Identification of ancillary fim genes affecting fimA expression in Salmonella typhimurium.

Regulation of the gene, fimA, encoding the major fimbrial subunit of S. typhimurium S6704 was examined by using a lambda fimA-lacZ lysogen. Transformation of the lambda fimA-lacZ lysogen with various derivatives of the recombinant plasmid that encodes type 1 fimbrial expression, pISF101, indicated that two regions of this plasmid alter beta-galactosidase production. One plasmid is a deletion resulting in the loss of a 28-kDa polypeptide downstream of fimA, while the other plasmid encodes a 24- and a 27-kDa polypeptide. Northern (RNA) blot analyses indicated that the steady-state fimA mRNA levels of these transformants were high. In addition, phenotypic expression of type 1 fimbriae by agar-grown cultures is observed only in those transformants bearing plasmids which show increased beta-galactosidase and fimA mRNA levels.


Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers.

DNA heterogeneity among members of the genus Brucella was demonstrated with the arbitrarily primed polymerase chain reaction (AP-PCR). Simple, reproducible genomic fingerprints from DNA of 25 different Brucella strains were generated with five arbitrarily chosen primers, alone and in pairs, with the PCR. Reaction conditions were optimized for each primer. Several DNA segments were amplified in each sample with all of the primers. PCR products that are not shared among all strains act as polymorphic markers. Polymorphism was apparent for each primer. The Brucella strains can be distinguished according to the banding patterns of their amplified DNA on agarose gels, and the differences can be diagnostic of specific strains. To determine genetic relatedness among the Brucella strains, similarity coefficients were calculated. Statistical analysis of the similarity coefficients revealed the degrees of relatedness among strains of the genus Brucella.


Dopaminergic inhibition of DNA synthesis in pituitary tumor cells is associated with phosphotyrosine phosphatase activity.

Dopaminergic D2 receptor agonists, such as bromocriptine, are potent anti-proliferative agents in the treatment of human pituitary adenomas. We have reproduced the anti-proliferative effect of dopamine in an established pituitary cell line stably transfected with the rat D2 dopamine receptor cDNA. We found that dopaminergic inhibition of DNA synthesis parallels the stimulation of a phosphotyrosine phosphatase activity. Both actions are blocked by pertussis toxin and by the phosphotyrosine phosphatase inhibitor, vanadate. We suggest that the anti-proliferative action of dopamine is mediated, at least in part, by the dopaminergic stimulation of a phosphotyrosine phosphatase.


Characterization and developmental expression of chick aortic lysyl oxidase.

The complete primary structure of chick lysyl oxidase was determined by recombinant DNA techniques. The nucleotide sequence of contiguous chick lysyl oxidase cDNA clones contained an open reading frame of 1260 bases which encodes a predicted protein of 420 amino acid residues (48,150 Da). In comparison to the deduced primary structure of rat lysyl oxidase, the chick enzyme is larger in size and exhibits a strong conservation of sequence within the latter two thirds of the molecule (92% identity) and a high degree of divergence in the first 150 amino acid residues (60% identity allowing for several insertions in both sequences). The developmental steady-state levels of lysyl oxidase mRNA together with the mRNAs encoding two of the enzyme's substrates (tropoelastin and type I collagen) increased between 8 and 16 days of embryonic development. Although levels of lysyl oxidase mRNA increased during aortic embryogenesis, the specific activity of the enzyme remained fairly constant suggesting that lysyl oxidase activity increases in direct proportion to total protein synthesis and cell number. In situ hybridization showed that the spatial expressions of lysyl oxidase and tropoelastin transcripts differ suggesting that the enzyme and substrate genes are differentially regulated within the cells of the arterial wall.


Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone.

We expressed human MDR1 cDNA isolated from the human adrenal gland in porcine LLC-PK1 cells. A highly polarized epithelium formed by LLC-GA5-COL300 cells that expressed human P-glycoprotein specifically on the apical surface showed a multidrug-resistant phenotype and had 8.3-, 3.4-, and 6.5-fold higher net basal to apical transport of 3H-labeled cortisol, aldosterone, and dexamethasone, respectively, compared with host cells. But progesterone was not transported, although it inhibited azidopine photoaffinity labeling of human P-glycoprotein and increased the sensitivity of multidrug-resistant cells to vinblastine. An excess of progesterone inhibited the transepithelial transport of cortisol by P-glycoprotein. These results suggest that cortisol and aldosterone are physiological substrates for P-glycoprotein in the human adrenal cortex and that substances that efficiently bind to P-glycoprotein are not necessarily transported by P-glycoprotein.


Inorganic pyrophosphatase from bovine retinal rod outer segments.

Rod outer segments from bovine retina contain a higher level of intracellular inorganic pyrophosphatase (EC 3.6.1.1) activity than has been found in any other mammalian tissue; the specific activity in extracts of soluble outer segment proteins is more than 6-fold higher than in extracts from bovine liver and more than 24-fold higher than in skeletal muscle extracts. This high activity may be necessary to keep inorganic pyrophosphate concentrations low in the face of the high rates of pyrophosphate production that accompany the cGMP flux driving phototransduction. We have begun to explore the role of inorganic pyrophosphatase in photoreceptor cGMP metabolism by 1) studying the kinetic properties of this enzyme and its interactions with divalent metal ions and anionic inhibitors, 2) purifying it and studying its size and subunit composition, and 3) examining the effects of pyrophosphate on rod outer segment guanylyl cyclase. Km for magnesium pyrophosphate was 0.9-1.5 microM, and the purified enzyme hydrolyzed > 885 mumol of PPi min-1 mg-1. The enzyme appears to be a homodimer of 36-kilodalton subunits when analyzed by gel electrophoresis and density gradient centrifugation, implying that kcat = 10(3) s-1, and kcat/Km = 0.7-1 x 10(9) M-1 s-1. The enzyme was inhibited by Ca2+ at submicromolar levels: 28% inhibition was observed at 138 nM [Ca2+], and 53% inhibition at 700 nM [Ca2+]. Imidodiphosphate acted as a competitive inhibitor, with Ki = 1.2 microM, and fluoride inhibited half-maximally approximately 20 microM. Inhibition studies on rod outer segment guanylyl cyclase confirmed previous reports that pyrophosphate inhibits guanylyl cyclase, suggesting an essential role for inorganic pyrophosphatase in maintaining cGMP metabolism.


Characterization of a stable, reactivatable complex between chaperonin 60 and mitochondrial rhodanese.

Efficient formation of the cpn60-rhodanese complex can be achieved by mixing unfolded rhodanese with excess cpn60 at low temperature. By employing these conditions, a stable and highly reactivatable complex is formed. The complex is not formed when native enzyme is used. Concentrations of NaCl, as high as 0.75 M, do not have any effect on the formation or disruption of the binary complex. cpn60-bound rhodanese contains an exposed hydrophobic surface, as detected by the binding of the fluorescent reporter, 1-anilinonaphthalene-8-sulfonic acid. The intrinsic fluorescence of cpn60-bound rhodanese reports that the average tryptophan is in an intermediate environment between that found in unfolded and native states. This form of rhodanese has an accessibility to quenching by acrylamide or iodide that is intermediate between the unfolded and native forms of the enzyme. Protease susceptibility studies show that rhodanese bound to cpn60 exhibits a trypsin digestion pattern similar to the native enzyme, although it is more rapidly proteolyzed. The results suggest that the conformation of cpn60-bound rhodanese resembles a native-like conformation, but with increased flexibility. Further, only intact rhodanese or enzyme lacking its N-terminal sequence can interact with cpn60 and form a stable binary complex. The protein fragment corresponding to the rhodanese N-terminal sequence did not form part of a stable complex with cpn60.


Long-term sensitization training in Aplysia leads to an increase in the expression of BiP, the major protein chaperon of the ER.

Long-term memory for sensitization of the gill- and siphon-withdrawal reflexes in Aplysia californica requires RNA and protein synthesis. These long-term behavioral changes are accompanied by long-term facilitation of the synaptic connections between the gill and siphon sensory and motor neurons, which are similarly dependent on transcription and translation. In addition to showing an increase in over-all protein synthesis, long-term facilitation is associated with changes in the expression of specific early, intermediate, and late proteins, and with the growth of new synaptic connections between the sensory and motor neurons of the reflex. We previously focused on early proteins and have identified four proteins as members of the immunoglobulin family of cell adhesion molecules related to NCAM and fasciclin II. We have now cloned the cDNA corresponding to one of the late proteins, and identified it as the Aplysia homolog of BiP, an ER resident protein involved in the folding and assembly of secretory and membrane proteins. Behavioral training increases the steady-state level of BiP mRNA in the sensory neurons. The increase in the synthesis of BiP protein is first detected 3 h after the onset of facilitation, when the increase in overall protein synthesis reaches its peak and the formation of new synaptic terminals becomes apparent. These findings suggest that the chaperon function of BiP might serve to fold proteins and assemble protein complexes necessary for the structural changes characteristic of long-term memory.


Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy.

The distribution of poly(A) RNA has been visualized in single cells using high-resolution fluorescent in situ hybridization. Digital imaging microscopy was used to quantitate the signal in various cellular compartments. Most of the poly(A) signal remained associated with the cellular filament systems after solubilization of membranes with Triton, dissociation of ribosomes with puromycin, and digestion of non-poly(A) RNA with ribonuclease A and T1. The actin filaments were shown to be the predominant cellular structural elements associating with the poly(A) because low doses of cytochalasin released about two-thirds of the poly(A). An approach to assess the extent of colocalization of two images was devised using in situ hybridization to poly(A) in combination with probes for ribosomes, membranes, or F-actin. Digital imaging microscopy showed that most poly(A) spatially distributes most significantly with ribosomes, slightly less with F-actin, and least of all with membranes. The results suggest a mechanism for anchoring (and perhaps moving) much of the cellular mRNA utilizing the interaction between actin filaments and poly(A).


Human aortic endothelial cells express the type I but not the type II receptor for interleukin-1 (IL-1).

