrenergic responsiveness is closely related to protein biosynthesis. The decreased beta-adrenergic responses in diabetic rat gastrointestinal tract seem to result from a decrease in the number of beta-adrenoceptors. It is most likely that the decreased gastrointestinal beta-adrenergic responsiveness is related to an impairment in the turnover of beta-adrenoceptors as a consequence of diabetes and that insulin has a beneficial effect on the impaired receptor turnover.


Studies on the interaction between formoterol and salmeterol in guinea-pig trachea in vitro.

The actions of and interaction between formoterol and salmeterol were studied on guinea-pig trachea in vitro. Tracheal strip preparations were contracted by 1 mumol/l carbachol giving a near maximal contraction. Salmeterol in concentrations from 0.1 to 3 mumol/l relaxed the tracheal smooth muscle by about 30 per cent of the maximum relaxation produced by theophylline. Formoterol caused a concentration-dependent and almost complete relaxation with a pD2 of 8.56. In the presence of salmeterol there was a rightward shift of the concentration-response curve for formoterol. The pA2 for salmeterol was estimated to 7.42. Similar experiments with isoprenaline indicated that salmeterol has a low affinity for beta 1-adrenoceptors. Formoterol and salmeterol both inhibited in a concentration-dependent manner the contractions evoked by stimulation of the vagus nerve in a tracheal tube preparation. The degree of inhibition decreased with increasing stimulation frequency. Complete inhibition was attained with salmeterol, but not with formoterol, at the highest frequency employed (45 Hz). The inhibiting effect of 10 mumol/l salmeterol was not blocked by 10 mumol/l sotalol, a beta-adrenoceptor antagonist. It is concluded that salmeterol, in comparison to formoterol, is a partial beta 2-adrenoceptor agonist and has, at high concentrations, an additional unspecific inhibitory action.


Lack of effect of certain histamine H2-receptor blockers on the glucuronidation of 7-hydroxy-4-methylcoumarin by human liver microsomes.

Contrary to the general belief that cimetidine and ranitidine spare glucuronidation of drugs, some authors have indicated that both cimetidine and ranitidine could inhibit the glucuronidation of paracetamol (acetaminophen) by cultured rat hepatocytes. Thus, we tested the effect of three histamine H2-receptor blockers (cimetidine, ranitidine and famotidine) on the glucuronidation of 7-hydroxy-4-methylcoumarin (7-OH-4-MC) by human liver microsomes. None of the drugs studied produced significant inhibition of the glucuronidation of 7-OH-4-MC when used at concentrations up to 1.5 mM. Thus, even the new H2-receptor blocker, famotidine, spares glucuronidation. These findings further support the previous reports which suggest that glucuronide conjugation in humans is spared by H2-receptor blockers.


Myocardial ultrastructure in the isolated rabbit heart exposed to dopamine, dobutamine, isoprenaline, G-strofanthin, xamoterol and hypoxia.

The isolated spontaneously beating heart was used for comparing the effects of hypoxia and positive inotropic drugs on myocardial ultrastructure. Hypoxia gives a significant decrease in the volume fractions of mitochondrial cristae relative to the total mitochondrial volume (Vvmcristae) and a significant increase in the volume fraction of mitochondrial matrix relative to the total mitochondrial volume (Vvmmatrix), but changes in volume fractions of mitochondria (Vvmitochondria) and myofibrils (Vvmyofibrils) were absent. Significant changes in ultrastructure in hearts treated with dopamine (0.6 microM), dobutamine (90 nM), G-strofanthin (0.25 microM), xamoterol (32 nM) and isoprenaline (0.15 microM or 15 nM) were absent. Furthermore, myocardial effects in the isolated rabbit heart without exposure to any treatment showed a significantly decrease in oxygen consumption after 90 min. and a significant decrease in frequency of contractions after 120 min. perfusion time, but no change in contractility was seen. We conclude that this experimental model is useful in studies of positive inotropic drugs.


[Polymerase chain reaction (PCR) in microbiology. Value and practical applications].

The gene amplification reaction (PCR) developed in 1985 is a molecular biology instrument which is used to amplify genomic restriction fragment lengths of micro-organisms found in biological samples. At the moment, its applications to direct microbiological diagnosis remain to be determined precisely, and its strict technique is awaiting standardization. However, it is a powerful tool for molecular typing used to follow up an antiviral therapy or to analyse retrospectively paraffin-embedded tissue banks in search of viral or bacterial nucleic acids.


Molecular cloning and functional expression of a brain-specific somatostatin receptor.

The PCR and conventional library screening were used to clone the brain-specific somatostatin receptor rSSTR-4 from a rat genomic library. The deduced amino acid sequence encodes a protein of 384 amino acids and displays structural and sequence homologies with members of the G protein-receptor superfamily. The amino acid sequence of rSSTR-4 is 60% and 48% identical to that of somatostatin receptors SSTR-1 and SSTR-2, respectively, two recently cloned subtypes. Competition curve analysis of the binding properties of the receptor transiently expressed in COS-1 cells revealed a higher apparent affinity for somatostatin 14 than for somatostatin 28. In contrast, the somatostatin analogs SMS 201-995, IM 4-28, and MK-678 failed to displace specific binding in transfected cells. These characteristics resemble the pharmacological binding properties of the previously described brain-specific somatostatin-receptor subtype. Examination of the tissue distribution of mRNA for rSSTR-4 revealed expression limited to various brain regions with highest levels in the cortex and hippocampus. Thus, based on the pharmacology and tissue localization of this receptor, we conclude that rSSTR-4 represents a brain-specific somatostatin receptor.


Isolation of transglutaminase-reactive sequences from complex biological systems: a prominent lysine donor sequence in bovine lens.

The transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13)-catalyzed cross-linking of proteins in biological systems can often be inhibited by inclusion of small primary amines or glutamine-containing peptides, which act as site-specific blockers of the relevant acceptor (i.e., glutamine) and donor (i.e., lysine) functionalities of the natural substrates. Compounds such as dansylcadaverine and dansyl-epsilon-aminocaproyl-Gln-Gln-Ile-Val are particularly useful in sorting out acceptor-donor relationships among lens crystallins. Apart from its fluorescent properties, the dansyl hapten offered special advantages as a "handle" for the rapid isolation of transglutaminase targets even in the complex system of lens cortical homogenate. The dansylated peptide was incorporated into bovine lens proteins under the influence of the Ca(2+)-activated intrinsic transglutaminase and, after digestion by endoproteinase Glu-C, the tracer-containing fragments were isolated by affinity chromatography on an anti-dansyl antibody column. The major fluorescent peak was isolated by HPLC and sequenced by Edman degradation, which yielded phenylthiohydantoin amino acid derivatives for the first 10 cycles, EKPAVTAAPK, and none for the next 2. The sequence, corresponding to residues 165-174 of alpha B-crystallin, unambiguously identifies the known carboxyl-terminal domain, EK-PAVTAAPKK, as the prominent lysine-donating fragment in bovine lens.


Enhancement of human immunodeficiency virus (HIV)-specific CD4+ and CD8+ cytotoxic T-lymphocyte activities in HIV-infected asymptomatic patients given recombinant gp160 vaccine.

Twenty-six human immunodeficiency virus (HIV)-infected asymptomatic patients with CD4+ lymphocytes > 400 per mm3 were randomly allocated to a range of doses of recombinant gp160 or a control (recombinant hepatitis B vaccine) on a double-blind basis. Each patient received an injection at 0, 4, 12, 24, 36, and 48 weeks. Treatment assignments were decoded when all patients reached 28 weeks of the study period. HIV-1-specific CD4+ and CD8+ cytotoxic T lymphocyte (CTL) activities were assessed in vitro before vaccination and 2 weeks after each injection. There were significant increases in major histocompatibility complex-restricted HIV-1 Env-specific CD4+ and CD8+ CTL activities in 18 of 21 gp160 vaccinees. No control-injected patients showed a significant change. Neither gp160 nor control recipients showed significant changes in HIV-1 Gag- and Pol-specific CTL activities. HIV-1 Env-specific CD4+ and CD8+ CTL precursor frequencies were also measured in three vaccinees before and at 24 weeks after vaccine was started. CTL precursor frequencies also increased in both CD4+ and CD8+ populations. This study shows that this gp160 vaccine is immunogenic in enhancing HIV-1 Env-specific cytotoxic T-cell-mediated immunity in HIV-seropositive individuals.


