Effect of anti-CD4 antibody treatment on inflammatory arthritis in MRL-lpr/lpr mice.

MRL-lpr/lpr mice develop an inflammatory arthritis in association with other manifestations of autoimmunity. Although a variety of immune cell disturbances have been described in these mice, the relationship of these abnormalities to the pathogenesis of arthritis has not yet been determined; the role of T cells is especially unclear since synovial hypertrophy and joint erosions have been noted in some studies in the absence of a significant T cell infiltrate. Therefore, to determine if T cells are required for arthritis in MRL-lpr/lpr mice, we evaluated the effects of prolonged treatment with a monoclonal anti-CD4 antibody. Knee joints from treated mice had markedly reduced arthritis compared to saline-treated control animals as measured by the degree of synovial hypertrophy and inflammation. Nephritis in these mice was concomitantly reduced. In contrast, rheumatoid factor levels were not affected by CD4+ cell depletion, despite significant effects on anti-DNA. These results indicate that in MRL-lpr/lpr mice anti-CD4 therapy can inhibit arthritis, suggesting an important role of T cells in the pathogenesis of this lesion.


BASREP: a method for maintaining euglycemia during somatostatin suppression of pancreatic secretion.

A glucagon infusion algorithm has been developed to reestablish basal glycemia when pancreatic insulin and glucagon secretion are inhibited by somatostatin (SRIF). When insulin alone is infused intraportally during SRIF to replace endogenous hormone release, hypoglycemia is generated by the combined actions of both peptides. In the presence of SRIF infusion, the normal physiologic response to hypoglycemia, i.e. stimulation of glucagon secretion and glucagon-induced increase in hepatic glucose production, has been prevented. Our computer algorithm, "BASREP", was designed to mimic the normal pancreatic counterregulatory response by substituting endogenous alpha-cell secretion with exogenous intraportal infusion. Sequential measurements of glucose concentration are analyzed with a minimal mathematical model of glucose disappearance, adapted to include a variable to describe glucagon stimulation of hepatic glucose production. Based upon the observed change in plasma glucose, BASREP computes after every sample the infusion rate of glucagon necessary to stimulate glucose production and maintain desired glucose level. This method minimizes instabilities and should prove useful in future investigations of glucose metabolism.


Interaction between corticosteroid and beta-agonist drugs. Biochemical and cardiovascular effects in normal subjects.

The aim of this study was to investigate whether the administration of prednisone potentiates any of the acute biochemical and cardiovascular effects of high-dose inhaled beta-agonist drugs. These agents are known to cause dose-related changes in plasma potassium and glucose, as well as ECG changes in heart rate, corrected QT interval (QTc), T wave, and U wave. On theoretical grounds, the concomitant use of systemic corticosteroids might enhance these actions. Twenty-four healthy subjects were randomized to receive one of three treatments: salbutamol 5 mg or fenoterol 5 mg or normal saline solution. Each drug was administered twice, 30 min apart by nebulizer, and the procedure was repeated after each subject had received prednisone 30 mg daily for one week. Plasma potassium and glucose levels were measured, and ECGs were obtained after each treatment, together with 12-h Holter monitoring for arrhythmias. Changes in plasma potassium and glucose following nebulized beta-agonist were significantly greater after treatment with prednisone. Baseline potassium level fell from 3.75 mmol/L (95 percent CI 3.61, 3.89) to 3.50 mmol/L (95 percent CI 3.36, 3.64), and thereafter all values were significantly lower at each time point (p = 0.003). The lowest mean plasma potassium was obtained 90 min after fenoterol administration with prednisone pretreatment: 2.78 mmol/L (95 percent CI 2.44, 3.13). Increases in heart rate and QTc interval following both beta-agonist drugs were significant, but T-wave amplitude reductions did not reach significance. Prednisone treatment did not significantly alter the cardiovascular responses. Supraventricular and ventricular ectopic activity was related to beta-agonist use, but no potentiating effect was noted following steroid treatment. We conclude that the acute biochemical effects of beta-agonist administration are augmented by prior treatment with prednisone, but this is not the case for ECG effects. However, the degree of hypokalemia noted as a result of this drug interaction may be of clinical significance in the hypoxic conditions of acute airways obstruction.


Rapid onset of action of inhaled formoterol in asthmatic patients.

Twelve patients with stable asthma (mean age, 39 years; asthma duration, 11 years; mean forced expiratory volume in 1 s, 65 percent of predicted; and reversibility, 31 percent) were studied in a double-blind crossover trial. The patients were studied during three test days. Airway resistance and specific airway conductance (Raw and SGaw) were measured using a body plethysmograph and pulse rate, blood pressure, tremor, and subjective effects were recorded before and 1, 3, 5, 10, 15, 30, 60, and 120 min after the test doses. A baseline Raw variability of +/- 20 percent was allowed between the test days. Formoterol 12 micrograms, 24 micrograms, and terbutaline 500 micrograms were given in a spacer (Nebulator) in a randomized double-blind crossover manner as two puffs with a 30-s interval in between. The effect of formoterol 12 micrograms on Raw was significantly better than terbutaline after 3, 5, 10, 60, and 120 min. Formoterol 24 micrograms was significantly better than terbutaline as soon as 3 min after inhalation and at every point in time after that. Formoterol 24 micrograms tended to be better than formoterol 12 micrograms but the differences were not significant at any point in time. All three treatments were well-tolerated. No differences were observed for pulse rate, blood pressure, tremor, or palpitations. The overall onset of bronchodilatation after formoterol 12 and 24 micrograms was faster than after terbutaline 500 micrograms. The tolerability of formoterol was good.


Variable expression of P-glycoprotein in normal, inflamed, and dysplastic areas in ulcerative colitis.

Screening programs for the detection of cancer in ulcerative colitis are inexact and not always successful in finding early, curable cancers. P-glycoprotein is a membrane-based, energy-dependent protein found in varying degrees within normal human tissue. P-glycoprotein is overexpressed in malignant tumors, particularly colorectal cancer, and is known to convey resistance to certain anticancer drugs by acting as a membrane "pump." The purpose of this study was to determine the expression of this protein in inflamed and premalignant colonic epithelium, compare its expression with normal controls, and assess its potential use as a screening tool for high-risk patients with ulcerative colitis. Using immunohistochemical techniques, the colons of 21 patients (10 with dysplasia) with ulcerative colitis were stained with monoclonal antibody C-219 (MAbC219) specific for P-glycoprotein. P-glycoprotein was expressed in 38 percent of normal areas, 71 percent of inflamed areas (P = 0.0156), and 70 percent of dysplastic areas. Comparing the level of expression when progressing from normal to inflamed areas within a given patient, 11 patients (52 percent) showed increased expression, 8 (38 percent) showed equal expression, and only 2 (10 percent) showed decreased expression (P = 0.0225). Comparing expression when progressing from inflamed to dysplastic areas (10 patients), 7 showed equal expression and 3 showed increased expression (P = 0.25). Increasing duration of disease was associated with a significant increase in P-glycoprotein expression, but only in histologically normal areas. Duration of disease had no effect on P-glycoprotein expression in inflamed or dysplastic areas. Similarly, when surgery was performed for elective reasons, there was a significant overexpression of P-glycoprotein, but only in histologically normal areas. Our findings suggest that the increase in P-glycoprotein expression from normal to inflamed and dysplastic areas reflects the premalignant nature of ulcerative colitis and occurs early in the course of the disease. Further research needs to be done to determine its role in cancer surveillance.