Endothelial cells (EC) are very responsive to the proinflammatory cytokine interleukin-1 (IL-1). EC are induced by IL-1 to secrete chemotactic factors and to increase expression of cell surface adhesion molecules leading to increased leukocyte adhesion. Activated EC further contribute to the inflammatory response by secreting additional cytokines. IL-1 interacts with EC through high-affinity cell-surface receptors. However, the low number of receptors present on EC has made characterization difficult. Further, recent evidence has suggested diversity in the responses of EC from different regions of the vascular system. Interested in the effect of IL-1 on early atherosclerotic lesion formation, we have characterized the IL-1 receptors on human aortic endothelial cells (HAEC). Using a direct binding assay, we found that HAEC have 1,000-3,000 IL-1 receptors per cell and bind IL-1 alpha with a Kd of 3.5 x 10(-10) M. We found that a monoclonal antibody specific for the type I receptor completely blocks IL-1 alpha binding. The blocking antibody also completely inhibits the IL-1 induced increase in intracellular adhesion molecule 1 (ICAM-1) expression by HAEC. Using solution hybridization and ribonuclease protection with an antisense probe, a sensitive method for detection of low abundance mRNA species we found that HAEC as well as human umbilical vein EC (HUVEC) have significant levels of mRNA for the type I IL-1 receptor. To test whether HAEC might also contain transcripts for the type II IL-1 receptor, we compared levels of mRNAs by polymerase chain reaction (PCR) amplification of cDNAs reverse-transcribed from total RNA. We found only transcripts for the type I receptor and not the type II receptor in HAEC. Based on this data, we conclude that aortic endothelial cells respond to IL-1 through the type I receptor.


Noradrenergic modulation of noxious heat-evoked fos-like immunoreactivity in the dorsal horn of the rat sacral spinal cord.

The tail-flick withdrawal reflex commonly is used to study spinal nociceptive mechanisms; noradrenergic agonists administered intrathecally inhibit the tail-flick reflex in a dose-dependent manner. The objectives of the present study were: (1) to use fos-like immunoreactivity as a marker for neuronal activity to examine the population of neurons in the spinal cord dorsal horn that are engaged by activation of nociceptive tail afferents, and (2) to determine whether fos-like immunoreactivity can be modulated by intrathecally administered alpha adrenoceptor agonists. Neurons demonstrating heat-evoked fos-like immunoreactivity were identified bilaterally in the sacral spinal cord in superficial and deep dorsal horn laminae. Heat-evoked fos-like immunoreactivity was inhibited dose-dependently by intrathecal norepinephrine (NE). The inhibition was attenuated significantly by: (1) phentolamine (PHEN), a nonselective alpha adrenoceptor antagonist; (2) yohimbine (YOH), an alpha-2 adrenoceptor antagonist; and (3) prazosin (PRAZ), an alpha-1 adrenoceptor antagonist. Thus, both spinal alpha-1 and alpha-2 adrenoceptors mediate the inhibition of heat-evoked fos-like immunoreactivity produced by intrathecal NE. ST-91, an alpha-2 adrenoceptor agonist, also inhibited significantly the expression of fos-like immunoreactivity; the inhibition was antagonized by YOH. In the absence of noxious heat, intrathecal NE dose-dependently evoked the expression of fos-like immunoreactivity in the superficial dorsal horn, which was antagonized by PHEN and PRAZ, but not by YOH, suggesting that the effect is mediated by spinal alpha-1 adrenoceptors.


Memory and naive CD4+ lymphocytes in multiple sclerosis.

Helper-inducer (CD29+CD4+) and suppressor-inducer (CD45RACD4+) T-cells have been recently renamed as memory and naive T-cells, respectively. We measured cells expressing these phenotypes in peripheral blood of 46 definite multiple sclerosis (MS) patients [32 relapsing-remitting (RR-MS), 14 secondary progressive (P-MS)] and controls. CD25+ (interleukin-2-receptor-positive) cells were also evaluated in the same groups of patients. RR-MS patients showed increased levels of CD29+CD4+ and CD25+ cells compared with controls. This finding was more evident in RR-MS patients during the attack than during the stable phase of the disease. In P-MS patients we found a reduction of CD45+CD4+ cells compared with either RR-MS patients or control subjects. Our results show that RR-MS and P-MS are characterized by two different T-cell subpopulations. This finding supports the hypothesis that during the evolution from RR-MS and P-MS changes occur in the immunological status of the patients.


Effect of modification of the basic residues of dynorphin A-(1-13) amide on kappa opioid receptor selectivity and opioid activity.

A series of dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues containing lysine or N epsilon-acetyllysine (Lys(Ac)) was prepared by solid-phase peptide synthesis and evaluated for opioid receptor affinity in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI). Substitutions were made at positions 6, 7, 9, 11, and 13, the basic amino acids in the C-terminus of the peptide, in order to assess the individual contributions of these residues to the kappa opioid receptor affinity and selectivity of Dyn A-(1-13)NH2. While substitutions of Lys(Ac) for Arg in position 6 did not affect kappa receptor affinity, it enhanced affinity for mu and delta receptors and therefore caused a loss of kappa receptor selectivity. When Lys(Ac) was substituted for Arg9, kappa opioid receptor affinity was enhanced and kappa receptor selectivity was retained. Replacement for Arg7, Lys11, or Lys13 by Lys(Ac) resulted in both decreased affinity and selectivity for kappa receptors. These results demonstrate the importance of Arg6 to the receptor selectivity profile of Dyn A-(1-13)NH2 and indicate that, of the five basic residues in the C-terminus, only Arg9 can be replaced by a nonbasic residue without substantial loss of kappa opioid receptor selectivity.


Prolactin bioassay and hyperprolactinemia.

The Nb2 rat lymphoma bioassay (BA) for prolactin (PRL) was performed in 26 subjects with hyperprolactinemia, 17 of whom had radiologic evidence of a pituitary adenoma. All subjects were treated with the long acting dopamine agonist CV 205-502. The radioimmunoassay (RIA) PRL significantly decreased with treatment but the BA/RIA PRL remained essentially the same, indicating that the relative bioactivity was unaffected.


Novel piperidine sigma receptor ligands as potential antipsychotic drugs.

sigma receptor ligands represent a new class of potential antipsychotic drugs. This paper presents the structure-activity relationships leading to novel disubstituted piperidine sigma ligands, which have little or no affinity for dopamine D2 receptors. Selectivity for sigma sites over dopamine D2 or serotonin 5-HT2 receptors appears to be governed by the chemical nature of the piperidine nitrogen substituent, its distance from the basic nitrogen, and its orientation relative to the other piperidine substituent. Several of these compounds have good oral potency in some animal models used to evaluate potential antipsychotic drugs. The N-cyclopropylmethyl ketones and ethers (e.g. 6i (DuP 734), 6q, 18a, and 18n) have the best in vivo potency. Compounds 6i (DuP 734) and 6q did not cause catalepsy in the rat, even at very high doses. On the basis of the pharmacology profiles of these sigma ligands, we propose these compounds may be effective antipsychotic drugs, which do not induce extrapyramidal side effects or tardive dyskinesia.


[The pattern of the expression of homeo box-containing genes in the mesenchyme of the mammalian limb bud as a coordinate network for subsequent morphogenetic processes].

Hypothesis is developed about mechanisms of determination of limb bud of mammals as a result of expression of homeo box containing genes (hox genes) of known up to date four hox families. Spatial pattern of the expression plays a role of coordinate system for subsequent processes of morphogenesis and differentiation of the limbs. Propositions of the hypothesis are realized in a computer dynamic model. The results of modeling are compared with known data on features of the expression of hox genes in the mesenchyme of developing limb buds in mammals and birds.


Synthesis and pharmacological characterization of 2-(4-chloro-3-hydroxyphenyl)ethylamine and N,N-dialkyl derivatives as dopamine receptor ligands.

2-(4-Chloro-3-hydroxyphenyl)ethylamine (4) and some derivatives were synthesized as dopamine (DA) receptor ligands. Amine 4 retains the dopaminergic pharmacophore 2-(3-hydroxyphenyl)-ethylamine, and the chlorine atom replaces the "para" hydroxyl group of DA. The derivatives 18a-e were obtained by introducing on the nitrogen of amine 4 the n-propyl and 2-phenylethyl or 3-phenylpropyl groups which can be accommodated by the D-2 receptor lipophilic sites 3C and pi 3, respectively. The affinity and selectivity of these compounds for D-1 and D-2 subtypes was determined in radioligand competition assays for the DA receptors of rat striatum membranes using [3H]SCH 23390 (D-1 selective) and [3H]spiperone (D-2 selective) as radioligands. The amine 4 shows about 7-fold lower affinity than DA for both sites and is not able to discriminate between the two subtypes of DA receptors. The introduction of two n-propyl groups (18a) on the nitrogen atom reduces by one-half and doubles the affinity for D-1 and D-2 binding sites, respectively. The substitution of an n-propyl group with different alkylphenyl groups, to give compounds 18b-e, increases the affinity for the D-2 subtype from 19-fold to 36-fold. These compounds have the same affinity at the D-2 site as the DA agonist N-n-propyl-N-(2-phenylethyl)-2-(3-hydroxyphenyl)-ethylamine (2a) and are about 20 times more selective than DA for this binding site. In the assay for D-2 receptor mediated inhibition of adenylate cyclase activity, all the tested compounds behaved as D-2 agonists; N-n-propyl-N-[2(4-hydroxyphenyl)ethyl]- (18d) and N-n-propyl-N-(2-phenyl-ethyl)-2-(4-chloro-3-hydroxyphenyl)ethylamine (18b) were more effective than DA or 2a. On the other hand, all compounds were less effective than DA in stimulation of adenylate cyclase activity in rat striatal homogenates, a kind of effect which is mediated by the D-1 subtype of DA receptors. These results suggest that the nitrogen substitution enhances the affinity and selectivity for the D-2 receptor. In the adenylate cyclase assay, the compounds behave as potent D-2 agonists.


Crystallization and preliminary crystallographic analysis of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

Aspartate-beta-semialdehyde dehydrogenase catalyzes the NADPH-mediated reductive dephosphorylation of beta-aspartylphosphate at a branch point in the biosynthesis of several amino acids. The enzyme from Escherichia coli has been crystallized by the vapor diffusion method from Tris buffer (pH 8.5) using polyethylene glycol 4000 as a precipitant. The crystals are orthorhombic and have the symmetry of space group P222(1), with unit cell dimensions of a = 177.8 A, b = 59.9 A, c = 118.65 A, and alpha = beta = gamma = 90 degrees. The dimensions and space group are indicative of two enzyme dimers (40 kDa per subunit) in the asymmetric unit. The crystals show strong diffraction, and a native data set has been collected to 2.5 A resolution.


The management of hyperthyroidism due to Graves' disease in the former USSR in 1991: results of a survey.