Toward a cytogenetically based physical map of the wheat genome.

Bread wheat (Triticum aestivum L. em Thell) is well suited for cytogenetic analysis because the genome, buffered by polyploidy, can tolerate structurally and numerically engineered chromosomes for analysis over infinite generations. This feature of polyploidy can be used in developing a high-resolution, cytogenetically based physical map of the wheat genome. We show that numerous deletions, observed in the progeny of a monosomic addition of a chromosome from Triticum cylindricum in wheat, result from single breakpoints and a concomitant loss of distal fragments. Breakages occurred in euchromatic and heterochromatic regions. Forty-one deletions for chromosomes 7A, 7B, and 7D, and a set of genetically mapped DNA probes, were used to construct physical maps. Recombination was low in proximal chromosomal regions and very high toward the distal ends. Deletion mapping was more efficient than genetic mapping in resolving the order of proximal loci. Despite variation in size and arm ratio, relative gene position was largely conserved among chromosomes 7A, 7B, and 7D and a consensus group 7 physical map was constructed. Several molecularly tagged chromosome regions (MTCRs) of approximately one to a few million base pairs were identified that may be resolved by long-range mapping of DNA fragments. Thus, a cytogenetically based physical map may be used to integrate chromosome and DNA-based maps. The MTCRs may simplify strategies for cloning of agronomically useful genes despite the genetic complexity and the large genome size of wheat.


Thymic depletion and peripheral activation of class I major histocompatibility complex-restricted T cells by soluble peptide in T-cell receptor transgenic mice.

Injection of mice transgenic for a class I major histocompatibility complex-restricted T-cell receptor with a soluble peptide antigen from influenza virus nucleoprotein results in clonal depletion of double-positive immature thymocytes in the thymus and activation of mature T cells in the periphery, accompanied by a transient up-regulation of the T-cell receptor and CD3 and CD8 coreceptor molecules.


Up-regulation of intercellular adhesion molecule 1 transcription by hepatitis B virus X protein.

Intercellular adhesion molecule 1 (ICAM-1), a counter-receptor for lymphocyte function-associated antigen 1 on T cells, is critically important to a wide variety of adhesion-dependent leukocyte functions, including antigen presentation and target cell lysis. ICAM-1 expression by hepatocytes is increased in areas of inflammation and necrosis during chronic hepatitis B. Whether induction of ICAM-1 is due to the effect of inflammatory cytokines or involves a direct effect of the hepatitis B virus (HBV) remains unknown. In the present study, transfection of the HBV genome into human hepatoma cell lines resulted in enhanced expression of ICAM-1 protein and RNA in the absence of inflammation. Results of subgenomic transfections indicated that the HBV X protein (pX) induced ICAM-1 expression. Nuclear run-on assays showed that pX induced the ICAM-1 gene by increasing its rate of transcription. Although both pX and interferon gamma induced transcription of ICAM-1, addition of interferon gamma to cells expressing pX did not show an additive or synergistic effect. These results indicate that pX can directly regulate expression of ICAM-1 and may participate in the immunopathogenesis of HBV infection.


Tat-responsive region RNA of human immunodeficiency virus type 1 stimulates protein synthesis in vivo and in vitro: relationship between structure and function.

The Tat-responsive region (TAR) sequence is present at the 5' end of human immunodeficiency virus 1 mRNAs and as a cytoplasmic form of 58-66 nucleotides. TAR RNA blocks the activation and autophosphorylation of the double-stranded RNA-activated protein kinase in vitro. We show here that TAR RNA also prevents the double-stranded RNA-mediated inhibition of translation in a cell-free system. Mutagenic and structural analyses of TAR RNA indicate that a stem of at least 14 base pairs is required for this activity, whereas the loop and bulge required for transactivation by Tat are dispensable. Truncation of the RNA to 68 nucleotides results in the loss of translational rescue ability, suggesting that the short cytoplasmic TAR RNA produced by viral transcription in vivo may not have the capability to suppress activation of the kinase. However, because longer TAR transcripts stimulate expression in a transient assay in vivo, the TAR structure at the 5' end of viral mRNAs could still exert this function in cis.


Identification and genetic mapping of a homeobox gene to the 4p16.1 region of human chromosome 4.

A human craniofacial cDNA library was screened with a degenerate oligonucleotide probe based on the conserved third helix of homeobox genes. From this screening, we identified a homeobox gene, H6, which shared only 57-65% amino acid identity to previously reported homeodomains. H6 was physically mapped to the 4p16.1 region by using somatic cell hybrids containing specific deletions of human chromosome 4. Linkage data from a single-stranded conformational polymorphism derived from the 3' untranslated region of the H6 cDNA placed this homeobox gene more than 20 centimorgans proximal of the previously mapped HOX7 gene on chromosome 4. Identity comparisons of the H6 homeodomain with previously reported homeodomains reveal the highest identities to be with the Nk class of homeobox genes in Drosophila melanogaster.


Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes.

Recently, two subunits of a large cytosolic protease and two putative peptide transporter proteins were found to be encoded by genes within the class II region of the major histocompatibility complex (MHC). These genes have been suggested to be involved in the processing of antigenic proteins for presentation by MHC class I molecules. Because of the high degree of polymorphism in MHC genes, and previous evidence for both functional and polypeptide sequence polymorphism in the proteins encoded by the antigen-processing genes, we tested DNA from 27 consanguineous human cell lines for genomic polymorphism by restriction fragment length polymorphism (RFLP) analysis. These studies demonstrate a strong linkage disequilibrium between TAP1 and LMP2 RFLPs. Moreover, RFLPs, as well as a polymorphic stop codon in the telomeric TAP2 gene, appear to be in linkage disequilibrium with HLA-DR alleles and RFLPs in the HLA-DO gene. A high rate of recombination, however, seems to occur in the center of the complex, between the TAP1 and TAP2 genes.


CD8 T cells can protect against an intracellular bacterium in an interferon gamma-independent fashion.

Specific T-cell immunity to Listeria monocytogenes is thought to occur through the action of lymphokines which activate phagocytes to ingest and kill microorganisms. Interferon gamma (IFN-gamma) has been shown to be an effective mediator of this type of macrophage activation in vivo and in vitro. The monoclonal antibody H22.1 efficiently neutralizes endogenous IFN-gamma, exacerbates disease in a mouse model of L. monocytogenes infection, and inhibits the in vivo protective activity of a Listeria antigen-specific CD4 T-cell line. In contrast, in vivo protection by Listeria-immune CD8 T cells is not inhibited by the neutralizing anti-IFN-gamma monoclonal antibody. These results suggest that CD8 T cells can protect against an intracellular pathogen in an IFN-gamma-independent manner.


Variation across species in the size of the nuclear genome supports the junk-DNA explanation for the C-value paradox.

The amount of DNA in the nuclear genome (the DNA C-value) of eukaryotes varies at least 80,000-fold across species, and yet bears little or no relation to organismic complexity or to the number of protein-coding genes. This phenomenon is known as the C-value paradox. One explanation for the C-value paradox attributes the size of the nuclear genome to 'junk' (typically non-coding) genetic elements that accumulate until the costs to the organism of replicating excess DNA select against it. Across species, organisms that develop at a slower rate should tolerate more junk DNA. Alternatively, junk DNA may function as a nucleo-skeleton to maintain the volume of the nucleus at a size proportional to the volume of the cytoplasm in the cell. Across species, the DNA C-value is predicted to vary with the nuclear and cytoplasmic volumes of cells. Previous studies have not been able to distinguish between the skeletal-DNA and junk-DNA explanations for the C-value paradox. We report a study of DNA content in 24 salamander species which does. The size of the nuclear genome is correlated with developmental rate even after the effects of nuclear and cytoplasmic volume have been removed. However, genome size is not correlated with cytoplasmic volume after controlling for developmental rate. These results support the view that junk DNA accumulates in the nuclear genome until the costs of replicating it become too great, rather than that it functions as a nucleo-skeleton.