Akinesia: a syndrome common to parkinsonism, retarded depression, and negative symptoms of schizophrenia.

A distinct hypokinetic syndrome appears to exist across several different neuropsychiatric diagnoses, involving (1) slowed motor activity with difficulty initiating and sustaining behaviors, (2) anhedonia with depressed mood and reduced affective range, and (3) cognitive impairment. Specifically, three well-recognized states--parkinsonism, retarded depression, and the negative symptoms of schizophrenia--prominently feature the components of this syndrome, and reduced dopamine turnover in the brain has been hypothesized to play a part in the pathophysiology of each. While aspects of this conceptualization remain controversial, it generates testable hypotheses that could have implications for the understanding and treatment of these states.


Gold salts and somatostatin: a new combined analgesic treatment for psoriatic arthritis.

In a group of patients affected with psoriatic arthritis the effects of the association between gold salts (GS) and somatostatin (SOM), in comparison with two groups treated with SOM and GS respectively, were investigated. Sixty patients with psoriatic arthritis were selected and randomly allocated in three groups of twenty patients each. Group 1 received SOM infusion (250 micrograms/h for 96 h) and was assessed at baseline and 1, 15, 30, 60, 90 and 120 days after; Group 2 received intramuscular GS and was assessed at baseline, four months later, and then every month for four months; Group 3 received GS for 8 months; at the fourth month SOM was infused (as in Group 1) and the patients assessed at baseline four months later and then as Group 1. Assessment was made with the Ritchie index, pain scale and morning stiffness evaluation. Growth hormone was assayed in Group 1 every 4 h for 24 h the day before and the day after SOM infusion. The association between GS and SOM demonstrated a particular analgesic activity, effective on joint pain and tenderness, that lasted for all four months of follow-up. SOM showed a good response only after 15 and 30 days, and GS proved to be effective at about the sixth month of treatment. Side effects were reported in Group 1 (abdominal cramps, mild erythrodermia and supraventricular arrhythmia). A growth hormone circadian rhythm was found in psoriatic patients both before and after SOM treatment. The beneficial effect of the SOM/GS combination is demonstrated in psoriatic arthritis.(ABSTRACT TRUNCATED AT 250 WORDS)


Akathisia: clinical phenomenology and relationship to tardive dyskinesia.

Akathisia and tardive dyskinesia (TD) are disorders of movement that are often associated with administration of antipsychotic medication. We surveyed 196 outpatients in a schizophrenia clinic, all receiving antipsychotic medication, for the presence of these disorders. Clinical global ratings of akathisia were reliable. Akathisia was found in 36% of patients, and TD in 23.5%. Akathisia was disproportionately common in patients receiving high-potency neuroleptics. The data affirmed recent revisions in the dose-equivalence formulas used with fluphenazine decanoate. Akathisia and TD did not seem to be interrelated. Because akathisia is common and often limits medication dose and contributes to noncompliance, psychiatrists must take this into account when prescribing antipsychotic medication.


[The prevention of Pneumocystis carinii pneumonia by pentamidine inhalation].

167 HIV-positive patients (155 men, 12 women; mean age 31 [18-61] years) with CD4 lymphocyte counts below 250/microliter every 4 weeks received 300 mg pentamidine per aerosol inhalation during out-patient visits, as prophylaxis against Pneumocystis carinii. 89 patients were clinically in the AIDS stage and 33 in the AIDS-related complex (ARC) stage. 29 patients had a lymphadenopathy syndrome, while 16 were asymptomatic. 130 patients received primary prophylaxis, while 37 who had previously had an attack of Pneumocystis carinii pneumonia were given pentamidine as secondary prophylaxis. During a mean observation period of 8 months three patients developed Pneumocystis carinii pneumonia (1.7%): their CD4 lymphocyte count was under 20/microliters. Pentamidine inhalation reduced the incidence of a first attack of pneumonia to 0.18% per month and recurrence to 0.32% per month. These figures confirm the great effectiveness of primary and secondary prophylaxis with pentamidine inhalation.


Lysosomal acid phosphatase is not involved in the dephosphorylation of mannose 6-phosphate containing lysosomal proteins.

The mannose 6-phosphate (Man6P) residues that are necessary for the targeting of newly synthesized lysosomal proteins are dephosphorylated after delivery of lysosomal proteins to lysosomes. To examine the role of lysosomal acid phosphatase (LAP) for the dephosphorylation of Man6P residues in lysosomal proteins, the phosphorylation of endogenous lysosomal proteins and of internalized arylsulfatase A was analyzed in mouse L-cells that overexpress human LAP. Non-transfected L-cells dephosphorylate endogenous lysosomal proteins slowly (half time approximately 13 h) as well as internalized arylsulfatase A. A more than 100-fold overexpression of LAP in these cells did not affect the dephosphorylation rate. Control experiments showed that the internalized arylsulfatase A and overexpressed LAP partially colocalize and that under in vitro conditions purified LAP does not dephosphorylate arylsulfatase A. Taken together, these results indicate that LAP is not the mannose 6-phosphatase that dephosphorylates lysosomal proteins after their delivery to lysosomes.


Immunocytochemical evidence for in vitro release of glutamate and GABA from separate nerve terminal populations in the rat pontine nuclei.

A quantitative electron microscopic immunocytochemical method was used to study the synaptic handling of glutamate and GABA in slice preparations from the rat pontine nuclei. Slices were subjected to a depolarizing stimulus (55 mM K+, 20 min) in the presence of a physiological or low Ca(2+)-concentration. Depolarization at physiological [Ca2+] evoked a depletion of glutamate-like immunoreactivity from nerve terminals that contain round vesicles and establish asymmetric synaptic contacts. When depolarization was induced in the presence of only 0.1 mM Ca2+ (10 mM Mg2+ added), the loss of glutamate was significantly reduced or abolished, indicative of a Ca(2+)-dependent component of glutamate release. By means of a double-labeling immunocytochemical method we could identify a population of nerve terminals that displayed strong GABA-like immunoreactivity, and a level of glutamate-like immunoreactivity that was low but yet clearly above background level. This type of terminal contains elongated or pleomorphic vesicles and establishes symmetric synaptic contacts. In these terminals, depolarization evoked a Ca(2+)-dependent depletion of GABA-like immunoreactivity, but failed to change the level of glutamate-like immunoreactivity. The present study demonstrates that two different types of nerve terminal in the rat pontine nuclei contain releasable pools of glutamate and GABA, respectively, and that the GABA-releasing terminals also contain a non-releasable pool of glutamate. The glutamate of the latter pool could act as precursor of GABA.


Locus coeruleus bursts induced by glutamate trigger delayed perforant path spike amplitude potentiation in the dentate gyrus.