A survey of the current management of Graves' disease was performed in the USSR among members of All Union Endocrine Society. The questionnaire was based on the format used previously for a survey of members of European Thyroid Association. The aim of a similar survey in the USSR was to obtain a comprehensive pattern of management of Graves' disease in Soviet endocrinology clinics and to compare medical attitudes in the former USSR to those in other European countries. One hundred and twenty questionnaires were mailed with 55 returned (46%). The responses originated from 33 cities, representing major endocrinology centers of the former Soviet Republics. Initial diagnosis was conducted both in hospitals (55%) and ambulatory care settings (45%). Thyroid scintigraphy was requested by 42.3% of the respondents; a majority of them (90%) used 131I. Thyroid ultrasonography was performed in more than 50% of cases. Measurements of cholesterol, total T4 and T3 were the most frequent laboratory tests requested to confirm the diagnosis. For the treatment with ATD, methimazole was the exclusive choice (PTU is not currently in use in the USSR). Beta-blocking agents were prescribed by a majority of respondents. For the long term treatment, a combination procedure of MMI and thyroid hormones was clearly preferred by almost 3/4 of the respondents. A fixed period of treatment was preferred by 62% of the respondents, with a duration of therapy of 18-24 months. Surgery for treatment of the index patient was chosen by only 6%, and radioiodine by 3%. The number of responses was too limited to attempt any characterization of the two latter modalities.


[Flow cytometric techniques for measurement of proliferating cell nuclear antigen (PCNA)].

Flow cytometric techniques have been developed for measurement of proliferating cell nuclear antigen (PCNA), which allows studies on the proliferative capacity of cells and tissues. PCNA-DNA dual staining procedures, for both fixed and unfixed cells, and analytical method by FCM are presented. We recommend performing either the fixed or unfixed method. Our studies using the MCF7 human breast adenocarcinoma cell line and the A549 human lung squamous cell carcinoma cell line, in the exponential growth phase, revealed that both ethanol and acetone were satisfactory fixatives, in contrast to methanol and paraformaldehyde, and PI with a concentration of 25 micrograms/ml was most suitable compared with 50 and 100 micrograms/ml.


[Necessity and limitations of effectiveness of oral beta-stimulants, theophylline and oral antiallergic drugs in asthma treatment].

Oral beta-stimulants, theophylline and oral antiallergic drugs are frequently used for asthma treatment in Japan. However, inhaled beta-stimulants and inhaled steroids are becoming the first and second choice of drugs in asthma treatment in European countries, and these three categories of drugs are becoming third or fourth-line therapeutic choices. Accordingly, we investigated the effectiveness of these drugs, which is limited by their pharmacological actions and effects. As found in this study many cases of bronchial asthma cannot be well controlled by their agents, either alone or in combination. The necessity of these drugs for the treatment of chronic bronchial asthma was investigated. Substitution of inhaled beta-stimulant for oral beta-stimulant was possible in 8 of 15 cases. However, in 7 cases, substitution was impossible because of the patient's complaints, and it could not be concluded that oral beta-stimulants are necessary for asthma treatment. Theophylline was substituted by inhaled corticosteroids in 6 of 8 cases. Oral antiallergic drugs were substituted by inhaled corticosteroids in 5 of 6 cases. These results do not conclusively indicate that theophylline and oral antiallergic drugs are not necessary for asthma treatment.


[Assessment and management of acute severe asthma].

In the management of acute severe asthma it is very important to start the treatment as soon as possible, by appropriate evaluation of the physical status and signs of airflow obstruction. We propose a guideline to be used by patients with asthma, emergency car crews, physicians and nurses to evaluate the severity and to choose the appropriate management of acute asthma, including intubation and mechanical ventilation, by the assessment of clinical features, as well as blood gas analysis and pulmonary function test. Several researchers have demonstrated that the additional administration of aminophylline to inhaled or subcutaneous beta 2-agonist bronchodilator during the first 4 hours of an attack provides no additional benefit compared to the administration of beta 2-agonist alone. In our retrospective study of 68 episodes of acute severe asthma in the last 5 years at our institute, however, the additional administration of aminophylline with beta 2-agonists was clearly shown to be effective with infrequent minor side effects.


Randomised controlled trial of Doppler ultrasound screening of placental perfusion during pregnancy.

We have done a randomised controlled trial to assess the effect on primary management and outcome of routine doppler ultrasound examinations of the umbilical and uterine arteries during pregnancy. Over 9 months, 2600 women with singleton pregnancies were recruited from a general obstetric population. Of 2475 women who delivered in hospital after 20 weeks' gestation, 1246 had been allocated at random to receive standard antenatal care with routine doppler examinations. The first doppler ultrasound was done at 19-22 weeks' gestation, and thereafter examinations were monthly if the pregnancy was considered high risk (192) or once at 32 weeks if considered low risk (1054). The control group of 1229 women received standard, antenatal care without doppler ultrasonography. The study groups did not differ in number of antenatal admissions or cardiotocographs, gestational age at delivery, method of delivery, frequency of deliveries with fetal distress, or need for resuscitation or admission to the neonatal intensive care unit. More perinatal deaths occurred in the doppler group (17 vs 7, relative risk 2.4, 95% Cl 1.00-5.76), but only 1 of 11 normally formed stillbirths and none of the 4 normally formed neonatal deaths after 24 weeks' gestation had an abnormal umbilical-artery doppler examination. We did not demonstrate any improvement in neonatal outcome by routine doppler ultrasound screening of a general obstetric population.


Antinuclear autoantibodies in women with silicone breast implants.

Clinical syndromes resembling autoimmune diseases have been reported in women who have had breast augmentation procedures. To see whether there is a humoral immune response in these diseases that is similar to the immune response in their idiopathic counterparts, we assessed the immunological specificity of antinuclear antibodies (ANAs) and certain epidemiological features in 24 patients, all of whom (with 1 exception) had received silicone gel breast implants. ANA specificities were identified by indirect immunofluorescence, immunodiffusion, western blot analysis, and immunoprecipitation of radiolabelled intracellular proteins. Of 11 patients who had symptoms and signs that met criteria for defined autoimmune diseases, 7 had scleroderma or subsets of this disorder and the others had systemic lupus erythematosus, rheumatoid arthritis, or overlapping autoimmune diseases. High ANA titres were present in 10 of these 11 patients and the ANA specificities were similar to those found in the idiopathic forms of the corresponding autoimmune diseases. Trauma, with resultant rupture of implants, accelerated onset of symptoms. 13 other patients had autoimmune disorders of a less clearly defined nature and low titres of ANAs whose specificities could not be identified. ANAs are associated with the development of autoimmune complications in women with silicone breast implants. Further studies are needed to see whether this relation is one of cause and effect and whether ANAs might be early serological markers preceding development of autoimmune symptoms.


Effects of dual-chamber pacing with short atrioventricular delay in dilated cardiomyopathy.

Mitral or tricuspid regurgitation of long duration may so shorten the ventricular filling time in dilated cardiomyopathy that stroke volume is limited. We assessed the effects of changing the atrioventricular interval during temporary or permanent dual-chamber DDD pacing in twelve dilated cardiomyopathy patients with short ventricular filling times due to regurgitation. We measured ventricular filling time and cardiac output with doppler echocardiography and exercise capacity on a treadmill, at baseline and with the best atrioventricular delay during pacing. The durations of both mitral and tricuspid regurgitation were significantly shorter at the shorter atrioventricular interval (mean reductions 85 [95% CI 60-110] ms and 110 [75-150] ms, respectively; p < 0.001 for both). There were consequent increases in left-ventricular and right-ventricular filling times (65 [35-95] ms and 90 [60-120] ms, p < 0.001). For each 50 ms reduction in atrioventricular delay, left-ventricular filling time increased by 35 ms in six subjects with presystolic mitral regurgitation and right-ventricular filling time by 30 ms in nine subjects with presystolic tricuspid regurgitation. At the short atrioventricular interval, cardiac output was greater than baseline (by 1.1 [0.8-1.4] l/min, p < 0.01) and there were rises in exercise duration (104 [45-165] s, p < 0.05) and maximum oxygen consumption (2.1 [1.5-2.7] ml kg-1 min-1, p < 0.05). There was a decrease in the Likert visual analogue score of breathlessness at peak exercise (8.6 [SD 2.1] vs 4.9 [3.1], p < 0.01). Although from a small sample, these findings suggest that DDD pacing with a short atrioventricular delay may have therapeutic potential in patients with dilated cardiomyopathy, even in the absence of conventional indications for pacemaker implantation.


High frequency of antibodies to Mycoplasma penetrans in HIV-infected patients.

Mycoplasma penetrans, a novel mycoplasma isolated from HIV-1-infected patients with AIDS, has pathogenic properties associated with in-vivo virulence. Enzyme-linked immunosorbent assay and western blotting detected a more than 100 times higher frequency of antibodies to the mycoplasma in serum from HIV-1-infected patients with AIDS (40%) than from HIV-negative controls (0.3%). Serum from 20% of HIV-1-infected, symptom-free individuals also had M penetrans specific antibodies. The antibodies' major immunoreactivity was directed against P35 and P38, the two main lipid-associated membrane protein antigens of the organism. Patients attending sexually transmitted disease clinics had a low frequency of antibody (0.9%). None of 178 HIV-negative patients with different non-AIDS diseases, many associated with immune dysfunction and/or low white cell counts, tested positive for the antibodies. M penetrans, apparently not a commensal and not a simple opportunist, is uniquely associated with HIV-1 infection and AIDS.


Nonsense mutation of glucokinase gene in late-onset non-insulin-dependent diabetes mellitus.

A nonsense mutation at codon 186 in exon 5 of the gene for glucokinase, an enzyme important for glucose-induced insulin secretion, was identified in a Japanese patient with late-onset non-insulin-dependent diabetes mellitus (NIDDM). All affected members of her family were heterozygous for the mutation and had late-onset NIDDM or impaired glucose tolerance, whereas unaffected members showed normal glucose tolerance. The early insulin response to oral glucose was impaired in affected relatives, but was normal in those unaffected. These findings suggest that the glucokinase mutation raises the set-point of pancreatic beta cells for glucose-induced insulin secretion, leading to abnormal glucose tolerance in some patients with late-onset NIDDM.


Insemination of HIV-negative women with processed semen of HIV-positive partners.

Many HIV-discordant couples want to have children so much that they are willing to abandon condom-protected sexual intercourse irrespective of the risks. Previous testing in our laboratory showed that gradient centrifugation followed by a swim-up procedure effectively removed HIV-1-infected cells from the semen of HIV-seropositive men. 85 HIV-discordant couples were screened for fertility; 29 women were found suitable for a timed insemination course with the processed semen of their HIV-seropositive partner. None of the inseminated women seroconverted, and 17 pregnancies were achieved in 15 women. All 10 babies born to these mothers remain HIV seronegative. The findings may help in the counselling of such couples and also give them hope of having healthy babies.


Inherited idiopathic dilated cardiomyopathy with multiple deletions of mitochondrial DNA.

Idiopathic dilated cardiomyopathy (DCM) is often familial, but the pathogenetic mechanisms of DCM are unknown. We report a woman and her son who both died of DCM. The son's cardiac and skeletal muscles showed a high proportion of mitochondrial DNA (mtDNA) with multiple large deletions by Southern-blot hybridisation and polymerase chain reaction analyses. Amplification of the mother's cardiac mtDNA from 20-year-old paraffin-embedded sections showed that she also had deletions of mtDNA. These data suggest that a subgroup of inherited DCMs is associated with mtDNA mutations.