Supply of O2 regulates O2 demand during utilization of reserves of poly-beta-hydroxybutyrate in N2-fixing soybean bacteroids.

A liquid reaction medium containing dissolved air and oxyleghaemoglobin, but no energy-yielding substrate, was supplied to bacteroids confined in a stirred flow reaction chamber. The relative oxygenation of the leghaemoglobin in the chamber was determined automatically by spectrophotometry of the effluent solution, and the concentrations of free, dissolved O2 ([O2]) and rates of O2 consumption were calculated. Dissolved CO2 and NH3 from N2 fixation were determined in fractions of the effluent solution. Bacteroids utilized endogenous reserves of poly-beta-hydroxybutyrate (PHB), which were depleted by 9.2% during a typical 5 h-long experiment. Stepwise increases in flow rate (increasing supply of O2) initially produced a drop in O2 demand and resulted in a rise in [O2] and a decline in N2 fixation. Subsequently, O2 demand rose (presumably because of increased mobilization of substrate from PHB) and [O2] declined to a low level. N2 fixation was fully restored, or even enhanced, within 15-20 min of establishment of a new, steady [O2]. This pattern of regulation by O2 supply was completely eliminated by adding low concentrations (20-50 microM) of oxidizable substrate (succinate, malate, ethanol) to the reaction medium. During endogenous activity, rates of CO2 evolution were proportional to, but less than, rates of O2 consumption up to 5.4 nmol O2 min-1 mg-1, above which CO2 evolution exceeded O2 consumption. These and other features of endogenous activity are discussed in relation to sustaining N2 fixation by nodules in vivo.


Low-level visual processing of biological motion.

Biological motion displays depict a moving human figure by means of just a few isolated points of light attached to the major joints of the body. Naive observers readily interpret the moving pattern of dots as representing a human figure, despite the complete absence of form cues. This paper reports a series of experiments which investigated the visual processes underlying the phenomenon. Results suggest that (i) the effect relies upon responses in low-level motion-detecting processes, which operate over short temporal and spatial intervals and respond to local modulations in image intensity; and (ii) the effect does not involve hierarchical visual analysis of motion components, nor does it require the presence of dots which move in rigid relation to each other. Instead, movements of the extremities are crucial. Data are inconsistent with current theoretical treatments.


DNA fingerprinting: parentage studies in natural populations and the importance of linkage analysis.

It has been suggested that a full linkage analysis is a prerequisite for confident paternity testing, by using DNA fingerprinting, in natural populations. These fears are based on a confusion between linkage and linkage disequilibrium and a misplaced assumption that linkage between bands will necessarily reduce the effective number of paternal-specific bands. Several methods for detecting linkage without resorting to the analysis of large sibships are considered, for example, by analysing half-sibships, by band-association, and by altering the experimental conditions used. Even if linkage is present, the magnitude of its effects are unlikely to undermine the accuracy of the technique, given the average levels of variability being detected. We conclude that the effects of linkage are only likely to present a problem when sample sizes are very small or when closely related individuals are being tested together.


Limited MHC polymorphism in the southern elephant seal: implications for MHC evolution and marine mammal population biology.

Genes of the major histocompatibility complex (MHC) are highly polymorphic in most terrestrial mammal populations so far studied. Exceptions to this are typically populations that lack genome-wide diversity. Here I show that two populations of the southern elephant seal (Mirounga leonina) have low DNA restriction fragment length polymorphism at MHC loci when compared with terrestrial mammals. Limited studies on MHC polymorphism in two cetacean species suggest this is a feature of marine mammal populations in general. MHC polymorphism is thought to be maintained by balancing selection, and several types of disease-based and reproductive-based mechanisms have been proposed. For the three marine mammal species examined, the low MHC polymorphism cannot be explained by low genome-wide diversity, or by any reproductive-based selection pressure. It can, however, be explained by diminished exposure to pathogenic selection pressure compared with terrestrial mammals. Reduced exposure to pathogens would also mean that marine mammal populations may be susceptible to occasional pathogen-induced mass mortalities.


Cerebral visual motion blindness: transitory akinetopsia induced by transcranial magnetic stimulation of human area V5.

The perception of visual motion can be selectively and reversibly compromised by transcranial magnetic stimulation (TMS) of a small region of cortex, roughly 1 cm in diameter and corresponding in position to human area V5. The reversible inactivation of a small and specialized visual area which receives its predominant input from area V1 and sends a powerful return (re-entrant) input back to it allowed us to study for the first time the backward influence of area V5 on area V1. In contrast to the complete and temporary visual motion blindness which occurs during stimulation of V5, a less-prominent interference with the perception of visual motion occurs at 70-80 ms after the onset of the visual stimulus when TMS is applied to V1. Because V5 is critical for the perception of coherent motion, and because an intact re-entry of signals from V5 to V1 is essential for the conscious perception of visual motion, the results obtained with stimulation of V1 must be caused by a disruption of the re-entrant signals from V5 to V1.


Reversal of female mate choice by copying in the guppy (Poecilia reticulata).

Ever since Fisher (1958) formalized models of sexual selection, female mate choice has been assumed to be a genetically determined trait. Females, however, may also use social cues to select mates. One such cue might be the mate choice of conspecifics. Here we report the first direct evidence that a female's preference for a particular male can in fact be reversed by social cues. In our experiments using the Trinidadian guppy (Poecilia reticulata), this reversal was mediated by mate-copying opportunities, such that a female (the 'focal' female) is given the opportunity to choose between two males, followed by a period in which she observes a second female (the 'model' female) displaying a preference for the male she herself did not prefer initially. When allowed to choose between the same males a second time, compared with control tests, a significant proportion of focal females reversed their mate choice and copied the preference of the model female. These results provide strong evidence for the role of non-genetic factors in sexual selection and underlie the need for new models of sexual selection that explicitly incorporate both genetic and cultural aspects of mate choice.


Is clutch size in birds affected by environmental conditions during growth?

Only environmental conditions occurring at the time of breeding have been shown to affect clutch size in birds, even though conditions experienced during growth are known to affect body size or egg size. We show here that environmental conditions experienced during early life can affect clutch size in captive zebra finches (Taeniopygia guttata) and wild great tits (Parus major). Not only do factors outside the immediate breeding season affect clutch size, but clutch size control mechanism is permanently influenced by conditions experienced during ontogeny.


Functional importance of a highly elastic ligament on the mammalian diaphragm.

The diaphragm of mammals is a musculotendinous dome separating the thoracic and abdominal cavities. With no skeletal elements to stretch it, the diaphragm has the problem of positioning its muscle fibres at a length appropriate for the onset of an inspiratory contraction. This is achieved through a negative intrapleural pressure, resulting from the opposing elastic recoil of the ribcage and lungs, which sucks the diaphragm into the thorax and extends the muscle fibres. A consequence of this negative pressure is that the diaphragm muscle is under tension when inactive during expiration. This is an unusual condition for skeletal muscles, which can suffer irreversible changes when stretched to long length, or they may respond by growing longer. We now describe a highly elastic and resilient diaphragmatic ligament which sets a sarcomere length enabling the muscle to use its full operating range, reduces stress on the diaphragm muscle fibres, and assists shortening of the diaphragm muscle at the onset of inspiration by means of elastic recoil.


17 alpha,20 beta-dihydroxy-4-pregnen-3-one 20-sulphate is a potent odorant in precocious male Atlantic salmon (Salmo salar L.) parr which have been pre-exposed to the urine of ovulated females.

Electrophysiological recordings from the olfactory epithelium have shown that 17 alpha,20 beta-dihydroxy-4-pregnen-3-one 20-sulphate (17,20 beta-P-sulphate; a conjugate of the oocyte-maturation-inducing steroid in teleosts) is a potent odorant in precocious male Atlantic salmon (Salmo salar L.) parr. However, the olfactory epithelium of these fish only appeared to be responsive to the steroid after stimulation with the urine of ovulated female Atlantic salmon. Immature fish did not respond at any time. Stimulation with urine from immature and precocious male Atlantic salmon parr did not make the olfactory epithelium of precocious male salmon parr responsive to the steroid. 17,20 beta-P-sulphate was found in the urines of ovulated females, precocious male parr and mature male Atlantic salmon. The findings are discussed in relation to the possible role of 17,20 beta-P-sulphate in the physiology of Atlantic salmon.