Glutamate pressure ejections in the vicinity of locus coeruleus (LC) neurons have been shown to produce both short and long-lasting potentiation of perforant path (PP) evoked population spike amplitude in the dentate gyrus (DG). These effects of LC-glutamate activation resemble those produced by direct application of NE in vitro or in vivo. The present study monitored the cellular response of LC neurons to local glutamate ejections concomitant with stimulation of the PP evoked potential. Double barrel micropipettes or 33 ga cannula-electrode assemblies permitted LC unit recording and glutamate ejection at or near the same site in urethane anesthetized rats. Glutamate ejections produced a burst of LC activity lasting 250-400 ms and followed by a depression of unit activity lasting 4.6 min. The maximal spike potentiation produced by LC activation was 158%. The first spike to exceed the control range occurred 34 s after the LC burst. Comparable silent intervals in LC unit activity induced by systemic clonidine were not accompanied by population spike amplitude potentiation. The mean duration of potentiation was 4.4 min except in four cases where responses remained potentiated for the duration of the experiment. The duration of potentiation was not correlated with the termination of LC depression. LC units recovered to baseline rates following glutamate induced depression of activity. The occurrence of potentiation appeared to require that glutamate activation reach a critical number of LC neurons since small glutamate ejections could produce a local burst without eliciting potentiation. Long-lasting changes were also related to larger glutamate volumes (100 nl).(ABSTRACT TRUNCATED AT 250 WORDS)


Correlation of MDR1/P-170 expression with daunorubicin uptake and sensitivity of leukemic progenitors in acute myeloid leukemia.

Prior studies have shown that multidrug resistance gene products may be detected in acute myeloid leukemia (AML) cells, and are associated with poor response to therapy. We studied whether P-170 expression was associated with in vitro daunorubicin (DNR) accumulation and sensitivity of leukemic clonogenic cells (CFU-L) to DNR in 16 newly diagnosed AML samples. P-170 expression was assessed by indirect immunofluorescence using the monoclonal antibody MRK16. DNR cellular content was measured by flow cytometry after short incubation with increasing concentrations of DNR, and was not correlated with P-170 expression, although there was a trend for higher values in P-170-negative samples. The sensitivity of CFU-L was studied in a semisolid culture assay by calculating the dose of DNR inhibiting the growth of 90% of CFU-L (D90). The D90 was significantly higher in P-170-positive than in P-170-negative samples (mean = 1.68 +/- 0.42 microgram/ml versus 0.97 +/- 0.35 micrograms/ml respectively, p less than 0.005). Eight of 9 cases achieving complete remission (CR) after intensive chemotherapy were P-170-negative, whereas 7 of 7 nonresponders were P-170-positive (p less than 10(-5)). D90 was significantly lower for patients achieving CR than for those who did not achieve CR (1.12 +/- 0.55 micrograms/ml versus 1.59 +/- 0.37 micrograms/ml, p = 0.04). It is concluded that P-170 expression is correlated with in vitro resistance of clonogenic cells to DNR and may be one mechanism of resistance to chemotherapy.


Spontaneous conversion of PrPC to PrPSc.

Octa-repeats of prion proteins (PrP) contain histidine and tryptophan residues which are known to function as ligands for transition metals. It is proposed that the spontaneous conversion of the PrPC (cellular) isoform into PrPSc (scrapie) isoform may be triggered by the coordination of these metals.


Pre-translational regulation by glucocorticoid of fatty acid and phosphatidylcholine synthesis in type II cells from fetal rat lung.

Exposure to fibroblast-conditioned cortisol-containing medium increased fatty acid synthase activity and fatty acid synthase, acetyl-CoA carboxylase and ATP citrate lyase mRNA abundance in fetal type II alveolar epithelial cells. Both fibroblast conditioning and cortisol in the medium were required for maximal effect on the mRNA levels, indicating involvement of mesenchymal-epithelial interaction in the cortisol effects. The observed effects provide evidence for an earlier hypothesis that increased activity of CTP:phosphocholine cytidylyltransferase in lung tissue caused by glucocorticoid is due to increased fatty acid synthesis. However, evidence suggesting pre-translational regulation of this enzyme by glucocorticoid was also found.


Putative nucleotide binding sites of guinea pig liver transglutaminase.

Three peptides corresponding to glycine-rich internal sequences of the guinea pig liver transglutaminase molecule were synthesized. These were peptide 1 (amino acid residues 520-544), peptide 2 (amino acid residues 345-367) and peptide 3 (amino acid residues 45-69). All of the synthetic peptides demonstrated significant binding ability for both ATP and GTP. Peptide 1 was the best protector of transglutaminase activity from both ATP and GTP inhibition, while peptides 2 and 3 protected the activity only from GTP inhibition. The data shown here lead us to propose putative binding site(s) for ATP and GTP guinea pig liver transglutaminase.


The apical sorting signal on human aminopeptidase N is not located in the stalk but in the catalytic head group.

Human aminopeptidase N carries an apical sorting signal on its ectodomain necessary for its correct transport to the apical membrane in Madin-Darby canine kidney cells. To determine whether the apical sorting signal is localized in the serine/threonine rich stalk or in the catalytic head group, anchor/stalk-minus aminopeptidase N, consisting of the hemagglutinin signal peptide and the catalytic head group of human aminopeptidase N, was expressed in MDCK cells. Anchor/stalk-minus aminopeptidase N was secreted mainly to the apical side. The catalytic head group of human aminopeptidase N thus carries an apical sorting signal.


One step purification and characterization of the pyrrolidone carboxyl peptidase of Streptococcus pyogenes over-expressed in Escherichia coli.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp), an enzyme which selectively removes pyrrolidone carboxylic acid (PCA) from some PCA-peptides and -proteins, was demonstrated in bacteria and in plant, animal and human tissues. In this paper we describe the purification to homogeneity of the enzyme of Streptococcus pyogenes, over-expressed in Escherichia coli. This was achieved, for the first time in one step, by hydrophobic interaction chromatography. Analysis under non-denaturing conditions revealed a molecular mass of 85 kDa and in the presence of sodium dodecyl sulfate gave a molecular mass of 23.5 kDa. Investigations on enzymatic properties showed that the Pcp over-expressed in E. coli disclosed properties similar to those found for the enzyme extracted from S. pyogenes or for some other Pcps studied previously. Thus the over-expressed enzyme should serve as a suitable source for N-terminal unblocking prior to some PCA protein sequencing.


Phosphorylated cystatin alpha is a natural substrate of epidermal transglutaminase for formation of skin cornified envelope.

Both keratohyalin granules (KHG) and cornified envelopes were stained histochemically in an indirect immunofluorescent study by antiphosphorylated cystatin alpha antibody, indicating that phosphorylated cystatin alpha is a component of the cornified envelope proteins. When phosphorylated cystatin alpha (P-cystatin alpha) was incubated with epidermal transglutaminase (TGase) and Ca2+ ions, polymerized protein was produced by formation of epsilon-(gamma-glutamyl)lysine cross-linking peptide bonds between lysine residues of cystatin alpha and glutamine residues of suitable protein(s) in the enzyme preparation. However, phosphorylated and non-phosphorylated cystatins were polymerized to similar extents by the TGase. Immunofluorescent and immunoelectron microscopic observations revealed that P-cystatin alpha could be detected in vivo in the KHG and cornified envelopes. Treatment of sphingosine, a specific inhibitor of protein kinase C, markedly suppressed the incorporation of cystatin alpha into KHG. Thus phosphorylation of cystatin alpha by protein kinase C may play an important role in targeting cystatin alpha into KHG.


[A hypothalamic hormone-somatostatin--from endocrinology to neurophysiology].