Creatinine clearance during cimetidine administration for measurement of glomerular filtration rate.

Creatinine clearance inaccurately estimates true glomerular filtration rate (GFR) because of tubular secretion of creatinine. We studied the ability of oral cimetidine, a blocker of tubular creatinine secretion, to improve the accuracy of measuring creatinine clearance. Clearances of inulin and endogenous creatinine were simultaneously measured in 16 patients with renal disease before administration of cimetidine and during 8 successive 3 h clearance periods with cimetidine 400 mg as priming dose followed by 200 mg every 3 h. At baseline, creatinine relative to inulin clearance (ClC/Cll) ranged from 1.14 to 2.27. With cimetidine, ClC/Cll approached unity in 8 patients (mean 1.02 [SD 0.03]), but considerably exceeded unity in 8 others (1.33 [0.14]). Plasma cimetidine/creatinine ratio was smaller in this second group, due to significantly higher renal clearance of cimetidine (333 [136] vs 165 [89] ml/min, p = 0.01). In a further study, cimetidine dose and, consequently plasma cimetidine concentration, was increased in 6 additional patients who had incomplete inhibited previously. This increased dose completely inhibited tubular creatinine secretion in the third until the sixth hour, so that creatinine clearance equalled GFR. Provided an adequate dose of cimetidine is given, 24 h creatinine clearance during administration of drug measures GFR accurately in patients with renal disease. However, because of the maximum daily dose of cimetidine that is advised, short clearance times (3 h) are recommended.


Phenotype and genotype heterogeneity in autosomal dominant polycystic kidney disease.

It is now clear that mutations of at least two genetic loci can lead to autosomal dominant polycystic kidney disease (ADPKD). We have compared the clinical features of ADPKD caused by mutations at the PKD1 locus (linked to the alpha-globin complex on chromosome 16) with those of disease not linked to the locus (non-PKD1). We identified 18 families (285 affected members) with mutations at PKD1 and 5 families (49 affected individuals) in which involvement of this locus could be dismissed. Non-PKD1 patients lived longer than PKD1 patients (median survival 71.5 vs 56.0 years), had a lower risk of progressing to renal failure (odds ratio 0.35, 95% CI 0.13-0.92), were less likely to have hypertension (odds ratio adjusted for age and family of origin 0.29, 0.11-0.80), were diagnosed at an older age (median 69.1 vs 44.8 years), and had fewer renal cysts at the time of diagnosis. Although most of the PKD1 families were ascertained through clinics treating patients with renal impairment, no non-PKD1 family was identified through this source. Non-PKD1 ADPKD has a much milder phenotype than that linked to PKD1. Partly as a result of this difference in severity, the reported prevalence of this genotype is probably an underestimate.


Early versus delayed neonatal administration of a synthetic surfactant--the judgment of OSIRIS. The OSIRIS Collaborative Group (open study of infants at high risk of or with respiratory insufficiency--the role of surfactant.

Although exogenous surfactants are of known efficacy in the prevention and treatment of respiratory distress syndrome (RDS), questions remain about the best regimens. During 1990-91, 6774 babies were recruited to an international multicentre trial to assess when administration of Exosurf, a synthetic surfactant, should be started and how often it should be given. The clinical outcome is known for 6757 (99.7%) infants. 2690 babies, judged to be at high risk of RDS when less than 2 hours of age, were randomly allocated to either early administration or delayed selective administration; 96% versus 73% received surfactant, at median ages of 118 and 182 min. The risk of death or dependence on extra oxygen at the expected date of delivery was 16% (95% CI 25% to 7%) lower among infants allocated early administration. Early administration was also associated with a 32% lower risk of pneumothorax. These 2690 infants were further randomised in a factorial design to either two doses of surfactant 12 hours apart, or the option of third and fourth doses at 12-36 hour intervals if signs of RDS persisted or recurred. 4067 other infants who later developed RDS were also recruited to this comparison, giving a total of 3376 infants allocated up-to-four doses (of whom, 45% received more than two) and 3381 allocated two doses. The outcome was similar in the two groups in respect of death, long-term oxygen dependence, and other major morbidity, even in secondary analyses restricted to infants who met the criteria for additional administration. There were more reports of poorly tolerated administration in the up-to-four doses group but no clear increase in serious morbidity, such as pulmonary haemorrhage. The OSIRIS trial suggests that early administration of surfactant to an estimated 32 babies, when compared with treatment of established RDS, would prevent 1 baby from dying and another from being dependent on extra oxygen long-term, but would entail the additional use of surfactant in 8 of these babies. It provides no evidence that a regimen including the option of third and fourth doses when signs of RDS persist or recur is clinically superior to a regimen of two doses.


Prognostic significance of cytoplasmic p53 oncoprotein in colorectal adenocarcinoma.

Mutation of p53, a tumour-suppressor protein, leads to overexpression of the protein and loss of its tumour-suppressive properties. In some tumours (eg, breast) p53 expression is related to well-known prognostic factors, but findings in colorectal tumours are equivocal. We have used the polyclonal antibody CM1 to investigate nuclear and cytoplasmic p53 expression in colorectal tumours and to assess their relations with prognosis. Of 293 colorectal adenocarcinomas, 71 (24%) showed p53 expression in the nucleus alone, 30 (10%) showed p53 in the cytoplasm alone, and 43 (15%) showed both nuclear and cytoplasmic expression. Nuclear p53 expression showed no relation with survival or Dukes' stage of the tumour. However, the frequency of cytoplasmic expression increased with advancing Dukes' stage (chi 2 for trend 11.18, 1 df, p = 0.0008) and cytoplasmic expression was associated with poor survival (rate ratio 2.3 [95% CI 1.6-3.3], p < 0.0001). Among tumours of Dukes' stage A-C, cytoplasmic expression showed prognostic value independent of nuclear staining, grade of differentiation, and Dukes' stage (2.3 [1.4-3.7], p = 0.0021). We conclude that cytoplasmic expression of p53 may be a useful biological indicator of prognosis in colorectal adenocarcinoma.


Effect of extended cold ischaemia with UW solution on graft function after liver transplantation.

Studies in animals on the use of UW solution in liver transplantation have shown an inverse relation between cold ischaemia time (CIT) and graft function. There are few clinical data on this relation in human beings. We have investigated the effect of extended cold ischaemia in a prospective study. We assessed early graft function and subsequent outcome for 306 consecutive elective liver transplantations; for analyses, grafts were grouped according to CIT (< 12 h group A, > or = 12 h group B), since a preliminary study identified 12 h as a significant cut-off point. Initial graft function was better in group A than group B, as shown by maximum alanine aminotransferase activity (mean 623 [805] vs 946 [1148], p = 0.02), bile production on days 1-3 (p < 0.05), maximum serum bilirubin by day 10 (206 [166] vs 244 [163] mumol/l, p = 0.04), and frequencies of primary non-function (1 [0.4%] vs 4 [7%], p = 0.006) and hepatocyte necrosis on routine biopsy sample after reperfusion (18% vs 31%, p = 0.04). Long-term outcome was also better in group A than group B; graft and patient survival rates were higher and fewer retransplantations were needed. These findings suggest that cold ischaemia in UW solution for longer than 12 h is a risk factor for graft function and patient survival. We recommend that the limit of the safe CIT be reconsidered.


Diabetes mellitus associated with a pathogenic point mutation in mitochondrial DNA.

Family studies of diabetes mellitus (DM) show that patients are more likely to have affected mothers than affected fathers. Since the inheritance of mitochondrial (mtDNA), unlike nuclear DNA, is exclusively maternal, could it be that defect(s) in mtDNA account for some cases of DM? Such defects have been associated with rare neurological syndromes, in some of which DM has been an accompanying feature. We have looked for glucose intolerance and for a previously known point mutation of mtDNA in a family, some of whose members have a multisystem disorder with DM but not neurological involvement. DNA samples were obtained from fourteen family members. The point mutation (affecting position 3243 in the tRNA leucine mitochondrial gene) was found in all three diabetic patients and post mortem tissues in the proband; it was also found in seven offspring of female patients. It was not found in the two children of the male proband. The contribution of this mutation to DM in general is not known but clinicians ought to be aware of the possibility, especially in families with multisystem disease and maternal transmission.


Meningococcal septicaemia in a C6-deficient patient and effects of plasma transfusion on lipopolysaccharide release.

Patients whose blood is deficient in the terminal component of complement have an increased susceptibility to meningococcal infection. However, mortality from meningococcal infection is lower in these patients than in immunocompetent subjects. We studied a C6-deficient patient with meningococcal sepsis who received fresh frozen plasma (FFP). The patient's initial plasma endotoxin, C6, and terminal-complement-complex concentrations were low, but rose sharply after treatment with FFP. Samples of the patient's serum taken shortly after admission did not cause endotoxin release from Escherichia coli J5 in vitro, but endotoxin-releasing activity was restored in serum samples taken after infusion of FFP. It is possible that C6-deficient patients have reduced mortality from meningococcal infection because their serum cannot cause acute release of endotoxin from the invading organism and extensive tissue damage is thus avoided.


Haemoglobin in gut lavage fluid as a measure of gastrointestinal blood loss.

To detect and measure occult gastrointestinal bleeding, we have measured haemoglobin concentrations (by HemoQuant) in the clear fluid obtained after whole-gut lavage. In subjects with healthy gastrointestinal tracts, lavage-fluid haemoglobin concentrations were 0.5-5.1 mg/L, equivalent to daily blood loss of 0.1-1.1 mL. High concentrations were found for patients with colorectal cancer, severe diverticular disease, and rectal varices, in seven of sixteen patients with active inflammatory bowel disease, and in four patients with iron-deficiency anaemia thought to be due to gastrointestinal bleeding. In these four patients, estimated blood loss ranged from 2.6-24.5 mL per day. This method could have various research and clinical applications.


Usefulness of consensus conferences: the case of albumin.

The usefulness of consensus conferences has been questioned. We have assessed the impact of a 1989 consensus conference about the treatment of hypovolaemia on prescribing practice at our hospital. Data available from the blood bank enabled us to follow albumin use and costs by department between 1987 and 1991. Medical journals, direct mail, and meetings were used to disseminate the recommendations of the consensus conference, which were that the only indications for albumin are massive haemorrhage, plasmapheresis, massive hypoalbuminaemia, and volume expansion in pregnancy; for hypovolaemia caused by septic shock or vasoplegia, fluid gelatins and crystalloids should be used. In the year after the conference, and in subsequent years, albumin use and costs were 40% lower than before the conference, even though total medical expenditure continued to rise and numbers of patients admitted did not change. We believe that consensus conferences can be a useful means of improving medical practice. The features that ensured success in this programme were the small number of prescribers, the homogeneous setting, and the frequent restatement of the recommendations.