Anterograde and retrograde effects of synapse formation on calcium currents and neurite outgrowth in cultured leech neurons.

The aim of our experiments has been to analyse how formation of chemical synapses affects the distribution of calcium (Ca2+) currents and neurite outgrowth of leech Retzius cells. Previous results showed that Ca2+ currents measured in the initial process or 'stump' of postsynaptic cells were significantly smaller than those in corresponding sites on presynaptic neurons. In the present experiments, neurons were plated together in close apposition as pairs or as triads, with the tip of one Retzius cell touching the soma of another. Ca2+ currents from selected areas of the neuronal surfaces were measured by loose-patch recording before and after the formation of chemically mediated synaptic connections, which developed in about 8 h. With three cells arranged in a row, the last of the series, which was purely postsynaptic (i.e. with no target), also showed a dramatic reduction in Ca2+ currents in its initial segment, compared with the currents seen in either the first cell (purely presynaptic) or the second cell of the chain (which was both postsynaptic to the first cell and presynaptic to the third). This suggests that retrograde as well as anterograde effects on Ca2+ currents occurred as a result of synapse formation: the Ca2+ currents in the middle cell did not decrease although a synapse had been formed on it. To test for additional consequences of synapse formation, neurite outgrowth was measured in postsynaptic cells and in single cells plated on an extract of extracellular matrix containing laminin (ECM-laminin). After 48 h, the total length of neuritic outgrowth in postsynaptic cells was only about one third of that in single cells.(ABSTRACT TRUNCATED AT 250 WORDS)


A model of bipedal locomotion on compliant legs.

Simple mathematical models capable of walking or running are used to compare the merits of bipedal gaits. Stride length, duty factor (the fraction of the stride, for which the foot is on the ground) and the pattern of force on the ground are varied, and the optimum gait is deemed to be the one that minimizes the positive work that the muscles must perform, per unit distance travelled. Even the simplest model, whose legs have neither mass nor elastic compliance, predicts the changes of duty factor and force pattern that people make as they increase their speed of walking. It predicts a sudden change to running at a critical speed, but this is much faster than the speed at which people make the change. When elastic compliance is incorporated in the model, unnaturally fast walking becomes uncompetitive. However, a slow run with very brief foot contact becomes the optimum gait at low speeds, at which people would walk, unless severe energy dissipation occurs in the compliance. A model whose legs have mass as well as elastic compliance predicts well the relationship between speed and stride length in human walking.


Suppression of superoxide release from human monocytes by somatostatin-related peptides.

Somatostatin and octreotide share with vasoactive intestinal peptide the property of having an inhibitory effect on leukocyte functions. While there are studies reporting the inhibitory effect of the latter on respiratory burst in human monocytes, no such reports are available about similar inhibitory effects of the former. The aim of the present study was to investigate such effects of somatostatin and octreotide on human monocytes. Release of superoxide anion from monocytes was measured by superoxide dismutase-inhibitable reduction of cytochrome c in vitro. Somatostatin 1-14, somatostatin 1-28 and octreotide inhibited release of superoxide anion from stimulated monocytes. Formylpeptide-stimulated reduction of cytochrome c was inhibited by 1 mumol/l of octreotide and somatostatin 1-14 by about 50% and 35%, respectively. The effect was dose-dependent with half-maximal effective peptide concentrations at about 10 nmol/l. Somatostatin 1-28, which is the major form found in circulating plasma, also antagonized formylpeptide-stimulated respiratory burst activity; when directly compared to the effect of 1 mumol/l of somatostatin 1-14, somatostatin 1-28 was significantly more active (P less than 0.05). Our observations suggest that somatostatin-related peptides have a regulatory role in oxygen radical metabolism and a mediator role in the neuro-immune axis.


Insertion and internalization of vasoactive intestinal peptide (VIP) receptors in murine CD4 T lymphocytes.

The specific binding of vasoactive intestinal peptide (VIP) to murine lymphocytes was investigated. CD4 T cells from mesenteric lymph nodes (MLN) bound more 125I-VIP than did unseparated MLN lymphocytes at 37 degrees C, but not at 4 degrees C. The differences between the amount of 125I-VIP bound by the CD4 T cells and unseparated MLN lymphocytes at 37 degrees C depended upon a difference in the amount of the ligand that was internalized by the cells. The rate of insertion of unoccupied VIP receptors from the cytoplasm into the cell membrane (370 receptors/cell/min), the rate constants for internalization of ligand occupied VIP receptors (0.55 min-1) and unoccupied VIP receptors (0.11 min-1), and the rate constant for the elimination of internalized VIP (0.07 min-1) by CD4 T cells were evaluated. These results provide new understanding of the behaviour of VIP receptors on lymphocytes and indicate a mechanism by which CD4 T lymphocytes can homologously regulate their surface expression of VIP receptors in the presence of ambient VIP.


Anxiolytic-like effect of neuropeptide Y (NPY), but not other peptides in an operant conflict test.

The peptide messengers neuropeptide Y (NPY), growth hormone-releasing hormone (GHRH), atrial natriuretic peptide (ANP) and beta-endorphin (BEND) were tested in an animal model of anxiety, the Geller-Seifter conflict test. Rats were subjected to a multiple schedule consisting of three components: in the first component, lever-pressing produced food-reward ('unpunished responding'). The second component was a time-out period, during which lever-pressing had no consequences. During the third component, lever-pressing produced food-reward, but was also punished by an incremental foot-shock ('punished responding'). After establishing a stable baseline of both unpunished and punished responding, animals were injected with various doses of NPY, GHRH, ANP, BEND, or with saline into the lateral cerebral ventricle, and testing was repeated. While changes in unpunished responding can reflect alterations in performance factors or motivational strength, increases in punished responding have previously been shown to be highly specific for anxiety-reducing drugs, such as the benzodiazepines. NPY markedly and dose-dependently increased punished responding. A smaller increase of unpunished responding was also seen. These results add further support to the hypothesis that NPY may be an endogenous anxiolytic. GHRH, ANP and END did not affect punished responding.


Polyamines are involved in retinoic acid-mediated induction of tissue transglutaminase in human peripheral blood monocytes.

The differentiation of human peripheral blood monocytes (HPBM) into macrophages, when cultured in vitro, has been associated with an increase in the expression of tissue transglutaminase (TGc). Retinoic acid (RA) addition to 5-day-old cultured monocytes, 36 h later induced about 5-folds increase of TGc content. The preliminary exposure of cultured monocytes to alpha-difluoromethylornithine (DFMO) significantly reduced TGc induction caused by RA. DFMO alone does not induce significant changes in the time-course of TGc activity. In cultured monocytes exposed to DFMO, putrescine and spermidine, but not spermine were significantly depleted. The supplementation of putrescine (1 mM) or spermidine (0.5 mM) to culture medium reversed the inhibiting effect of DFMO on RA-mediated induction of TGc. However, the addition of polyamines in the absence of RA or DFMO did not mimic the induction of TGc by RA. We conclude that TGc induction by RA during in vitro maturation of monocytes to macrophages may be modulated by polyamine availability.


TNF alpha-mediated tissue damage in mouse footpads primed with mycobacterial preparations.

Tissue sites involved in certain types of inflammation become sensitive to the destructive effect of a subsequent injection of tumour necrosis factor alpha (TNF alpha). To try to further delineate the cascade of effector and regulatory events controlling this activity, a new model is described and its main properties characterized. C57BL/GrFa mice received mycobacterial products subcutaneously in the footpads. Recombinant TNF alpha was injected 24 h later into the same sites. To assess the tissue-destructive effect of TNF alpha in these "primed" footpads, swelling and haemoglobin content of injected footpads were measured, 16 h and 20 h respectively after the injection of TNF alpha. When loaded with either Escherichia coli LPS (10 micrograms) or Mycobacterium vaccae soluble sonicate (17 micrograms), footpads were reactive to the subsequent injection of 1 microgram recombinant TNF alpha, as assessed by both swelling and haemoglobin content. When C57BL/GrFa mice received 10(9) autoclaved M. vaccae subcutaneously in the back 10 days before the footpad was "primed" with soluble M. vaccae sonicate, the destructive effect of TNF alpha was significantly enhanced, becoming 5-10-fold greater than that seen in sites "primed" with an optimal dose of LPS. This higher reactivity was abrogated by a single dose of anti-CD4 given just before the injection of TNF alpha. This local reactivity to TNF alpha of skin sites loaded with mycobacterial products is compared to the local LPS-dependent Shwartzman reaction, and the relevance of this assay as a model with which to delineate the mechanisms of tissue damage in tuberculosis is discussed.