The densest distribution of somatostatin (SRIF) neuron perikarya is localized in the hypothalamic periventricular nucleus (Pe) close to the third ventricle, from which many fibers are projected to the median eminence. The release of SRIF in the neurohemal organ into the anterior pituitary modulates GH secretion from pituitary somatotrophs. When SRIF input from the hypothalamus to rat anterior pituitary is reduced by either neurosurgery or SRIF antiserum iv injection, the responsiveness of the pituitaries to human GH releasing factor (hGRF) in an in vitro perifusion system is markedly attenuated. Moreover, SRIF pretreatment facilitates the GH release response of dispersed anterior pituitary cells to hGRF. The long lasting SRIF effect to sensitize somatotrophs appears to take place beyond cAMP formation or as an unknown distal effect. These findings indicate that SRIF neurons in the Pe play a role in maintaining the pituitary responsiveness to GRF in addition to the original action to inhibit GH secretion. Neuronal networks between Pe-SRIF neurons, and intra- and extrahypothalamic nuclei are identified by Pe stimulation test on GRF-GH secretion. In addition to the physiological role in maintaining pituitary responsiveness, Pe SRIF neurons have a wide influence on specific SRIF receptor binding in various brain regions as well as in the anterior pituitary. Shortly after lesioning the Pe neurons, there is a continuous increase in plasma GH level with a transient increase in specific binding of 125I-Tyr 11-SRIF-14 to the anterior pituitary. Furthermore, there is a similar but a little longer increase in binding of the radioligand to some brain areas such as the cerebral cortex, hippocampus, and amygdala nuclei. However, neuronal connections between the SRIF neurons and nuclei which are up-regulated by the lesioning have not been fully proven. When the labeled ligand is infused into the lateral ventricle, it is rapidly and widely distributed in many periventricular structures in the lateral and third ventricles. These findings suggest that SRIF produced in the Pe neurons is transported to other brain areas via cerebrospinal fluid in addition to neuronal connections for modulating the activity of neurons which have SRIF receptors. Thus, hypothalamic Pe SRIF neurons have dualistic roles for controlling anterior pituitary function and modulating CNS neuron activity.


A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo.

To obtain gene sequences controlling the early steps of amphibian neurogenesis, we have performed differential screening of a subtractive cDNA library prepared by a novel PCR-based method from a single presumptive neural plate of a Xenopus laevis late-gastrula embryo. As a result we have isolated a fragment of a novel homeobox gene (named XANF-1, for Xenopus anterior neural folds). This gene is expressed predominantly in the anterior part of the developing nervous system. Such preferential localization of XANF-1 mRNA is established from its initially homogenous distribution in ectoderm of early gastrula. This change in the expression pattern is conditioned by a differential influence of various mesoderm regions on ectoderm: anterior mesoderm activates XANF-1 expression in the overlying ectoderm, whereas posterior axial and ventral mesoderm areas inhibit it. The data obtained demonstrate for the first time that selection of genes for specific expression in the CNS of the early vertebrate embryo is affected not only by chordamesoderm (a neural inductor) but also by ventral mesoderm.


DNA fingerprinting and analysis of population structure in the chestnut blight fungus, Cryphonectria parasitica.

We analyzed DNA fingerprints in the chestnut blight fungus, Cryphonectria parasitica, for stability, inheritance, linkage and variability in a natural population. DNA fingerprints resulting from hybridization with a dispersed moderately repetitive DNA sequence of C. parasitica in plasmid pMS5.1 hybridized to 6-17 restriction fragments per individual isolate. In a laboratory cross and from progeny from a single perithecium collected from a field population, the presence/absence of 11 fragments in the laboratory cross and 12 fragments in the field progeny set segregated in 1:1 ratios. Two fragments in each progeny set cosegregated; no other linkage was detected among the segregating fragments. Mutations, identified by missing bands, were detected for only one fragment in which 4 of 43 progeny lacked a band present in both parents; no novel fragments were detected in any progeny. All other fragments appeared to be stably inherited. Hybridization patterns did not change during vegetative growth or sporulation. However, fingerprint patterns of single conidial isolates of strains EP155 and EP67 were found to be heterogenous due to mutations that occurred during culturing in the laboratory since these strains were first isolated in 1976-1977. In a population sample of 39 C. parasitica isolates, we found 33 different fingerprint patterns with pMS5.1. Most isolates differed from all other isolates by the presence or absence of several fragments. Six fingerprint patterns each occurred twice. Isolates with identical fingerprints occurred in cankers on the same chestnut stems three times; isolates within the other three pairs were isolated from cankers more than 5 m apart. The null hypothesis of random mating in this population could not be rejected if the six putative clones were removed from the analysis. Thus, a rough estimate of the clonal fraction of this population is 6 in 39 isolates (15.4%).


Effect of wing scalloping mutations on cut expression and sense organ differentiation in the Drosophila wing margin.

A number of wing scalloping mutations have been examined to determine their effects on the mutant phenotype of cut mutations and on the expression of the Cut protein. The mutations fall into two broad classes, those which interact synergistically with weak cut wing mutations to produce a more extreme wing phenotype than either mutation alone and those that have a simple additive effect with weak cut wing mutations. The synergistically interacting mutations are alleles of the Notch, Serrate and scalloped genes. These mutations affect development of the wing margin in a manner similar to the cut wing mutations. The mutations inactivate the cut transcriptional enhancer for the wing margin mechanoreceptors and noninnervated bristles and prevent differentiation of the organs. Surprisingly, reduction of Notch activity in the wing margin does not have the effect of converting epidermal cells to a neural fate as it does in other tissues of ectodermal origin. Rather, it prevents the differentiation of the wing margin mechanoreceptors and noninnervated bristles.


Epistasis and the genetic divergence of photoperiodism between populations of the pitcher-plant mosquito, Wyeomyia smithii.

Parallel crosses between each of two southern (ancestral) and one northern (derived) population of the pitcher-plant mosquito, Wyeomyia smithii, were made to determine the genetic components of population divergence in critical photoperiod, a phenological trait that measures adaptation to seasonality along a climatic gradient. Joint scaling tests were used to analyze means and variances of first- and second-generation hybrids in order to determine whether nonadditive genetic variance, especially epistatic variance, contributed to divergence in critical photoperiod. In both crosses, digenic epistatic effects were highly significant, indicating that genetic divergence cannot have resulted solely from differences in additively acting loci. For one cross that could be tested directly for such effects, higher order epistasis and/or linkage did not contribute to the divergence of critical photoperiod between the constituent populations.


A genetic map of the mouse suitable for typing intraspecific crosses.

We report the construction of a genetic linkage map of the mouse, consisting entirely of genetic markers that can be rapidly typed by polymerase chain reaction and that show a high degree of polymorphism among inbred laboratory strains. Specifically, the map contains 317 simple sequence length polymorphisms at an average spacing of 4.3 cM and is detectably linked to approximately 99% of the mouse genome. In typical crosses between inbred laboratory strains, about 50% of the markers are polymorphic, making it straightforward to follow inheritance in almost any cross.


Transition from rapid processive to slow nonprocessive polyadenylation by vaccinia virus poly(A) polymerase catalytic subunit is regulated by the net length of the poly(A) tail.

The mRNA of vaccinia virus, like that of eukaryotes, possesses a poly(A) tail. VP55, the catalytic subunit of the heterodimeric vaccinia virus poly(A) polymerase, was overexpressed and purified to near homogeneity. VP55 polyadenylated a 30-mer primer representing the 3' end of a vaccinia virus mRNA bimodally: 30-35 adenylates were added in a rapid, processive, initial burst, after which polyadenylation decelerated dramatically and became nonprocessive. Polyadenylation of variants of the 30-mer primer, which contained preformed 3'-oligo(A) extensions, showed that the transition between the two modes of polyadenylation was regulated by the net length of the 3'-oligo(A) tail rather than the number of adenylate additions catalyzed by VP55. Primers comprising oligo(A) alone were polyadenylated only if they were greater than 34 nucleotides in length and, then, only in the slow nonprocessive mode. These data support a dynamic model whereby the mode of polyadenylation by VP55 is regulated by sequences within the 3' 30-35 nucleotides of the mRNA: Polyadenylation is rapid and processive until a net 3'-oligo(A) length of 30-35 nucleotides is achieved. Consistent with this, excess oligo(A) did not compete with the 30-mer primer for rapid processive polyadenylation. The primer specificity of VP55 may contribute to the selective polyadenylation of newly formed mRNA.