Geographic distribution of physicians in Japan.

In the 1970s, Japan's physician manpower policy was to increase the number of medical students and medical schools to ease the difficulties caused by shortage of doctors and their maldistribution. 34 medical schools were established during this time, which eventually doubled the number of newly certificated physicians from about 4000 to about 8000 per year by the mid 1980s. To examine the success of this policy, we analysed the numbers of physicians in relation to the population in all municipal bodies (n = 3268) in Japan. Between 1980 and 1990, the number of practising physicians increased by about 37%, and the ratio of physicians per 100,000 population increased from 127 to 165 throughout the country. However, analyses by the Lorenz curve and the Gini coefficient indicated that the inequality in physician distribution did not improve. While municipal bodies with a population over 30,000 gained proportionally more physicians, most communities with fewer than 10,000 residents showed little gain. Our findings have important implications for policy changes now being considered by the government to plan the future supply of physician manpower in Japan. A policy that will alleviate physician maldistribution also needs to be devised.


Analysis of the transcriptional activity of the hut promoter in Bacillus subtilis and identification of a cis-acting regulatory region associated with catabolite repression downstream from the site of transcription.

Levels of transcripts initiated at a hut promoter in Bacillus subtilis were analysed. The addition of histidine to the culture medium increased the level of the transcript sixfold. In the presence of histidine and glucose together, the level of the transcript was reduced to the level in the absence of induction. Furthermore, addition of a mixture of 16 amino acids to cultures of induced cells and of catabolite-repressed cells decreased levels of the transcript 16-fold and 2.6-fold, respectively. Thus, it appears that at least three regulatory mechanisms associated with induction, catabolite repression, and amino acid repression, control the transcriptional activity of the hut promoter. Expression of the hut promoter-lacZ fusions that contained various regions of the hutP gene and deletion analysis of the hutP region revealed a cis-acting sequence associated with catabolite repression that was located between positions +204 and +231 or around position +203.


Mono- and bi-phasic Salmonella typhi: genetic homogeneity and distinguishing characteristics.

Several lines of evidence indicate a relatively low genetic heterogeneity in the natural Salmonella typhi population. However, some S. typhi isolates found in Indonesia express, instead of the usual fliC-d flagellin gene, a different flagellar gene fliC-j. In addition, Indonesian strains may have a second flagellar antigen fliC-z66. We have previously suggested, on the basis of the flagellar antigen constitution, that S. typhi evolved in an isolated human population in Indonesia. In order to test this hypothesis, we have gathered S. typhi isolates from around the world and tested the genetic heterogeneity among them. In general, polymorphism was greater in isolates from the Far East, as was indicated by Southern hybridizations with rDNA and fliC DNA probes. Gene fliC-j was not found in S. typhi isolates, other than those from Indonesia. However, the one-clone origin of S. typhi was indicated by a common DNA fingerprint pattern and by the occurrence, in the 5' end region of the fliC gene, of 10 scattered nucleotides that differ from the corresponding 10 nucleotides in other fliC alleles studied. These nucleotides were present in all isolates tested but did not change the amino acid sequence of the flagellin polypeptide.


Characterization of a Bordetella pertussis fimbrial gene cluster which is located directly downstream of the filamentous haemagglutinin gene.

The biosynthesis of fimbriae is a complex process requiring multiple genes which are generally found clustered on the chromosome. In Bordetella pertussis, only major fimbrial subunit genes have been identified, and no evidence has yet been found that they are located in a fimbrial gene cluster. To locate additional genes involved in the biosynthesis of B. pertussis fimbriae, we used TnphoA mutagenesis. A PhoA+ mutant (designated B176) was isolated which was affected in the production of both serotype 2 and 3 fimbriae. Cloning and sequencing of the DNA region harbouring the transposon insertion revealed the presence of at least three additional fimbrial genes, designated fimB, fimC and fimD. The transposon was found to be located in fimD. Analysis of PhoA activity indicated that the fimbrial gene cluster was positively regulated by the bvg locus. A potential binding site for BvgA was observed upstream of fimB. FimB showed homology with the so-called chaperone-like fimbrial proteins, while FimC was homologous with a class of fimbrial proteins located in the outer membrane and presumed to be involved in transport and anchorage of fimbrial subunits. An insertion mutation in fimB abolished the expression of fimbrial subunits, implicating this gene in the biosynthesis of both serotype 2 and 3 fimbriae. Upstream of fimB a pseudogene (fimA) was observed which showed homology with the three major fimbrial subunit genes, fim2, fim3 and fimX. The construction of a phylogenetic tree suggested that fimA may be the primordial major fimbrial subunit gene from which the other three were derived by gene duplication. Interestingly, the fimbrial gene cluster was found to be located directly downstream from the gene coding for the filamentous haemagglutinin, an important B. pertussis adhesin, possibly suggesting co-operation between the two loci in the pathogenesis of pertussis.


Cloning and characterization of fxp, the flexible pilin gene of Aeromonas hydrophila.

The flexible pilus of Aeromonas hydrophila is a morphologically and biochemically unique organelle which binds eukaryotic cell surfaces and whose expression is induced by specific physiochemical conditions. fxp, the structural gene coding for the flexible pilus subunit, was localized on a 7.6kb plasmid of A. hydrophila strain AH26. A putative Shine-Dalgarno sequence and -10 and -35 regions were identified, a signal peptide sequence delineated, and the coding sequence compared with other bacterial sequences and found to be unique. Plasmid and chromosomal DNA was prepared from 66 other Aeromonas strains and 12 strains from other bacterial genera and examined by Southern blot hybridization using a labelled fxp oligonucleotide and the 7.6kb plasmid as probes. No hybridizing sequences were identified except in the original strain, AH26. It is proposed that fxp codes for a highly evolved organelle, possibly widely distributed in nature, but that it is carried on a genetic element that is rapidly lost from most strains upon in vitro cultivation and storage.


Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes.

In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.


Relative roles of signals upstream of AAUAAA and promoter proximity in regulation of human immunodeficiency virus type 1 mRNA 3' end formation.

At least two mechanisms have been implicated in regulating poly(A) site use in human immunodeficiency virus type 1 (HIV-1): inhibition of basal signals within 500 nucleotides (nt) of the cap site, leading to specific suppression of the 5' poly(A) site, and stimulation of basal signals by long terminal repeat U3 sequences, leading to specific activation of the 3' poly(A) site. We determined the relative contributions of these mechanisms in a HeLa cell transcription/processing reaction and by transient transfection analysis. In vitro, the efficiency of basal signals is equivalent close to (270 nt) and far from (1,080 nt) the promoter and is stimulated at least 30-fold in both positions by upstream U3 sequences. In vivo, U3 sequences also enhance processing at both positions. There are two additional effects when the poly(A) site is close to the cap site: at least a 15-fold reduction in total RNA levels and a 5-fold decrease in relative levels of RNA processed at the HIV-1 site in constructs containing U3. Both effects are overcome by insertion of upstream splicing signals in an orientation-dependent manner. Splicing appears to influence poly(A)+ RNA levels by two distinct mechanisms: stabilizing nuclear transcripts and directly stimulating 3' end formation. It is proposed that upstream elements play major roles in regulating poly(A) site choice and in controlling the subsequent fate of polyadenylated RNA. The impact of these findings on mechanisms of mRNA biogenesis in the HIV-1 provirus is discussed.


A controlled trial of aerosolized pentamidine or trimethoprim-sulfamethoxazole as primary prophylaxis against Pneumocystis carinii pneumonia in patients with human immunodeficiency virus infection. The Dutch AIDS Treatment Group.

Primary prophylaxis against Pneumocystis carinii pneumonia (PCP) is recommended for patients with human immunodeficiency virus (HIV) infection if their CD4 cell counts are below 200 per cubic millimeter (0.2 x 10(9) per liter). Either aerosolized pentamidine or trimethoprim-sulfamethoxazole (co-trimoxazole) is commonly prescribed for prophylaxis, but the relative efficacy and toxicity of these agents are unknown. We conducted a multicenter trial involving 215 HIV-infected patients with no history of PCP but with CD4 cell counts below 200 per cubic millimeter. The patients were randomly assigned to one of three regimens: aerosolized pentamidine once a month, 480 mg of trimethoprim-sulfamethoxazole once a day (80 mg of trimethoprim and 400 mg of sulfamethoxazole), or 960 mg of trimethoprim-sulfamethoxazole once a day (160 mg and 800 mg, respectively). The cumulative incidence of PCP was estimated by Kaplan-Meier survival analysis. After a mean follow-up of 264 days, 6 of the 71 patients in the pentamidine group had a confirmed first episode of PCP (11 percent), whereas none of the 142 patients in the two trimethoprim-sulfamethoxazole groups had PCP (P = 0.002). However, adverse events that required discontinuation of the medication were much more frequent in the trimethoprim-sulfamethoxazole groups (17 and 18 patients) than in the pentamidine group (2 patients). The adverse reactions occurred significantly sooner in the group given 960 mg of trimethoprim-sulfamethoxazole than in the group given 480 mg (mean time, 16 vs. 57 days; P = 0.02). For patients with HIV infection, trimethoprim-sulfamethoxazole taken once a day is more effective as primary prophylaxis against PCP than aerosolized pentamidine administered once a month, although adverse drug reactions are more frequent with trimethoprim-sulfamethoxazole.


Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160.

The envelope glycoprotein of human immunodeficiency virus (HIV) initiates infection by mediating fusion of the viral envelope with the cell membrane. Fusion activity requires proteolytic cleavage of the gp160 protein into gp120 and gp41 at a site containing several arginine and lysine residues. Activation at basic cleavage sites is observed with many membrane proteins of cellular and viral origin. We have recently found that the enzyme activating the haemagglutinin of fowl plague virus (FPV), an avian influenza virus, is furin. Furin, a subtilisin-like eukaryotic endoprotease, has a substrate specificity for the consensus amino-acid sequence Arg-X-Lys/Arg-Arg at the cleavage site. We show here that the glycoprotein of HIV-1, which has the same protease recognition motif as the FPV haemagglutinin, is also activated by furin.


Expression of Hox-7.1 in myoblasts inhibits terminal differentiation and induces cell transformation.