The treatment of the restless leg syndrome with or without periodic leg movements in sleep.

There are presently three main treatments for restless leg syndrome-periodic leg movements in sleep (RLS-PLMS). The benzodiazepines (especially clonazepam) are considered by most clinicians to be the treatment of choice in mild cases, especially in young subjects. In our experience, however, L-dopa and bromocriptine are more effective treatments, although no controlled studies have ever been conducted to compare their therapeutic benefits and the side effects of benzodiazepines and dopaminergic drugs. The use of opioids should be restricted to patients who have severe symptoms and who fail to respond to benzodiazepines or L-dopa. Propoxyphene was found less effective than L-dopa in decreasing PLMS, but some patients resistant to L-dopa may exhibit a masked therapeutic response to opioids. However, there is currently no method to predict the response to any treatment modality.


The beneficial effects of one treatment session and recording of nightmares on chronic nightmare sufferers.

Twenty subjects with chronic nightmares for 17.2 years mean duration were randomly divided into two groups: Rehearsal and Recording. At inception, subjects in both groups were instructed to write down their nightmares for one month. The Recording group received no other intervention. Rehearsal subjects received a single treatment group session teaching an imagery rehearsal technique to reduce nightmare frequency. At inception and three months follow-up, both groups were compared for nightmare frequency and for self-rated distress with scales (Symptom Checklist and Symptom Questionnaire) measuring anxiety, depression, hostility, somatization and total distress. Nightmare frequency decreased significantly in both groups: Rehearsal group-7.2 per month to 2.0 per month (72% reduction) (p < 0.006); Recording group-9.4 per month to 5.0 per month (47% reduction) (p < 0.02). There were no statistically significant differences in the nightmare frequency reductions between groups. All anxiety, depression, somatization, hostility and total distress scores decreased substantially in the Rehearsal group. Most changes were significant. Changes in the Recording group were inconsistent and not significant. Two brief case histories are presented.


Long-term double-blind study on the influence of dietary fibres on faecal bile acid excretion in juvenile ulcerative colitis.

A double-blind cross-over long-term trial (18 months) with randomized supplementation with wheat fibre or ispaghula for two periods of six months, separated by a six-month wash-out period with placebo, was performed in ten patients with juvenile ulcerative colitis to study the effect on faecal bile acid (BA) excretion. All patients were in remission since 0.5-2 years and orally treated with sulphasalazine. The average intake of either fibres was 16 g day-1. Faecal samples were collected (72 h) before and after each fibre period. Faecal water were prepared by centrifugation of faeces at 15,000 g for 2 h. BA in faeces and faecal water were studied using capillary column gas-liquid chromatography-mass spectrometry. Faecal excretion of total BA were not significantly changed by the two fibres. Supplementation with wheat fibre, but not with ispaghula, decreased the faecal concentration of total BA by 43% (p < 0.05), unconjugated BA by 41% (p < 0.01), and taurine conjugated BA by 58% (p < 0.05). Addition of wheat fibre decreased the concentration of chenodeoxycholic acid by 66% (p < 0.05) and isomers of cholic acid by 51% (p < 0.05) in faeces. The mean faecal water concentration of taurine-conjugated BA decreased by 55% when wheat fibre was added (p < 0.05) and the concentration of isomers of deoxycholic acid increased by 39% when ispaghula was supplemented (p < 0.05). The ratio isomeric deoxycholic acid to deoxycholic acid in faecal water increased significantly when wheat fibre was added (p < 0.05). The percentage distribution of secondary and ketonic BA was not influenced by the dietary fibre supplementation. The concentration of BA in faeces and faecal water decreased only by wheat fibre, suggesting that it is superior in obtaining an affect on faecal BA concentration.


Induction of mucosal and systemic immunity to a recombinant simian immunodeficiency viral protein.

Heterosexual transmission through the cervico-vaginal mucosa is the principal route of human immunodeficiency virus (HIV) infection in Africa and is increasing in the United States and Europe. Vaginal immunization with simian immunodeficiency virus (SIV) had not yet been studied in nonhuman primates. Immune responses in macaques were investigated by stimulation of the genital and gut-associated lymphoid tissue with a recombinant, particulate SIV antigen. Vaginal, followed by oral, administration of the vaccine elicited three types of immunity: (i) gag protein p27-specific, secretory immunoglobulin A (IgA) and immunoglobulin G (IgG) in the vaginal fluid, (ii) specific CD4+ T cell proliferation and helper function in B cell p27-specific IgA synthesis in the genital lymph nodes, and (iii) specific serum IgA and IgG, with CD4+ T cell proliferative and helper functions in the circulating blood.


Roles of SWI1, SWI2, and SWI3 proteins for transcriptional enhancement by steroid receptors.

The SWI1, SWI2, and SWI3 proteins, which are required for regulated transcription of numerous yeast genes, were found also to be essential for rat glucocorticoid receptor function in yeast; the receptor failed to activate transcription in strains with mutations in the SWI1, SWI2, or SWI3 genes. Certain mutations in genes encoding components of chromatin, identified as suppressors of swi mutations, partially relieved the SWI- requirement for receptor function. Immunoprecipitation of glucocorticoid receptor derivatives from wild-type (SWI+) yeast extracts coprecipitated the SWI3 protein; such receptor-SWI3 complexes were not detected in swi1- or swi2- mutant strains, implying that a complex of multiple SWI proteins may associate with the receptor. Prior incubation of a Drosophila embryo transcription extract with the yeast SWI3-specific antibody inhibited receptor function in vitro whereas the antibody had no effect if added after initiation complex formation. Thus, positive regulation by the glucocorticoid receptor in vivo and in vitro appears to require its interaction, at an early step, with one or more SWI proteins.


Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line.

The doxorubicin-selected lung cancer cell line H69AR is resistant to many chemotherapeutic agents. However, like most tumor samples from individuals with this disease, it does not overexpress P-glycoprotein, a transmembrane transport protein that is dependent on adenosine triphosphate (ATP) and is associated with multidrug resistance. Complementary DNA (cDNA) clones corresponding to messenger RNAs (mRNAs) overexpressed in H69AR cells were isolated. One cDNA hybridized to an mRNA of 7.8 to 8.2 kilobases that was 100- to 200-fold more expressed in H69AR cells relative to drug-sensitive parental H69 cells. Overexpression was associated with amplification of the cognate gene located on chromosome 16 at band p13.1. Reversion to drug sensitivity was associated with loss of gene amplification and a marked decrease in mRNA expression. The mRNA encodes a member of the ATP-binding cassette transmembrane transporter superfamily.


Thermal stability comparison of purified empty and peptide-filled forms of a class I MHC molecule.

A secreted form of a class I major histocompatibility complex (MHC) molecule was denatured and renatured in vitro in the absence of peptide. The resulting empty class I heterodimer was immunologically reactive and structurally similar to a heterodimer renatured in the presence of an appropriate restricted peptide. Thermal stability profiles indicated that the two forms of heterodimer differed in their resistance to denaturation by heat but that a significant portion of the empty class I heterodimers had a native conformation at physiological temperatures. Free energies calculated from these data gave a direct measure of the stabilization of the class I MHC molecule that resulted from peptide binding.


[Effect of opioid peptides on progesterone production by rat luteal cells in vitro].