The Ustilago maydis pyr3 gene: sequence and transcriptional analysis.

The pyr3 gene of Ustilago maydis encodes a 391-amino acid (aa) polypeptide. The sequence has identifies with dihydro-orotases (DHOases) from other organisms, but is most related to sequences of other monofunctional enzymes. The polypeptide contains the three domains conserved in other DHOases. The polypeptide encoded by the pyr3-1 allele has an aa change seven residues away from the C-terminal conserved domain. Transcription start point (tsp) is 58 nucleotides upstream from the start codon, and is in a region characterised by CTTT and CATC motifs. In the absence of TATA and CAAT boxes, these motifs might be important in transcriptional regulation. Gene disruption experiments suggest that the pyr3 gene product might have another function in addition to that in pyrimidine biosynthesis.


Sulphasalazine induced renal failure.

Two men with longstanding ulcerative colitis who were treated with sulphasalazine for several years and who developed chronic renal failure are reported. Renal biopsy specimens showed histological changes consistent with drug induced chronic intestinal nephritis. Extensive investigation made other causes of chronic renal failure unlikely. One of these patients underwent renal transplantation, the other has impaired but stable renal function.


Treatment of ulcerative colitis with fish oil supplementation: a prospective 12 month randomised controlled trial.

The effect of fish oil on the course of ulcerative colitis was investigated in a randomised blinded controlled study. Eighty seven patients received supplements of 20 ml HiEPA fish oil as triglyceride (4.5 g of eicosapentaenoic acid) or olive oil placebo daily for one year. The oils were given in addition to standard drug therapy and trial entry was stratified for disease activity. Fish oil significantly increased the eicosapentaenoic acid content of rectal mucosa to 3.2% of total fatty acids at six months, compared with 0.63% for patients on olive oil. This was associated with increased synthesis of leukotriene B5, and 53% suppression of leukotriene B4 synthesis by ionophore--stimulated neutrophils. Leukotriene B4 suppression persisted for at least two months after treatment was stopped. Treatment with fish oil resulted in measurable, but only limited clinical benefit. For patients entering the trial in relapse (n = 53), there was a significant reduction in corticosteroid requirement after one and two months treatment. There was a trend towards achieving remission (off corticosteroids) faster in the patients on fish oil, although differences were not significant. For patients in remission at trial entry or during the trial (n = 69), there was no significant difference in the rate of relapse by log rank analysis. We conclude that fish oil supplementation produces a modest corticosteroid sparing effect in active disease, but there is no benefit in maintenance therapy.


Effect of aminophenols (5-ASA and 4-ASA) on colonic interleukin-1 generation.

The effect of 5-ASA and 4-ASA, drugs used for the treatment of inflammatory bowel disease, on modulation of experimental colitis and on colonic generation of interleukin-1 was evaluated. Three weeks of treatment with 5-ASA or 4-ASA (50 micrograms/kg) and one week of treatment with 5-ASA significantly decreased colonic interleukin-1 generation and the extent and severity of inflammation in a rat model of colitis induced by trinitrobenzene sulphonic acid. Colonic biopsies were obtained from patients with active ulcerative colitis and organ cultured 24 hours in the absence or presence of the following drugs: sulphasalazine, sulphapyridine, 5-ASA and 4-ASA (25-100 micrograms/ml). Interleukin-1 content in tissue cultured in the presence of 5-ASA (100 micrograms/ml) was two-thirds of its content in tissue cultured in drug free medium and its release into the medium was decreased by 50%. Sulphasalazine 50 micrograms/ml significantly decreased by 33% the tissue content but did not affect interleukin-1 release and a higher dose was not more effective. Sulphapyridine and 4-ASA in doses up to 100 micrograms/ml did not affect either interleukin-1 colonic content or its release into the culture medium. We conclude that pharmacological suppression of colonic interleukin-1 generation may be one, although not the sole mechanism to explain the therapeutic efficacy of 5-ASA in the treatment of inflammatory bowel disease.


[Effects of ifenprodil on the adenosine triphosphatase of guinea pig liver mitochondria].

The effects of ifenprodil on adenosine triphosphatase (ATPase) activity were examined using guinea pig liver mitochondria. 1) Intact mitochondrial ATPase activity was stimulated by ifenprodil in a concentration-dependent manner, this effect being further potentiated with dinitrophenol. The stimulation by ifenprodil appeared with only ATP among four nucleotides as substrate. Mg2+ and Ca2+ attenuated the effect of ifenprodil. Ifenprodil abolished the KCN-induced inhibition. 2) Heat-treated mitochondrial ATPase activity, kept for 60 min at 50 degrees C, was decreased in a concentration-dependent manner by ifenprodil. The inhibitory effect of ifenprodil was abolished by Mg2+ and Ca2+. These results indicate that ifenprodil has two behaviors, acceleration of a latent ATPase and inhibition of an activated ATPase. These findings, together with our previous data, suggest that ifenprodil seems to affect the actions of Mg2+ and Ca2+ on mitochondrial ATPase by directly affecting the membrane, and these mechanisms may be involved in its anti-cyanide effect.


[Pharmacologic profile of urapidil. Consequences for use as an antihypertensive drug].

Urapidil, a phenylpiperazine-substituted derivative of uracil, is an example of an anti-hypertensive which lowers elevated blood pressure via at least two different mechanisms. As such, it belongs to the multifactorial or hybrid anti-hypertensives. Urapidil is a peripherally acting alpha 1-adrenoceptor antagonist, and, in this regard, is comparable with prazosin and its successor compounds (e.g. doxazosin). In addition, urapidil lowers elevated blood pressure via a central mechanism which is new and differs from that of the classical antihypertensives with a central nervous effect (clonidine, etc.). This additional mechanism favourably influences the side effect profile of urapidil.


[Compliance in therapy of schizophrenic patients with neuroleptics--an overview].

In connection with the efforts to improve the prophylaxis against relapse in schizophrenic patients, the topic of non-compliance to therapy with neuroleptics is gaining interest in scientific research. The importance of this problem has been underestimated in the past. Starting with a review of the current state of research, the article provides an overview of the various determinants of compliance. The conclusions made are discussed in terms of optimizing therapy possibilities. Central to this point are less psychopharmacological options but more process- related aspects of the doctor/patient relationship. Fully informed consent of the patient is aimed at; providing him with detailed information on the purposes and the side-effects of neuroleptic therapy is discussed in terms of its consequences regarding compliant behaviour.


Monoclonal antibody OPD4 detects neoplastic T cells but does not distinguish between CD4 and CD8 neoplastic T cells in paraffin tissue sections.