The terminal differentiation of myogenic cells initiates in the proximal portion of the limb bud whereas the distal region remains undifferentiated and proliferative. The apical ectodermal ridge maintains the progress zone in an undifferentiated state and induces proliferation of limb mesenchymal cells. Hox-7.1, a homeobox-containing gene, is expressed throughout the limb bud when limb outgrowth begins, whereas transcripts are later restricted to distal limb mesenchyme which is the proposed site of positional specification. Transplantation of proximal limb bud tissue into the distal portion of the limb results in a re-expression of Hox-7.1 in the transplanted mesenchyme. Similar grafts result in a positional reassignment to distal structures as well as de-differentiation of the grafted proximal tissue. Because of the association of Hox-7.1 expression with proliferative and undifferentiated cells, we tested whether Hox-7.1 regulates differentiation by transfection of Hox-7.1 complementary DNA into determined myogenic cells which represent one mesenchymal lineage in the limb. Here we report that forced expression of Hox-7.1 blocks terminal differentiation and results in a corresponding decrease in steady-state levels of MyoD1. Consistent with the association of Hox-7.1 with proliferation, Hox-7.1-expressing cells also acquire a transformed phenotype. Forced expression of Hox-8.1, a related Hox-gene, does not affect terminal differentiation indicating that the effects of Hox-7.1 are specific.


Effects of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline on the prolactin suppression induced by a series of full and partial dopamine D2 receptor agonists in male rats.

The effect of pretreatment with a high dose of the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (20 mg/kg, 24 h) on the intrinsic activity displayed by a series of full and partial dopamine D2 receptor agonists on prolactin regulating pituitary D2 receptors in male rats was studied. To increase baseline prolactin levels, gamma-butyrolactone in a dose inhibiting brain dopamine neurotransmission was given to all animals. In controls, i.e. rats not given EEDQ, supramaximal doses of all full and partial D2 receptor agonists tested decreased serum prolactin levels with greater than 80%. While the intrinsic activities of the dopamine precursor 1-DOPA and of the full agonists (+)-3-PPP, 5-OH-DPAT, B-HT 920 (talipexole), apomorphine, and NPA (R-(-)-N-n-propylnorapomorphine) were not affected by pretreatment with EEDQ, the effects of supramaximal doses of the partial agonists (-)-HW-165, TDHL (terguride), SDZ208-911, (-)-3-PP) (preclamol), and SDZ 208-912 were reduced to 66%, 74%, 59%, 100%, and 100%, respectively. The effect of EEDQ on the intrinsic activity displayed by the various agonists corresponds inversely to the intrinsic efficacy displayed by the drugs in other models of D2 receptor function with one exception only; thus, the prolactin suppressive effect of (-)-3-PPP was more effectively antagonized by EEDQ than would have been predicted from the intrinsic efficacy usually attributed to the drug. Since the dose of EEDQ used in the present study has previously been shown not to decrease D2 receptor density in the pituitary as measured using in vivo radioligand binding, it is suggested that alkylation of D2 receptors may change the conformation of the individual receptor complexes in a way that decreases the responsiveness to partial but not full agonists.


Electrophysiological and electrochemical analysis of the secretion of ATP and noradrenaline from the sympathetic nerves in rat tail artery: effects of alpha 2-adrenoceptor agonists and antagonists and noradrenaline reuptake blockers.

The aim of this study was to investigate whether or not nerve impulses release ATP and noradrenaline in parallel from the sympathetic nerve terminals of the rat tail artery. The extracellularly recorded excitatory junction current (EJC) was used to study, pulse by pulse, the release of ATP. An electrochemical method was used to study online the nerve stimulation-induced rise in the extracellular concentration of endogenous noradrenaline at the probe, a carbon fibre electrode (CF). This parameter, which does not directly represent noradrenaline release, but reflects release minus clearance, has been termed delta[NA]CF. The effects of a number of pharmacological agents on the EJCs were examined both at 0.1 and 2 Hz, and the effects on the EJC response to 100 pulses at 2 Hz compared with that on the delta[NA]CF response. Clonidine and xylazine were used as alpha 2-agonists, yohimbine and idazoxan as alpha 2-antagonists and desipramine and cocaine as blockers of noradrenaline reuptake. Most of these agents had unwanted side effects, especially at higher concentrations. However, clonidine and xylazine depressed at lower concentrations the EJC and delta[NA]CF responses to about the same extent; these effects were partially or completely reversed by yohimbine. Yohimbine or idazoxan did not affect the EJCs at 0.1 Hz but enhanced the EJC and delta[NA]CF responses to 100 pulses at 2 Hz to the same extent. All effects of desipramine (1 microM) seemed explainable as a result of block of noradrenaline reuptake, while cocaine (10 microM) in addition exerted an 'unspecific' depressant (probably local anesthetic) effect. Under control conditions, both agents depressed the EJC but dramatically enhanced the delta[NA]CF response to 100 pulses at 2 Hz. Addition of yohimbine prevented the depressant effect of desipramine on the EJCs completely and reduced that of cocaine, but increased their effects on the delta[NA]CF response. These results are compatible with the view that ATP and noradrenaline are released in parallel from the sympathetic nerve terminals of this tissue. The different, and under some conditions even opposite, effects of desipramine or cocaine on the EJC and delta[NA]CF responses are explainable in terms of the known post-secretory effects of these agents.


Activation of histamine H3 receptors produces presynaptic inhibition of neurally evoked cat nictitating membrane responses in vivo.

This study was undertaken in order to determine the potential role of prejunctional histamine H3 receptors in an in vivo adrenergic model system. Frequency-dependent nictitating membrane responses were elicited by sympathetic nerve stimulation in anesthetized cats. Systemic administration of the selective histamine H3 receptor agonist, (R)-alpha-methylhistamine (R alpha MeHA) produced a dose-related depression of amplitude of the evoked nictitating membrane responses with a threshold of about 10 micrograms/kg and maximal effect (50% depression at the lowest frequency; 0.5 Hz) seen at 100-300 micrograms/kg. Responses obtained with low frequency stimulation were more sensitive to depression by R alpha MeHA than were responses evoked with higher frequencies of stimulation. Larger doses of R alpha MeHA given to the same animals, failed to produce additional inhibition. R alpha MeHA depressed the amplitude of nictitating membrane responses evoked by either pre- or postganglionic nerve stimulation to an equivalent degree. This depressant action of R alpha MeHA was antagonized by pretreatment with the specific histamine H3 antagonist, thioperamide (3 mg/kg), but not by combined pretreatment with histamine H1 and H2 blockers chlorpheniramine (300 micrograms/kg) and cimetidine (5 mg/kg). Intravenous administration of adrenaline (1-30 micrograms/kg) also produced graded nictitating membrane responses that were not altered by subsequent administration of R alpha MeHA. These results suggest that histamine H3 receptors are involved in the modulation of neurally evoked noradrenaline release in the cat nictitating membrane by an inhibitory presynaptic action.(ABSTRACT TRUNCATED AT 250 WORDS)


Reversal by 3,3',5-triido-L-thyronine of the working memory deficit, and the decrease in acetylcholine, glutamate and gamma-aminobutyric acid induced by ethylcholine aziridinium ion in mice.

The effect of 3,3',5-triiodo-L-thyronine (T3) on working memory in ethylcholine aziridinium ion (AF64A)-treated mice was studied in a delayed non-matching to sample task using a T-maze. After behavioural testing was completed, mice were killed by microwave irradiation and regional brain levels of acetylcholine, aspartate, glutamate, glutamine, glycine, taurine, and gamma-aminobutyric acid (GABA) were measured by high-performance liquid chromatography with electrochemical detection. Treatment with AF64A (7 nmol, i.c.v.) produced a deficit in working memory performance in the non-matching to sample task at 30 s delay, and decreased acetylcholine, glutamate, and GABA levels in the hippocampus, but not in the septum and cerebral cortex. Administration of T3 (0.3 mg/kg, p.o., once daily for 6 days) to AF64A-treated animals improved the deficit in working memory performance and reversed the decrease in acetylcholine, glutamate, and GABA levels in the hippocampus. These results indicate that the deficit in performance induced by AF64A can be improved by T3 administration.


Functional changes in neuropeptide Y- and somatostatin-containing neurons induced by limbic seizures in the rat.

The influence of sustained epileptic seizures evoked by intraperitoneal injection of kainic acid on the gene expression of the neuropeptides somatostatin and neuropeptide Y and on the damage of neurons containing these peptides was studied in the rat brain. Injection of kainic acid induced an extensive loss of somatostatin and, though less pronounced, of neuropeptide Y neurons in the inner part of the hilus of the dentate gyrus. Neuropeptide Y-immunoreactive neurons located in the subgranular layer of the hilus, presumably pyramidal-shaped basket cells, were spared by the treatment. Although neuropeptide Y messenger RNA was not detected in granule cells of control rats, it was found there after kainic acid seizures at all time intervals investigated (12 h to 90 days after injection of kainic acid). High concentrations of neuropeptide Y messenger RNA were especially observed 24 h after injection of kainic acid. At this time neuropeptide Y messenger RNA was also transiently observed in CA1 pyramidal cells. Neuropeptide Y synthesis in granule cells in turn gave rise to an intense immunoreactivity of the peptide in the terminal field of mossy fibers which persisted for the entire time period (90 days) investigated. In addition, neuropeptide Y messenger RNA concentrations were also drastically elevated in presumptive basket cells located at the inner surface of the granule cell layer, especially at the "late" time intervals investigated (30-90 days after kainic acid). These data support the concept that extensive activation of granule cells by limbic seizures contributes to the observed neuronal cell death in CA3 pyramidal neurons and interneurons of the hilus. Consecutively, basket cells containing neuropeptide Y and presumably GABA might be activated and participate in recurrent inhibition of granule cells. Neuropeptide Y-immunoreactive fibers observed in the inner molecular layer at "late" time intervals after kainic acid may result either from collateral sprouting of mossy fibers or from basket cells extensively expressing the peptide. It is speculated that neuropeptide Y synthesized and released at a high rate from granule cells and basket cells may exert a protective action against seizures.


Morphine and the endogenous opioid dynorphin in the brain attenuate digoxin-induced arrhythmias in guinea pigs.