The effect of exogenous opioid peptides on progesterone production by incubated rat luteal cells was studied. beta-endorphin (beta-EP) stimulated progesterone production in a dose-dependant manner (10(-8)-10(-6) mol/L); dynorphin exhibited a stimulatory effect only at 10(-6) mol/L, while Met-enkephalin had no substantial effect at dose from 10(-10)-10(-6) mol/L. mu-opioid receptor agonists DAGO and morphine also stimulated progesterone production. The stimulatory actions of beta-EP, DAGO and morphine were reversed completely by naloxone. On account of the fact that the beta-EP level in rat plasma is lower than that in the ovary, it seemed that beta-EP may be an intra-ovarian luteotrophic factor and be involved in the regulation of progesterone production. This action of beta-EP may be mediated by mu-opioid subtype receptors.


[Effects of electrical lesioning of hippocampal CA3 region and anterior commissura hippocampi fornix on plasma insulin level and islet cells in the rat].

Bilateral electrical lesioning of the hippocampal CA3 region (HCA3-EL) or anterior commissura hippocampi (ACHF-EL) caused marked elevations in plasma basal levels of insulin. 2 weeks later, fasting blood glucose levels were also augmented with decreased glucose tolerance. In contrast, the secretory response of pancreatic B cells to glucose stimulation was markedly enhanced. Following intravenous glucose tolerance test (IVGTT), the relative amounts of glucagon-like and insulin-like immunoreactants were reduced in the pancreatic islets of both HCA3-EL and ACHF-EL rats in comparison with the controls. In the HCA3-EL group, the relative amounts of somatostatin-like immunoreactants and gross numbers of such immunostained cells in islets were also decreased as compared with the control. No difference was seen in pancreatic-polypeptide-like immunoreactivities as assessed by immunohistochemistry plus microphotometry method. The above results suggest strongly that HCA3 and ACHF exert a tonic inhibitory action on the insulin secretion in the rat.


Effect of H2 antagonists on outcome of simple closure for perforated duodenal ulcer.

The treatment of perforated duodenal ulcer is controversial. Since the advent of H2 antagonists, the number of ulcer operations has declined tremendously. We wanted to find out if the addition of a H2 antagonist after simple closure of a perforated duodenal ulcer would change the outcome and therefore reviewed 46 patients treated in this fashion. Our results show that this is a safe and effective way of treating patients with perforated duodenal ulcer.


Role of pancreatoduodenectomy in the management of primary duodenal wall gastrinomas in patients with Zollinger-Ellison syndrome.

The role of pancreatoduodenectomy in the surgical management of duodenal wall gastrinomas (DWGs) has not been well established. Recently DWGs have been recognized with increasing frequency, and several reports have emphasized that pancreatoduodenectomy can now be performed with a low operative morbidity and mortality for other conditions. The purpose of this study was to determine the indications, safety, and efficacy of pancreatoduodenectomy in the treatment of DWGs. Forty-five patients with Zollinger-Ellison syndrome were evaluated and surgically treated between 1960 and 1991; 15 (33%) of these had primary DWGs. Pancreatoduodenectomy was considered necessary for curative resection in six patients. Two of these patients had multiple gastrinomas as part of multiple endocrine neoplasia type 1 syndrome and underwent tumor excisions and total gastrectomy; both died of tumor-related complications (survival, 8.5 and 12 years). A third patient did not consent to pancreatoduodenectomy, underwent total gastrectomy and tumor excision, and also died of tumor-related complications (survival, 10 years). The remaining three patients underwent pancreatoduodenectomy. After pancreatoduodenectomies were performed, these three patients became and remained eugastrinemic with normal results from secretin stimulation tests (mean follow-up, 7.5 years). In patients with DWGs and Zollinger-Ellison syndrome, pancreatoduodenectomy should be considered the treatment of choice whenever complete tumor excision is not possible by a lesser procedure.


Gastrin-dependent inhibitory effects of octreotide on the genesis of gastric ECLomas.

The efficacy of octreotide in the regulation of endocrine tumor secretion and symptomatology has been well documented. Its effects on neuroendocrine tumor generation and cell proliferation are less well understood. The purpose of this study was to determine if blockade of somatostatin receptors by octreotide would alter gastrin levels and influence enterochromaffin-like (ECL) cell proliferation. The well-established gastric ECLoma model of the rodent, mastomys, was used. Animals received loxtidine (1 mg/kg/day), an irreversible H2 blocker, and subcutaneous slow release, octreotide pellet implants (150 or 300 micrograms/kg/day) or placebo pellets for a 4-month period. Control parameters for gastric mucosal thickness, plasma gastrin level, ECL cell density, and bromodeoxyuridine-positive cells were 517 +/- 20 microns, 46.1 +/- 11.4 pmol/L, 7.4 +/- 0.9 cells/visual field, and 13.8 +/- 2.6 cells/visual field, respectively. After loxtidine-placebo treatment all values were significantly increased (p < 0.05; 883 +/- 70 microns, 192.8 +/- 10.6 pmol/L, 97 +/- 16.2 cells/visual field, and 51.7 +/- 19.2 cells/visual field, respectively). High dose octreotide significantly inhibited all parameters (668 +/- 3.5 microns, 66.2 +/- 20.5 pmol/L, 37.0 +/- 8.0 cells/visual field, and 10.9 +/- 2.2 cells/visual field; p < 0.05). Low dose octreotide failed to significantly inhibit ECL cell density mucosal thickness, or cell proliferation. Irreversible H2 receptor blockade results in hypergastrinemia and ECL cell tumor generation. Hypergastrinemia, ECL cell hyperplasia, and cell proliferation are significantly inhibited by in vivo blockade of somatostatin receptors by administration of octreotide.


Long-term follow-up of a large North American kindred with multiple endocrine neoplasia type 2A.

Sixty-one patients with multiple endocrine neoplasia (MEN) type 2A (27 men; 34 women; mean age, 39 years) from a single kindred (76 affected, 49 at risk) were followed 1 month to 33 years (median, 14 years) after diagnosis for disease expression. Peripheral basal and pentagastrin-stimulated calcitonin, parathyroid hormone, and calcium levels were measured in 60 patients, 58 of whom had undergone previous thyroidectomy; the calcitonin concentration in four patients was concomitantly determined in the hepatic and internal jugular veins. Biochemical or radiographic screening for pheochromocytoma was performed in 58 patients. Recurrence of medullary thyroid carcinoma (MTC) developed in nineteen (33%) of 58 patients after they underwent thyroidectomies. In 14 of 19 patients regional metastases were inapparent at initial operation, and lymphadenectomy was not undertaken. Three patients underwent neck reexploration with removal of micrometastases after selective venous studies were performed. One patient, 33 years of age, died of MTC, and two patients who refused thyroidectomy are alive at 76 and 83 years of age. Fifteen patients are alive with disease 8 to 33 years after they underwent thyroidectomies. All patients identified by prospective screening remain pentagastrin negative. Pheochromocytoma developed in six patients (10%), and four patients have Hirschsprung's disease. Hyperparathyroidism was present in seven patients and did not occur in those with minimal MTC. These observations suggest that (1) the course of MTC in MEN 2A is highly variable, (2) early treatment of C-cell disease can be curative, (3) routine lymphadenectomy for occult micrometastases may be necessary for cure of MTC, and (4) the hyperparathyroidism of MEN 2A may not be a primary genetic event.


Hyperparathyroidism in multiple endocrine neoplasia syndrome.

We analyzed clinical findings and results of parathyroidectomy in 42 patients treated from 1936 to 1988 at the University of California, San Francisco (UCSF) for primary hyperparathyroidism and multiple endocrine neoplasia (MEN) syndrome to document results of parathyroidectomy and reasons for failed parathyroid operations. Of the 42 patients (38 had MEN 1 syndrome; 4 had MEN 2A syndrome), 40 patients were treated surgically: 29 had initial parathyroidectomy at UCSF; 11 were referred to UCSF because of MEN syndrome. Eight of these 11 patients required reoperation for persistent or recurrent hyperparathyroidism. Patients with hyperplasia were treated with subtotal parathyroidectomy; the glands of those patients with solitary or double adenomas were removed with or without biopsy of the normal appearing glands. Overall, in seven (50%) of 14 patients with hyperplasia, three (16%) of 19 patients with solitary adenoma, and one (14%) of seven patients with double adenomas, recurrent or persistent hyperparathyroidism developed. Failure in patients with hyperplasia was due to missed supernumerary glands (13%) and missed ectopic glands (33%). Failure occurred in patients with solitary (three patients) or double (one patient) parathyroid tumors because of unrecognized hyperplasia. None of the four patient with MEN 2A syndrome had persistent or recurrent disease, but hypoparathyroidism developed in one patient; hypoparathyroidism developed in three patients with MEN 1 syndrome. These data suggest that although many patients with primary hyperparathyroidism and MEN syndrome have multiple abnormal parathyroid glands, two populations of patients exist; one population has solitary or double adenomas and recurrence is uncommon, whereas the other population of patients has hyperplasia and persistent or recurrent disease is common.