Monoclonal antibody (MoAb) OPD4, reported to preferentially react with benign CD4 T cells in formalin-fixed tissue sections, was examined for its reactivity with 56 T-cell neoplasms after formalin or Bouin's fixation to determine if it also preferentially detects neoplastic CD4 T cells in paraffin tissue sections. Monoclonal antibody OPD4 did not preferentially detect neoplastic CD4 T cells, since it reacted with 22 of 38 (58%) CD4-positive compared with nine of 14 (64%) CD4-negative T-cell neoplasms. However, MoAb OPD4 appears to detect neoplastic T cells in Bouin's-fixed (11 of 20 cases [55%]) about as well as in formalin-fixed (20 of 32 cases [63%]) tissues. Since MoAb OPD4 does not preferentially react with neoplastic CD4 T cells, the utility of this MoAb as a pan-T-cell marker in routinely processed tissues was also explored and compared with that of Leu-22, UCHL-1, and CD3. All four antibodies reacted with approximately the same percentage of T-cell malignancies (51% to 57%). However, examination of different clinicopathologic groups and types of fixative highlighted differences. Monoclonal antibodies OPD4 and Leu-22 reacted with 62%, while CD3 detected only 41% of formalin-fixed, postthymic T-cell neoplasms. OPD4, UCHL-1, and CD3 each reacted with 55%, but Leu-22 recognized only 45% of Bouin's-fixed, postthymic T-cell malignancies. OPD4 reacted with none, but CD3 reacted with all four T-cell lymphoblastic lymphomas. Various antibody combinations were examined to determine an optimal panel for the recognition of T-cell neoplasms in paraffin sections. The combination of MoAbs OPD4 and Leu-22 detected 86% of postthymic T-cell neoplasms in formalin-fixed tissue sections. Furthermore, MoAb OPD4 appears to be relatively specific for T-cell neoplasms, detecting 31 of 56 (55%) T-cell malignancies, while only reacting with two of 39 (5%) B-cell neoplasms. Therefore, while not preferentially reactive with neoplastic CD4 T cells, MoAb OPD4 may be useful as a pan-T-cell marker of postthymic T-cell neoplasms in routinely processed, formalin-fixed tissues, especially when used in conjunction with MoAb Leu-22.


Identification of novel human T-cell receptor V beta gene segments by the anchored-polymerase chain reaction.

We have studied the diversity of the expressed human T-cell receptor (TCR) beta-chain repertoire by analysis of mRNA from unstimulated peripheral blood T-cells. The anchored-polymerase chain reaction (PCR) was used to isolate TCRB transcripts. Of 20 full or near full-length functional transcripts sequenced, two were novel TCRVB gene segments. They have strong sequence similarities to the known TCRBV5, and 8 subfamilies. Southern blot analysis and sequence-specific oligonucleotide hybridization confirmed: a) that these sequences are present in genomic DNA; b) their relationship to the known TCRBV families. A TCRBV sequence similar to a recently identified novel TCRBV24 subfamily was also found. We show by southern blotting that this sequence forms a single member subfamily, and by deletion analysis of T-cell lines, we have mapped this sequence to lie between the genes which encode the TCRBV8.1 and TCRBV5.3 gene segments. The results show that the anchored PCR is a powerful tool in the analysis of the TCR repertoire, which may contain more V gene segments than previously defined.


In vitro anti-tumor activity of anti-c-erbB-2 x anti-CD3 epsilon bifunctional monoclonal antibody.

With the aim of developing an effective cancer immunotherapy for common epithelial cancer, a new class of bifunctional antibody (BFA) was developed; one arm of this BFA recognized c-erbB-2 gene product, and the other arm recognized CD3 epsilon, a T-cell specific surface antigen. Application of this BFA with human peripheral blood lymphocytes exhibited specific anti-tumor activity in vitro on a breast tumor cell line, ZR-75-1, which expressed abundant c-erbB-2 gene product on its cell surface. These results indicate that BFA recognizing an oncogene product on cell surface is a potential new agent for cancer immunotherapy.


Novel complications with HTLV-1-associated myelopathy/tropical spastic paraparesis: interstitial cystitis and persistent prostatitis.

Lower urinary symptoms associated with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are common, but have been regarded as 'neurogenic' due to spinal involvements. However, in some cases, these symptoms are persistent, progressive, and not directly correlated with the severity of other neurologic symptoms of the lower spinal cord. These findings prompted us to locate organic lesions in the lower urinary tract and to correlate them with HTLV-1 infection. Among 35 HAM patients with lower urinary symptoms, we found 4 cases with the symptoms persistent and progressive: 3 with contracted bladder and another with persistent prostatitis. Histological or cytological examinations indicated local lymphocytic infiltrations in the lower urinary tract in all cases: 3 by the infiltration in the bladder and the other by a high concentration of lymphocytes in expressed prostatic secretions. Of 3 cases whose urinary samples were available, 2 showed significant increase in the concentration of urinary anti-HTLV-1 antibody of IgA class. The urinary IgA antibody of the third case was not elevated, but the sample had been obtained after resection of the affected bladder. None of the control cases showed significant anti-HTLV-1 IgA antibody in urine except for a case of gross hematuria due to chemotherapy directed against adult T-cell leukemia. We suggest inclusion of these processes into the spectrum of complications for HAM/TSP. The elevated excretion of anti-HTLV-1 of IgA class in urine may be an indicator of these complications.


Efficacy and effects on pulmonary function tests of weekly 600 mg aerosol pentamidine as prophylaxis against Pneumocystis carinii pneumonia.

A prospective study was designed to evaluate the efficacy and effects on pulmonary function tests of weekly 600 mg aerosolised pentamidine as prophylaxis against Pneumocystis carinii pneumonia (PCP) amongst two groups of patients infected with the human immunodeficiency virus. Group 1 (primary prophylaxis) consisted of patients with either diseases indicative of AIDS other than PCP or whose absolute CD4 positive lymphocyte count was below 200/mm3, and Group 2 (secondary prophylaxis) comprised patients with previous proven episodes of PCP. Fifty-five patients (30-Group 1, 25-Group 2) were studied over a period of 36 months, and no patients reached a study end point of either relapse or death due to PCP after a mean duration of treatment of 14.9 months (range 9-36 months). There were no significant differences between the pulmonary function tests (forced expiratory volume in the first second, forced vital capacity and carbon monoxide diffusion capacity) performed at the start and end of the study on both groups of surviving patients. Ten patients (18%) reported coughing and eight patients (15%) were documented to have bronchoconstriction, which was found to be preventable by prior administration of disodiumcromoglycate. The results showed that weekly 600 mg aerosolised pentamidine is effective and well tolerated for primary and secondary prophylaxis against PCP without additional adverse effects. Further prospective randomized trials are needed to determine whether doses higher than the current recommended 300 mg monthly dosage of aerosolised pentamidine provide more efficacy before such an alternative prophylactic treatment is generally adopted for patients who cannot tolerate other systemic agents.


Activation of the mouse proliferating cell nuclear antigen gene promoter by adenovirus type 12 E1A proteins.

A plasmid carrying the 5'-flanking region (-1584 to +47 with respect to the transcription initiation site) of the mouse proliferating cell nuclear antigen (PCNA) gene was fused with the chloramphenicol acetyltransferase (CAT) gene, and then cotransfected into mouse N18TG2 cells with expression plasmids for the adenovirus type 12 E1 genes. Expression of E1A gene products elevated the CAT expression by 5- to 9-fold, but expression of the E1B gene product did not. RNase protection analysis revealed that the activation of the PCNA gene promoter by E1A was at the transcription step. Both the 13S E1A and the 12S E1A activated the PCNA gene promoter, indicating that the activation domain of E1A resides in a common region(s) of 13S and 12S E1A products. The major target region of E1A was mapped within the 68 base-pair region (-21 to +47) of the PCNA gene, which includes consensus sequences for transcription factors PEA3 and E2F, although the upstream region (-83 to -21) including ATF(CREB)-binding consensus had an additional effect in the transactivation.