The effects of the opioid receptor agonists morphine and dynorphin on digoxin-induced arrhythmias were examined in guinea pigs that had received intravenous digoxin (50 mu/kg bolus plus 500 mu/kg/hr intravenously). Animals received either morphine (50 or 100 micrograms/kg) or dynorphin A(1-13) (50 or 100 micrograms/kg) or saline (the diluent) into the lateral cerebral ventricle (intracerebroventricularly) prior to digoxin. Morphine and dynorphin produced significant (P < 0.05) dose-dependent increases in the threshold of digoxin-induced arrhythmias. The mean digoxin dose at the development of fatal arrhythmias was 775 +/- 42 micrograms/kg in the control group but was significantly higher namely 958 +/- 45 micrograms/kg after 100 micrograms/kg of morphine ICV, and 984 +/- 47 micrograms/kg after 100 micrograms/kg of dynorphin A (1-13) intracerebroventricularly. In the absence of digoxin, the highest doses of each of these opioids did not produce arrhythmias. Changes in blood pressure and heart rate were unlikely explanations for the observed actions of these opioids as morphine accentuated the increase in blood pressure that accompanied digoxin while dynorphin was associated with a lower blood pressure with digoxin, despite similar effects on arrhythmias. In the control group, fatal digoxin-induced arrhythmias were ventricular tachyarrhythmias in two-thirds of cases and complete heart block in the rest. Morphine and dynorphin reduced the development of ventricular tachyarrhythmias. The role of the cholinergic system was explored, with morphine, utilizing atropine sulfate which crosses the blood brain barrier and atropine methylnitrate which does not enter the CNS. Atropine sulfate but not atropine methylnitrate reversed the effects of morphine on digoxin-induced arrhythmias.(ABSTRACT TRUNCATED AT 250 WORDS)


Kinetics and mechanism of degradation of a cyclic hexapeptide (somatostatin analogue) in aqueous solution.

A highly active cyclic hexapeptide analogue of somatostatin, Cyclo(N-Me-L-Ala-L-Tyr-D-Trp-L-Lys-L-Val-L-Phe), L-363,586, was found to improve the control of postprandial hyperglycemia in diabetic animals when given in combination with insulin. The compound is reported to be relatively stable in blood, nasal cavity, and intestinal lumen but undergoes rapid degradation in aqueous solution. The objective of this study was to elucidate the degradation mechanisms based on the kinetic data and the structure of the degradation products. Both pH and temperature had a profound influence on the instability of the peptide in aqueous solution. The data indicated that the peptide was most stable at a pH of about 4.7. The pH-rate profile exhibited specific acid catalysis at a pH less than 3.0 and base catalysis above pH 10.5. The kinetic pKa was determined to be 9.7. This pKa could be attributed to the tyrosine residue. The mechanisms of degradation under acidic and alkaline conditions appear to be different. Identification of the fragments obtained using mass spectrometry and amino acid sequencing suggest that the cyclic compound was cleaved to yield a linear fragment, which underwent further cleavage at both peptide linkages alpha to the tryptophanyl residue. The indole group of that residue is probably the potential nucleophile attacking the adjacent carbonyls. A rate equation for the degradation of the hexapeptide has been proposed.


Behavioral effects of dilazep on cholinergic, dopaminergic, and purinergic systems in the rat.

This study examined the effects of 1,4-bis[3-(3,4,5-trimethoxy benzoyloxy)-propyl] perhydro-1,4-diazepine (dilazep; Comelian) on central dopaminergic, cholinergic, and purinergic neuronal systems in rats. Intraperitoneal injections of dilazep (1-5 mg/kg) produced yawning responses, the most effective dose being 2 mg/kg. Dilazep potentiated physostigmine-induced yawning but not pilocarpine- and bromocriptine-induced yawning. Dilazep-induced yawning was not affected by low doses of haloperidol or sulpiride, but was completely inhibited by atropine, a muscarinic M1 receptor antagonist. Dilazep-induced yawning, as well as physostigmine-induced yawning, were markedly inhibited by pretreatment with SK & F 38393, a dopamine D1 receptor agonist, and were potentiated by SCH23390, a dopamine D1 receptor antagonist that alone does not elicit yawning. Caffeine, an adenosine receptor antagonist, inhibited dilazep- and physostigmine-induced yawning responses but N6-cyclohexyl adenosine (CHA) and N6-(L-phenylisopropyl, adenosine (L-PIA), adenosine A1 receptor agonists, were inactive. These results suggest that because the effects of dilazep on central cholinergic neurons are similar to those of physostigmine dilazep may potentiate indirectly the action of endogenous acetylcholine. Cholinergic neurons activated by dilazep may be modulated by postsynaptic dopamine D1 receptor activity but may not be affected by dopamine D2 receptor activity. Furthermore, the stimulatory effects of dilazep on cholinergic neuron may not be due to an inhibition of dopamine D1 receptors via purinergic (adenosine A1 receptor) stimulation by dilazep.


Anxiolytic-like effects of alpha-2-adrenoceptor agonists on conflict behavior in the rat: pre- versus postsynaptic receptor mechanisms.

The effects of acute pretest administration and chronic posttest administration of clonidine or the selective alpha 2-adrenoceptor agonist UK-14,304 on conflict behavior were investigated. In daily 10-min sessions, water-deprived rats were trained to drink water from a tube that was occasionally electrified (0.25 mA); electrification was signaled by a tone. Prior to treatment, subjects accepted 25-30 shocks/session (punished responding) and consumed approximately 12-15 ml/session (unpunished responding). Acute pretest administration of clonidine or UK-14,304 did not increase punished responding. In contrast, chronic posttest clonidine administration (40 micrograms/kg, IP, twice daily for 8 weeks) resulted in a robust and time-dependent increase in punished responding (60-70 shocks/session) relative to saline-treated controls. Moreover, the selective alpha 2-adrenoceptor agonist UK-14,304 also increased punished responding when administered chronically (1.0 mg/kg, BID). Administration of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine HCl (DSP4, 65 mg/kg, IP) significantly decreased punished responding in control conditioned suppression of drinking sessions. The anticonflict effect associated with chronic posttest clonidine treatment was not altered by DSP4 pretreatment. These findings suggest that chronic posttest alpha 2-adrenoceptor agonist treatment produces an anticonflict effect independent of its actions at presynaptic alpha 2-adrenoceptors.


Pertussis toxin-mediated ribosylation of G proteins blocks the hypnotic response to an alpha 2-agonist in the locus coeruleus of the rat.

Biologic responses mediated by adrenoceptors are transduced by a receptor-effector mechanism that involves a guanine nucleotide binding protein (G protein). Recently, we determined that the transduction mechanism for the hypnotic response to dexmedetomidine, a highly selective alpha 2-agonist, is located in the locus coeruleus (LC) of the rat. In this study, we examined the role of pertussis toxin-sensitive (PTX) G proteins in the LC for the hypnotic response to dexmedetomidine. The LC of rats were stereotactically cannulated and treated with PTX, 0.34 micrograms, or vehicle. Five days later, the hypnotic response to dexmedetomidine, 7 micrograms into the LC or 50 micrograms.kg-1 IP, was tested. On the following day, the LC was harvested and assayed to determine whether the G proteins had been ribosylated by pretreatment with PTX in vivo. Quantitative immunoblotting of G0 alpha, Gi alpha 1,2, and Gi alpha 3, the alpha-subunit of three PTX-sensitive proteins, was also performed. In vivo treatment with PTX into the LC blocked the hypnotic response to LC-administered dexmedetomidine and, to a lesser extent, IP-administered dexmedetomidine. The in vivo PTX treatment effectively ribosylated the G proteins. No alteration in the amount of the different species of PTX-sensitive alpha-subunit was produced by in vivo PTX treatment. These data suggest a pivotal role for PTX-sensitive G proteins in the LC in the hypnotic response to alpha 2-agonists in the rat.


Activation of alpha 2-adrenoceptors suppresses excessive wheel running in the semistarvation-induced hyperactive rat.

Male Wistar rats were housed in cages linked to running wheels and fed on a schedule designed to reduce their body weight by 20-30%. During this period of semistarvation, rats increased their daily running wheel activity (RWA) by up to 30 km/day. RWA could be kept at this level provided that body weight was kept constant. Different alpha-adrenergic agonists and antagonists were tested for their effects on RWA and it was found that RWA could be suppressed only by agonists that display high affinity for the alpha 2-receptor (clonidine and guanfacine). Neither antagonist had an effect on RWA. Clonidine's inhibiting effect on RWA was prevented by pretreatment with yohimbine, which also has high affinity for alpha 2-receptors. From these results, we conclude that semistarvation-induced hyperactivity can be blocked by alpha 2-agonists. In view of this result and those that were obtained in previous studies, a theoretical model for the development of semistarvation-induced hyperactivity will be presented.


Investigation of MDMA-related agents in rats trained to discriminate MDMA from saline.

To determine whether metabolite-related analogs of N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) produce stimulus effects similar to those of the parent compound, and to determine the structural requirements associated with the MDMA stimulus, several MDMA analogs were examined in tests of stimulus generalization using rats trained to discriminate 1.5 mg/kg MDMA from saline. Although several of the analogs produced up to 50-60% MDMA-appropriate responding, none [with the exception of N-methyl-1-(4-methoxyphenyl)-2-aminopropane (PMMA)] resulted in stimulus generalization. The partial generalization, coupled with the possible reduced ability of certain of the agents to penetrate the blood-brain barrier relative to MDMA, suggests that these agents are not behaviorally inactive. PMMA, although not a metabolite of MDMA, is closely related in chemical structure to MDMA and its metabolites; PMMA produces > 80% MDMA-appropriate responding and is approximately three times more potent (ED50 = 0.2 mg/kg) than MDMA itself (ED50 = 0.76 mg/kg). PMMA is a newer scheduled substance with an as yet unknown mechanism of action; however, on the basis of the stimulus generalization observed PMMA may share some behavioral and mechanistic similarity with MDMA. These results also indicate that an intact methylenedioxy ring, such as that found in MDMA but absent in PMMA, is not a prerequisite for MDMA-like activity and further support the notion that ring-opened MDMA metabolites may produce effects that contribute to the actions of MDMA.


Comparison of behavioral and central BDZ binding profile in three rat lines.

The performance of three widely used rat lines (Sprague-Dawley, Wistar, and Long Evans hooded) were evaluated in behavioral test systems that are sensitive to benzodiazepines. The in vivo effects of flunitrazepam and the brain [3H]Ro 15-1788 binding were determined and compared in these rat lines. The behavioral end points evaluated in this study were anxiolysis, measured using the automated elevated plus-maze; sedation by modification of locomotor activity; hyperphagia following food deprivation; protection for pentylenetetrazol-induced convulsions; and hypothermia. There were comparable results in the hypnotic, hypothermic, anticonvulsant, and feeding tests in these lines following flunitrazepam administration. However, the behavior of the Long Evans hooded rat was most amenable to the detection of drug-induced changes in the anxiety test. There was no difference in the maximum number of binding sites (Bmax) or the affinity (Ki) of the Ro 15-1788 or flunitrazepam binding in either the cerebellum or whole brain (minus cerebellum) in the three rat lines as determined by the competitive binding against [3H]Ro 15-1788. Thus, while these rat lines exhibited similar behavioral profiles in most tests the modest differences in the baseline responses and the ability to detect anxiolysis at lower doses of flunitrazepam observed with Long Evans hooded rats makes them particularly suited for these types of studies.


Raclopride decreases sucrose intake of rat pups in independent ingestion tests.