P-glycoprotein expression and multidrug resistance in adrenocortical carcinoma.

The response of adrenocortical carcinoma (ACC) to adjuvant chemotherapy has been disappointing with no significant impact on survival. The normal adrenal cortex has very high levels of P-glycoprotein, an energy-dependent efflux pump of a variety of structurally unrelated chemotherapeutic agents. P-glycoprotein has been implicated as a cause of multidrug resistance in a variety of neoplasms. The purpose of this study was to evaluate P-glycoprotein expression in ACC. Eleven patients with ACC had paraffin-embedded tumor evaluated for P-glycoprotein expression. These were analyzed by immunohistochemistry assay with a battery of four anti-P-glycoprotein antibodies (MRK-16, JSB-1, UIC-2, MDR). All eleven cases showed intense, predominantly membrane immunoreactivity for P-glycoprotein. In 10 of the cases, most tumor cells were immunoreactive with at least three antibodies, and six of 11 cases were positive for all four antibodies. In this small series no correlation existed between P-glycoprotein expression and tumor grade, stage of disease, or survival. All 11 cases of ACC studied showed P-glycoprotein expression, which was similar to the normal adrenal cortex. This possible mechanism of multidrug resistance may help explain the significant chemoresistance seen in ACC.


[The comparative efficacy of slow-acting (basic) preparations in psoriatic arthritis].

Overall 126 patients with verified and clinically active psoriatic arthritis (PA) were subjected to a randomized study of the efficacy of chrisanolum (Chr), sulfasalicylic drugs (SSD) (sulfasalazine and salazopyridazine) and methotrexate (MT) as compared to nonsteroidal anti-inflammatory drugs (NSAID). The treatment that lasted for a year was completed by 77 patients: in the group on NSAID, by 31, on Chr by 15, on SSD by 15, and on MT by 16. In the remainder, the treatment was discontinued because of side effects. The best clinical effect was recorded in patients on Chr. The improvement was observed in 73% of the patients, with a significant effect being attained in 60%. In the groups on SSD and MT, the improvement was observed in 80 and 69%, respectively. However, noticeable improvement was only recorded in 20 and 19%. SSD turned out more effective than MT. in the group on NAID, the improvement was ascertained but in 35% of the patients, with noticeable one being attained in 6%. According to Pearson's criterion chi 2, the results of the treatment with NAID alone were less potent than in the group given Chr (p < 0.001) and SSD (p < 0.05). The differences between the effect of the treatment with NAID and MT appeared nonsignificant (p > 0.1). Therefore, according to the diminution of the clinical efficacy in PA, the basic drugs may be distributed in the following way: Chr, SSD, MT. The side-effects in the group on NAID were. recorded in 37% of cases, in the group on Chr in 53%, on SSD in 33%, and on MT in 55%. This means that SSD were tolerated best of all.


Modulation of calciuria by cadmium pretreatment in rats with cadmium-metallothionein-induced nephrotoxicity.

One group of male Wistar rats (Group B) was pretreated by a daily subcutaneous injection with CdCl2 during 5 days with increasing doses (0.5, 1, 1, 2 and 2 mg Cd/kg). Another group of rats (Group A) was daily given normal saline subcutaneously for 5 days. On the second day after the last injection, a single s.c. injection of 109Cd-metallothionein (CdMT, 0.4 mg Cd/kg) was given to each animal in both groups. Urinary calcium, protein, metallothionein (MT), N-acetyl-beta-D-glucosaminidase (NAG) and gamma glutamyltransferase (gamma-GT) were measured. In Group A, calciuria, proteinuria, metallothioneinuria and enzymuria was induced by CdMT. Calciuria reached a peak during 0-6 h after the administration of CdMT, thus appearing earlier than other effects. Enzymuria was displayed at 6-12 h for gamma-GT and 12-24 h for NAG. A prominent increase of proteinuria appeared at 24-48 h after the challenge of CdMT. In Group B, no significant increase of urinary calcium, protein, or NAG was observed after the CdMT injection and urinary gamma-GT was only slightly elevated, thus demonstrating the protective action of pretreatment. This study demonstrates for the first time that calciuria, one of the signs of cadmium nephrotoxicity, can be prevented by cadmium pretreatment. Urinary MT increased slightly during the 4-5 days of CdCl2 pretreatment. This is in accordance with previous observations that cadmium pretreatment induces new synthesis of MT which is likely to constitute the background for the resistance to the CdMT challenge to the kidney.


Neurochemical effects in rats following gestational exposure to styrene.

Styrene was evaluated for the reproductive effects of pregnant rats and the neurochemical effects in the offspring of rats exposed during gestation. Pregnant Wistar rats were exposed to 0, 50, or 300 ppm styrene for 6 h/day during days 7 to 21 of gestation. No significant differences in the number of offspring delivered were observed between the exposed and control groups. Body weights at 1 day of age of the offspring whose mothers were exposed to styrene were significantly lower than those of the control group. Although, there were neither statistically significant differences of protein contents nor brain weights among styrene-exposed and their control offsprings of rats, analyses of neurotransmitter studies showed dose-dependent decreases of neuroamines, especially 5-HT (serotonin) and its metabolite 5HIAA (5-hydroxyindoleacetic acid) in the newborn offspring of styrene-exposed rats. The results suggest that gestational exposure to styrene at these concentrations does not produce apparent reproductive toxicity but affects the body weight of pups and causes lowering of the neurotransmitter levels in the brain.


Receptor classes and the transmitter-gated ion channels.

Transmitter-gated channels, which can be selective for cations or for anions, form an important class among the membrane receptors responsible for signal transduction. Thirteen principal types of these channels can now be recognized and most of these are available for analysis in recombinant form. It is instructive to contrast their characteristic structural features with those of the two other primary classes of the signal-transducing receptors of membranes.


Human cytomegalovirus in the pancreas of patients with type 2 diabetes: is there a relation to clinical features, mRNA and protein expression of insulin, somatostatin, and MHC class II?

Human cytomegalovirus (HCMV) was recently demonstrated in the pancreas of about half the patients with type 2 diabetes mellitus in the absence of mumps, rubella or Coxsackie B virus. The present study addresses the question as to whether type 2 diabetes with an HCMV-positive pancreas differs from those with HCMV-negative pancreases with respect to age, sex, treatment, duration of disease, volume densities of B-cells and D-cells, mRNA levels of insulin and somatostatin, islet amyloid peptide deposits and major histocompatibility complex (MHC) class I and class II gene transcription, and protein expression. HCMV-positive type 2 diabetic patients showed a tendency towards a shorter duration of disease and significantly increased levels of MHC class II on RNA. In addition, expression of MHC class II product (HLA-DR) was identified in duct epithelial cells and/or islet cells in 9 diabetic pancreases and in 2 non-diabetic glands. No MHC class I expression could be detected. No other clinical differences between HCMV-positive and HCMV-negative glands were found. All 10 HCMV-positive diabetics showed a strong expression of MHC class II mRNA in the pancreas. By immunocytochemistry, 4 of 10 demonstrated expression on the islets; three of ten also expressed MHC DR beta on ductal cells. This finding might be related to the viral infection, as only 2 of the 9 HCMV-negative patients were HLA-DR beta positive and none of the non-diabetic controls showed increased levels of MHC class II mRNA. These data suggest that HCMV infection in the pancreas is associated with type 2 diabetes. However, no conclusions as to a role of this virus in the aetiopathology of type 2 diabetes can be drawn at present.


Immunohistochemical localization of c-erbB-2 protein and epidermal growth factor receptor in normal surface epithelium, surface inclusion cysts, and common epithelial tumours of the ovary.