Pancreaticojejunostomy and pancreatic leakage after pancreaticoduodenectomy.

Some techniques of pancreaticojejunostomy have been introduced with reference to pancreatic leakage from the pancreaticojejunal anastomosis. Pancreatic leakage (+/- ) occurred in 21.4% of pancreaticojejunostomy patients and pancreatic leakage (+) in 9.5%. The frequency of pancreatic leakage tended to be higher in cases where the pancreatic duct was undilated and the remaining distal segment of the pancreas had a soft tissue. Pancreatic leakage led to secondary complication in none of the patients in our series, but further technical refinement of pancreaticojejunostomy is warranted to reduce the frequency of pancreatic leakage to zero.


Effector cell analysis of human multidrug-resistant cell killing by mouse-human chimeric antibody against P-glycoprotein.

A mouse-human chimeric monoclonal antibody (mAb), MH162, against P-glycoprotein was previously found to be more effective than an all-mouse mAb (MRK16) in lysis of multidrug-resistant (MDR) tumor cells by blood mononuclear cells. The present study was performed to identify the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against MDR cells. The ADCC reaction was assessed by a 6-h 51Cr release assay. Highly purified lymphocytes (greater than 99%), monocytes (greater than 99%) and neutrophils (greater than 96%) were obtained from peripheral blood of the same healthy donors. A comparison of these three effector cell populations showed no difference between MH162 and its all-murine counterpart MRK16 in MDR cell lysis by monocytes or neutrophils. But MH162 was more effective than MRK16 in lymphocyte-mediated lysis of the MDR cells. The lymphocytes responsible for this ADCC had CD16+ Fc receptors. Pretreatment of monocytes with colony-stimulating factors (IL-3, GM-CSF and M-CSF) caused significant increase in their MH162-mediated lysis of MDR cells. Another anti-P-glycoprotein chimeric mAb (MH171) was also more effective than its murine counterpart MRK17 in lymphocyte-mediated lysis of MDR cells. These findings suggest that mouse-human chimeric mAbs may be useful therapeutically for in vivo destruction of MDR cancer cells by the ADCC reaction.


The changing pattern of the splenic lymphocyte subsets in tumor-bearing mice after oral treatment with OK-432.

The aim of this study was to investigate the influence of oral administration of OK-432 on the tumor growth of tumor-bearing mice. In addition, the changing pattern of the splenic lymphocyte subsets of tumor-bearing mice was evaluated by flow cytometry. OK-432 at a dose of 0.1, 1 or 10 KE was administered orally every 3 days or every other day for 30 days to subcutaneously Meth A tumor-inoculated mice. The tumor growth was significantly inhibited in the 1 KE every 3 days group, in the 1 KE every other day group and in the 10 KE every 3 days group. In the 10 KE every other day group, OK-432 inhibited the tumor growth on days 10 and 20, while the agent did not show a marked inhibitory effect on day 30. The percentages of splenic L3T4-positive cells and splenic asialo GM1-positive cells were significantly increased in the 1 KE every other day group, while the Lyt2+/Thy1.2+ ratio was decreased. On the other hand, in the 10 KE every other day group, OK-432 showed no effect on the percentages of splenic L3T4-positive cells and Lyt2+/Thy1.2+ ratio on days 20 and 30. Our results suggest that the antitumor effect of oral administration of OK-432 may be correlated with the changing pattern of L3T4-positive cells and Lyt2+/Thy1.2+ ratio.


Skeletal muscle protein metabolism and serum growth hormone, insulin, and cortisol concentrations in growing steers implanted with estradiol-17 beta, trenbolone acetate, or estradiol-17 beta plus trenbolone acetate.

Skeletal muscle protein degradation, measured by urinary N tau-methylhistidine excretion, and circulating concentrations of growth hormone (GH), insulin (INS), and cortisol (CT) were monitored in steers before and after implantation with estradiol-17 beta (E2; 24 mg) and trenbolone acetate (TBA; 300 mg). Yearling crossbred steers (n = 43) were randomly assigned to four treatment groups in a 2 x 2 factorial arrangement: nonimplanted controls (C); TBA; E2; and TBA plus E2 (TBA+E2). A subgroup (Block 1) of 16 steers was bled on d -12, 31, and 72 after implanting. Deposition of skeletal muscle protein was markedly increased (P less than .001) by E2 and TBA+E2 treatment. This response occurred mainly within the first 40 d after implantation and declined (P less than .001) in concert with decreasing (P less than .01) concentration of serum E2. Anabolic steroid treatment did not affect the rate of skeletal muscle protein breakdown. There was no apparent relationship between reduced serum CT concentration (linear effect; P less than .01) in TBA-treated steers and skeletal muscle protein degradation rate. Blood concentration and pulse activity of INS were not affected by anabolic steroid administration. Both TBA- and TBA+E2-implanted steers displayed a linear decrease (P less than .05) in serum GH concentration over time, which was similar to C. Lowered mean GH concentration resulted from a reduction (TBA main effect; P less than .05) in pulse amplitude of GH. Unlike TBA, TBA+E2, and C, only E2 maintained serum GH concentrations over time. Although increased muscle protein deposition was evident in TBA+E2-treated steers, an obvious causal relationship between this response and circulating GH, INS, and CT was not revealed. These results do not support the concept that combined androgenic agent and estrogen administration effectively reduce bovine muscle protein degradation by static modulation of circulating endogenous anabolic and antianabolic hormones.


Further studies on some physical and biochemical characteristics of asaccharolytic pigmented Bacteroides of feline origin.

The ultrastructure of the appendages of 24 strains of asaccharolytic pigmented Bacteroides spp. of cats was studied by transmission electron microscopy. All strains examined by thin section showed abundant fimbriae, outer membrane vesicles and capsules. Negative staining showed fimbriae which varied from long, fine and wavy in Bact. salivosus and cat Group 2 to shorter, less abundant and thicker fimbriae in cat strains of Bact. gingivalis as well as type strains of Porphyromonas gingivalis and P. asaccharolytica. Capsular material was very thick amorphous in human P. gingivalis, cat strains of Bact. gingivalis and in P. assaccharolytica but fine and fibrillary in preparations of Bact. salivosus and cat Group 2 organisms. Wet india ink preparations showed a large capsule although those of Bact. salivosus and Group 2 appeared largest. Five-day Group 2 broth cultures featured a thick ropy growth consistent with a large accumulation of extracellular slime. Enzymatic activities of the 24 strains measured by API ZYM system as well as the conventional biochemical tests showed it was possible to differentiate reliably Bact. salivosus from the other cat strains of asaccharolytic pigmented Bacteroides species and from human P. gingivalis and P. endodontalis by a combination of these tests. These tests suggest that Bact. salivosus is unlikely to belong to the genus Prevotella. Its place within the genus Porphyromonas is still to be determined.


Effect of mechanical removal of pili on gliding motility of Myxococcus xanthus.