To investigate the role of dopaminergic activity at D2 receptors in the mediation of the positive reinforcing effect of sucrose on ingestion in preweanling rats, we tested the effects of the D2 antagonist, raclopride, on the intake of 10% sucrose of rats on postnatal days (PN) 7, 14, and 21. Intake was measured during independent ingestion tests in which pups licked sucrose from the floor of a beaker and during oral catheter tests in which sucrose was continuously infused through an anterior, sublingual, oral catheter. Rats were tested once to eliminate the possibility that repeated test experience would affect the response to raclopride. Pretreatment with raclopride resulted in decreased intake in independent ingestion (II) tests, but not in oral catheter (OC) tests on PN 7, 14, and 21. The inhibition of intake was not due to a generalized motor deficit because raclopride did not affect latency to eat, time-sampled activity scores, or latency to withdraw the hindlimb from a raised position. These results demonstrate that dopaminergic activity at D2 receptors is necessary for the positive reinforcing effect of sucrose that maintains ingestion in the II test but not in the OC test.


Stimulus effects of d-amphetamine 1: DA mechanisms.

As part of a continuing effort to assess the role of monoaminergic neuronal systems in the subjective effects of CNS stimulants, 10 rats trained to discriminate 1.0 mg/kg d-amphetamine from saline were treated with compounds that act through different dopaminergic mechanisms. In substitution (generalization) tests, 20 mg/kg of the dopamine (DA) uptake inhibitor GBR 12909 mimicked the training drug completely; at a dose of 15 mg/kg, GBR 12909 substituted for d-amphetamine incompletely. Neither the D1 agonist SK&F 38393 (1, 10 mg/kg) nor the D2 agonist quinpirole (LY 171555; 0.05-0.2 mg/kg) had amphetamine-like effects. When given in combination with the training drug, the D1 antagonist SCH 23390 blocked the amphetamine cue completely at a dose of 0.05 mg/kg but did not have significant effects at higher or lower doses; the D2 antagonist metoclopramide did not block d-amphetamine at any dose tested (1-5 mg/kg). These data indicate that: a) The discriminable effects of d-amphetamine are due, at least in part, to inhibition of DA uptake; b) direct stimulation of either D1 or D2 receptor sites is not sufficient to evoke d-amphetamine-like responding; and c) blockade of D1 receptors attenuates the subjective effects of d-amphetamine to a greater extent than blockade of D2 receptors.


Donor leukocyte migration following extremity transplantation in an experimental model.

In an effort to further define the immunologic mechanisms leading to acute composite-tissue allograft rejection, the migratory patterns of donor leukocytes were evaluated. Using a rat model, 52 orthotopic vascularized hindlimb transplants were performed in strains representing major histocompatibility mismatches. In order to evaluate the effect of allogeneic skin on limb rejection, all donor skin was removed in a second group of allografts. Recipient lymphoid organs were examined during the week following transplantation for antigen-presenting cells using a donor-specific class II monoclonal antibody. Donor leukocytes, with dendritic cell morphology, were identified in recipient spleen and lymph nodes draining the allograft. Significantly higher numbers of donor leukocytes were present during postoperative days 1 through 4 for both groups. Association of these important passenger leukocytes with host T-lymphocytes may represent the site of initiation of the immune response.


Seasonal relapses in affective disorder in the Tropics: a prospective follow-up of 12 patients.

Seasonal relapses of affective disorder are known. We report 12 patients who had season-linked relapses during a prospective follow-up period of 4 years. There were both winter and summer relapses of mania and depression. The centre is in the tropical zone, with lesser variation of sunshine and temperature than in more extreme latitudes. This may inference the pattern of relapse in affective disorder. Differences in relapses between tropical and temperate zones need to be investigated.


Influence of sympathetic and parasympathetic substances in clinical concentrations on human nasal ciliary beat.

The effect of autonomic substances on ciliary beat in mucosal biopsies from the normal human nose was studied. The influence on ciliary beat frequency (CBF) as well as on ciliary beat harmony was measured photoelectrically. The sympathetic beta-agonist isoprenaline was found to have a stimulatory effect on CBF, whereas the alpha-agonist xylometazoline reduced CBF and ciliary beat harmony. The parasympathetic agonist carbachol had a positive effect on CBF. The sympathetic antagonists timolol (beta) and phentolamine (alpha) as well as the parasympathetic antagonist atropine had no effect. The effect of the agonists on ciliary beat was absent when they were administered in combination with their specific antagonists. These experiments indicate that, in clinical concentrations, the autonomic substances modify ciliary beat by a direct effect on the ciliated cells.


Successful pancreatojejunal anastomosis for pancreatoduodenectomy.

A series of 100 patients underwent pancreatoduodenectomy for carcinoma of the head of the pancreas (36 patients), the bile duct (19 patients), the gallbladder (14 patients), the papilla of Vater (13 patients), the duodenum (seven patients), the stomach (seven patients) or the colon (one patient), or for chronic pancreatitis (two patients) and adenomyoma of the bile duct (one patient) between October 1983 and December 1990. The operative morbidity and mortality rates were 27 and 6 percent, respectively, and failure of the pancreatojejunal anastomosis was the most common post-operative complication (16 patients). Anastomotic leakage correlated significantly with the quality of the pancreatic remnant and the preoperative technique used (p < 0.01). The leakage rate in 34 patients with fibrotic pancreatic tissue was 5.8 percent (two of 34) and was significantly less than that in 66 patients with a normal pancreatic remnant (21.2 percent). Forty-seven patients underwent direct anastomosis between the main pancreatic duct and the jejunum, using mucosa to mucosa sutures and a pancreatic duct drain tube as well as a single layer of sutures between the pancreatic tissue and the jejunum. Only two patients had a pancreatic fistula (4.2 percent) and the incidence was significantly less than that in 53 patients who received the usual technique of double-layer sutures between the pancreatic remnant and the jejunum without a direct anastomosis to the main pancreatic duct (26.4 percent, p < 0.01).


Two new nonsense mutations in type Ia antithrombin III deficiency at Leu 140 and Arg 197.

Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing, the molecular basis of hereditary type Ia antithrombin III (AT III) deficiency was disclosed in two families. One mutation was a change from T to A in the codon of TTA for Leu 140 forming a stop codon of TAA, which was confirmed by mutated primer-mediated PCR-HindIII digestion. The application of this method demonstrated that all four affected members had the mutant allele in a heterozygous state and that none of unaffected subjects had this mutation. Another mutation in the second family was a change from C to T in the codon of CGA for Arg 197 also forming a stop codon of TGA, which was confirmed by PCR-HaeIII digestion. Based on these, it was concluded that the two new nonsense mutations in the AT III gene in a heterozygous state are the molecular basis of hereditary type Ia AT III deficiency.


Restriction fragment length polymorphism in four virulence-associated genes of Listeria monocytogenes.

We observed restriction fragment length polymorphism in 4 genes of Listeria monocytogenes associated with virulence. Using the polymerase chain reaction (PCR) and primers derived from published sequences, we amplified the following genes: hlyA coding for listeriolysin O, iap coding for a putative invasion-associated factor, mpl coding for a metalloprotease, and the prfA gene that positively regulates the hylA gene. PCR-amplified DNA were cut with several restriction endonucleases, and the restriction profiles from 29 strains, representing 6 serovars (serovars 1/2a, 1/2b, 1/2c, 3a, 3b and 4b) were compared. Based on these restriction profiles, the strains were categorized into 2 subgroups: one group contained all 10 strains of serovars 1/2a, 1/2c and 3a, the other group contained all 19 strains of serovars 1/2b, 3b and 4b. This division is in complete agreement with multilocus enzyme electrophoretic analysis data which divide the species into the same 2 subgroups. Whether the differences observed in the nucleotide sequences of the 4 virulence-associated genes for the 2 subgroups of L. monocytogenes represent salient variations in pathogenic mechanisms is not known.


[Growth hormone deficiency: diagnosis and treatment].

Growth hormone (GH) and thyroxine are the major growth promoting hormones in postnatal life. The pulsatile pattern of GH-release by the anterior pituitary is regulated by the interaction of stimulating and inhibiting hormones and influenced by physiological and pharmacological factors. GH promotes longitudinal bone growth mainly through activation of insulin-like growth factor I (IGF-I), but there may also be a direct effect. The most important clinical feature of growth hormone deficiency (GHD) is a low growth velocity resulting in short stature. In addition to retardation of bone maturation many GH-deficient children exhibit a number of typical, clinical characteristics. The diagnosis GHD can be confirmed by performing GH-stimulation tests and IGF-I measurements. However, the maximal GH-response to provocative stimuli is a poor indicator of the spontaneous GH-secretion. Classifications and etiology of GHD are given. Since 1958 GH-deficient children are treated with human GH with initially disappointing results regarding attained final height. Early diagnosis and treatment and optimization of GH-dose, mode of administration and injection frequency might improve final height. In The Netherlands the currently used GH-dose is 12-14 IU/m2/week in 6 or 7 daily doses administered subcutaneously. Ongoing retrospective and prospective studies serve to determine optimal treatment schedules. Up to now, important adverse effect of GH-therapy with the current GH-doses in GH-deficient children are not known. The beneficial effects of long-term GH-treatment of GHD adults is at present subject to ongoing investigation.(ABSTRACT TRUNCATED AT 250 WORDS)


Extracellular alkalinity exacerbates injury of cultured cortical neurons.

We have previously shown that extracellular acidity protects cultured fetal murine neocortical neurons from glutamate toxicity and combined oxygen-glucose deprivation injury, an action at least in part mediated by reduction in N-methyl-D-aspartate receptor activation. We now investigate the effect of extracellular alkalinity on both glutamate neurotoxicity and injury due to combined oxygen-glucose deprivation. The effects of extracellular alkalinity during injury induced by exposure of murine neocortical cultures to glutamate (0.5 mM for 5 minutes) or oxygen-glucose deprivation are characterized morphologically and quantitated by efflux of lactate dehydrogenase from both neurons and glia to the bathing medium. Calcium accumulation is measured with calcium-45. Moderate extracellular alkalinity is well tolerated by cortical cells but significantly potentiates both glutamate neuronal toxicity and oxygen-glucose deprivation neuronal injury. In contrast, glial viability in the face of combined oxygen-glucose deprivation is little affected by extracellular alkalinity. Increased accumulation of calcium-45 during oxygen-glucose deprivation in alkalotic medium and blockade of this increase by MK-801 is demonstrated. These observations suggest that alkaline pH can exacerbate excitotoxic neuronal injury, most likely because of increased N-methyl-D-aspartate receptor activation. Metabolic alkalosis of any etiology may sensitize neurons to ischemic injury and potentiate reperfusion injury.