The c-erbB-2 (HER-2/neu) protein is a membrane glycoprotein growth factor receptor showing molecular homology with the epidermal growth factor receptor (EGFR). We examined the immunohistochemical reactivity of monoclonal antibodies against both of these proteins in normal surface epithelium, surface inclusion cysts, and common epithelial tumours of the ovary. The ovarian tumours were classified as benign (16), borderline malignant (2), and malignant (19). Normal surface ovarian epithelium was weakly positive for both c-erbB-2 protein and EGFR. In surface inclusion cysts, however, the epithelial cells lining the lumen exhibited stronger staining for c-erbB-2 protein, but no staining for EGFR. All 16 benign ovarian tumours and the 2 borderline malignant ovarian tumours were positive for c-erbB-2 protein and negative for EGFR. Of the ovarian carcinomas, 13 of the 19 (68.4%) were positive for c-erbB-2 protein and negative for EGFR, while 4 showed positivity for both c-erbB-2 protein and EGFR. Two cases were negative for both proteins. Expression of both c-erbB-2 protein and EGFR was found in endometrioid carcinoma with squamous differentiation and in clinically advanced poorly differentiated serous carcinomas. Expression of c-erbB-2 protein appears to be increased and that of EGFR is reduced in the early stage of epithelial ovarian oncogenesis. The expression of EGFR with c-erbB-2 protein in ovarian carcinoma is related both to histological differentiation and/or advanced clinical stage.


Storage of glycoprotein in NCTR-Balb/C mouse. Lectin histochemistry, and biochemical studies.

A strain of Balb/C mice carrying a lysosomal storage disorder exhibits metabolic and phenotypic abnormalities similar to patients with sphingomyelin-cholesterol lipidoses type II (i.e., Niemann-Pick C and D). Their foamy cells, which belong to the reticuloendothelial system, stained intensely by periodate-Schiff (PAS) reagent and were resistant to predigestion with diastase. To identify the chemical nature of the PAS-positive storage material, we applied lectin histochemistry and biochemical methods. Paraffin embedded sections, and delipidated frozen tissue sections, were treated with biotinylated lectins and localized with avidin-biotin-peroxidase complex. Araldite-embedded semithin sections were incubated with biotinylated lectins followed by avidin-gold and were enhanced with silver. By both histochemical methods the affected foamy cells stained positively as follows: Concanavalia ensiformis agglutinin, Datura stramonium agglutinin, Griffonia simplicifolia-I, Lens culinaris agglutinin, peanut agglutinin, Ricinus communis agglutinin-I, wheat germ agglutinin (WGA), and succinylated-WGA. Biochemical analysis of liver extracts complemented the histochemical data and demonstrated accumulation of glycoproteins containing polylactosaminoglycans in affected mice. Our findings indicate that the storage material in NCTR-Balb/C mice is heterogeneous. The lipids that are extracted by organic solvents during the histologic preparations mask the occurrence of polylactosaminoglycan containing glycoproteins in native frozen sections.


Modulation of KB and A431 cell adhesion by epidermal growth inhibitory pentapeptide.

A pentapaptide, pyroGlu-Glu-Asp-Ser-GlyOH (EPP), isolated from mouse epidermis, inhibits mitoses and enhances differentiation in primary cultures and in transformed mouse epidermal cells in vitro (Elgjo et al. 1986 b). The present work demonstrates that EPP also modulates the adhesiveness of two human tumour cell lines (KB and A431) of epidermal origin to uncoated plastic and to plastic coated with fibrinogen or collagen type 1. The adhesion modulatory effect of EPP was observed over a broad range of concentrations (10(-12)-10(-6) M), and depended on the substrate the cells were growing on. Thus, when cells were seeded on plastic or collagen, the attachment to the substrate was suppressed at the highest concentrations of EPP, and stimulated at the lowest ones. The opposite concentration-response pattern was observed when fibrinogen was used as substrate.


Influence of estrogen and progesterone on the induction of mammary carcinomas by 7,12-dimethylbenz(a)anthracene in ovariectomized rats.

The influence of estrogen on mammary carcinogenesis was studied in female Sprague-Dawley rats ovariectomized at the age of 36 days and given injections of 17 beta-estradiol (group I:0, II:1, III:10, IV:100, V:1000 micrograms/2 days) between the ages of 36 and 250 days and a single oral dose of 20 mg of 7,12-dimethylbenz(a)anthracene (DMBA) at the age of 50 days. No palpable mammary carcinomas were detected up to the age of 135 days. At the age of 135 days, each group was divided into two subgroups (a and b). Rats of the second subgroup (Ib, IIb, IIIb, IVb and Vb) were given additional injections of progesterone (P; 4 mg/2 days) between the ages of 135 and 250 days. At the age of 250 days, the incidence of mammary carcinoma was significantly higher in rats from group IIIb than in groups Ib and IIIa, and that in group IVa was also higher than in group Ia. The incidence in group IVb was significantly lower than in group IVa. The carcinomas in group IIIb were palpable papillo-tubular adenocarcinomas and those in group IVa were secretory micro-adenocarcinomas. These results indicate that the induction of mammary carcinomas by DMBA is totally inhibited by ovariectomy and/or high doses of estrogen, but that mammary carcinomas are initiated by DMBA under hormonal conditions in which suitable levels of estrogen are present. They also suggest that the growth of DMBA-induced mammary carcinomas in the rats from group III were accelerated by additional injections of P and that those in rats from group IV were inhibited by additional P.(ABSTRACT TRUNCATED AT 250 WORDS)


Promoting effect of ovariectomy on hepatocellular tumorigenesis induced in mice by 3'-methyl-4-dimethylaminoazobenzene.

The treatment of female C57BL/6 x DS-F1 mice with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) at 10, 12, 14, 16 and 18 days of age resulted in the development of hepatocellular adenomatous nodules after 10 months of age. Ovariectomy in these mice at 1 month of age hastened the development of adenomatous nodules, which then first appeared at 8 months of age. The incidence of adenomatous nodules in females ovariectomized at the age of 1 month was much higher than that in intact females of the same age. These results showed that the ovaries exerted a suppressive effect on the development of adenomatous nodules. To determine the time from which the ovaries exert this suppressive effect, females were ovariectomized at 4, 6, 8, and 10 months of age, and the incidences of adenomatous nodules were compared at 10 and 12 months of age. Delayed ovariectomy after 8 months of age did decrease the incidence of adenomatous nodules at 10 and 12 months of age, but ovariectomy after 4 and 6 months of age did not. When the incidence of adenomatous nodules in females ovariectomized at 10 months of age was examined over the subsequent 6 months, it became significantly higher after 14 months of age compared with that in intact females. The results show that the ovariectomy has the promoting effect on the development of adenomatous nodules in the liver induced by 3'-Me-DAB after 6 months of age.


Visualization and rapid quantification of autoradiographic labeling in scanning electron microscopy applied to localization of receptor sites on the surface of whole cells.

The use of backscattered electron imaging (BEI) as a routine procedure for examining autoradiographic reactions in scanning electron microscopy (SEM) is described. This technique allows the determination of the number of receptor sites occupied by 125I-epidermal growth factor (EGF) on whole cells. The effect of 1.25 dihydroxyvitamin D3 (1,25 (OH)2D3) on the number of epidermal growth factor receptors (EGF-R) in the BT 20 human mammary carcinoma cell line (which is known to possess a very high number of EGF-R) has been evaluated with this method. To compare the silver grain density over the cells (controls and 1,25 (OH)2D3-treated cells) we used an image analysis system Quantimet 900. The results were compared with those of a previous study using transmission electron microscopy (TEM). This study confirmed the results obtained with TEM and showed the even distribution of receptors sites on a single cell and a large difference in the number of receptor sites from one cell to another. The use of BEI to visualize the autoradiographic reaction in SEM allowed the examination of a large surface with good contrast and resolution and eliminated artefacts not corresponding to the silver grains. It gave new information not delivered by quantitative TEM autoradiography and was easier and faster to use. The efficient use of SEM autoradiography combined with BEI could facilitate whole area distribution mapping of radioactive labeling.