Gliding motility of Myxococcus xanthus is governed by both the adventurous (A) and the social (S) motility gene systems. The presence of pili has previously been shown to be correlated with a genetically intact S-motility system (D. Kaiser, Proc. Natl. Acad. Sci. USA 76:5952-5956, 1979). The purpose of the present work was to study the direct effect of mechanical removal of pill on the social motility of M. xanthus. Depiliation resulted in (i) a loss of streaming motility of A- S+ mutants, i.e., strains which are able to move by virtue of the S-motility system only, (ii) no effect on motility in A+ S- mutants, i.e., strains capable of movement by the A-motility system only, and (iii) a retardation of streaming speed in the wild-type strain (A+ S+). Cell-cell cohesion, another characteristic of social behavior, was not affected by mechanical removal of pill. The observation that mechanical depiliation perturbed the motility of strains which rely on the S-motility system strongly supports a role for pili in social motility of M. xanthus.


Biosynthetic thiolase from Zoogloea ramigera. Mutagenesis of the putative active-site base Cys-378 to Ser-378 changes the partitioning of the acetyl S-enzyme intermediate.

The proposed active-site base Cys-378 of thiolase, responsible for deprotonation of acetyl-CoA, has been converted to a less acidic residue Ser-378 by mutagenesis. Comparison of the CD spectra and dimethyl suberimidate cross-linking experiments of the wild type, mutant Ser-378, and Gly-378 enzymes indicated that there have been no major conformational changes. The Ser-378 enzyme retains 0.1% of the Vmax of wild type in the direction of acetoacetyl-CoA thiolytic cleavage and 0.07% of the Vmax in the Claisen condensation direction. Analysis of the acetyl S-enzyme intermediate partitioning, that is capture of the acetyl enzyme by 1) the thiolate of coenzyme A relative to 2) the C-2 carbanion of acetyl-CoA, is changed to favor reaction 2 in the case of the Ser-378 mutant enzyme.


Evidence for synthesis of scrapie prion proteins in the endocytic pathway.

Infectious scrapie prions are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrP) which is designated PrPSc. A chromosomal gene encodes both the cellular prion protein (PrPC) as well as PrPSc. Pulse-chase experiments with scrapie-infected cultured cells indicate that PrPSc is formed by a post-translational process. PrP is translated in the endoplasmic reticulum, modified as it passes through the Golgi, and is transported to the cell surface. Release of nascent PrP from the cell surface by phosphatidylinositol-specific phospholipase C or hydrolysis with dispase prevented PrPSc synthesis. At 18 degrees C, the synthesis of PrPSc was inhibited under conditions that other investigators report a blockage of endosomal fusion with lysosomes. Our results suggest that PrPSc synthesis occurs after PrP transits from the cell surface. Whether all of the PrP molecules have an equal likelihood to be converted into PrPSc or only a distinct subset is eligible for conversion remains to be established. Identifying the subcellular compartment(s) of PrPSc synthesis should be of considerable importance in defining the molecular changes that distinguish PrPSc from PrPC.


Conalbumin-conjugated silica gel, a new chiral stationary phase for high-performance liquid chromatography.

A new chiral stationary phase using conalbumin (from chicken egg white) was developed for high-performance liquid chromatography. Chiral resolution of racemic azelastine, an antiallergic drug, was achieved on a conalbumin-conjugated silica gel column. The effects of the pH, the concentration of organic solvents and salts in the mobile phase, and the temperature on the capacity factor and resolution of racemic azelastine were examined. This column shows good stability and can separate optical isomers with an aqueous mobile phase. It should be very useful in studies on pharmacokinetics and in clinical chemistry.


Prazosin suppresses development of axonal damage in rats inoculated for experimental allergic encephalomyelitis.

The effectiveness of the alpha 1-adrenergic antagonist prazosin for preventing monoaminergic axonal damage in the spinal cords of rats that were inoculated for experimental allergic encephalomyelitis (EAE) was assessed using immunohistochemistry. Prazosin injections (2 mg, i.p.) given twice daily from day 7 to day 15 postinoculation significantly reduced paralysis, spinal cord inflammation and monoaminergic axonal damage compared to saline injections. A close positive correlation between severity of inflammation and severity of axonal damage was found for both prazosin- and saline-treated rats that were inoculated for EAE. These findings confirmed previous observations of suppression of the development of clinical signs of EAE by prazosin treatment and supported the hypothesis that some factor associated with spinal cord inflammation may be responsible for the bulbospinal monoaminergic axonal damage that occurs during EAE.


Receptor guanylyl cyclases.

Three different guanylyl cyclase cell receptors are known, but others will likely be discovered within the next few years. The general function of these receptors appear to relate to the regulation of fluid volume or fluid movement. New receptors, or possibly the currently known receptors, therefore, may be discovered in areas of the body where fluid volume regulation is important. Such fluids whose volume or composition might be regulated by guanylyl cyclase receptors include synovial fluid, uterine/oviductal luminal fluid, follicular fluid, aqueous humor, cerebral spinal fluid, seminiferous tubule luminal fluid, epididymal luminal fluid, seminal plasma, and airway luminal fluid. The function of the heterodimeric forms of guanylyl cyclase appear to relate to a primary regulation of nitric oxide (or similar molecules) concentrations, which are in turn regulated by a Ca2+/calmodulin-sensitive nitric oxide synthase.


Insulin and glucocorticoid dependence of hepatic gamma-glutamylcysteine synthetase and glutathione synthesis in the rat. Studies in cultured hepatocytes and in vivo.

We reported that glucagon and phenylephrine decrease hepatocyte GSH by inhibiting gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis (Lu, S.C., J. Kuhlenkamp, C. Garcia-Ruiz, and N. Kaplowitz. 1991. J. Clin. Invest. 88:260-269). In contrast, we have found that insulin (In, 1 microgram/ml) and hydrocortisone (HC, 50 nM) increased GSH of cultured hepatocytes up to 50-70% (earliest significant change at 6 h) with either methionine or cystine alone as the sole sulfur amino acid in the medium. The effect of In occurred independent of glucose concentration in the medium. Changes in steady-state cellular cysteine levels, cell volume, GSH efflux, or expression of gamma-glutamyl transpeptidase were excluded as possible mechanisms. Both hormones are known to induce cystine/glutamate transport, but this was excluded as the predominant mechanism since the induction in cystine uptake required a lag period of greater than 6 h, and the increase in cell GSH still occurred when cystine uptake was blocked. Assay of GSH synthesis in extracts of detergent-treated cells revealed that In and HC increased the activity of GCS by 45-65% (earliest significant change at 4 h) but not GSH synthetase. In and HC treatment increased the Vmax of GCS by 31-43% with no change in Km. Both the hormone-mediated increase in cell GSH and GCS activity were blocked with either cycloheximide or actinomycin D. Finally, when studied in vivo, streptozotocin-treated diabetic and adrenalectomized rats exhibited lower hepatic GSH levels and GCS activities than respective controls. Both of these abnormalities were prevented with hormone replacement. Thus, both in vitro and in vivo, In and glucocorticoids are required for normal expression of GCS.


Interaction between alpha 2-adrenergic and angiotensin II systems in the control of glomerular hemodynamics as assessed by renal micropuncture in the rat.

The hypothesis that renal alpha 2 adrenoceptors influence nephron filtration rate (SNGFR) via interaction with angiotensin II (AII) was tested by renal micropuncture. The physical determinants of SNGFR were assessed in adult male Munich Wistar rats 5-7 d after ipsilateral surgical renal denervation (DNX). DNX was performed to isolate inhibitory central and presynaptic alpha 2 adrenoceptors from end-organ receptors within the kidney. Two experimental protocols were employed: one to test whether prior AII receptor blockade with saralasin would alter the glomerular hemodynamic response to alpha 2 adrenoceptor stimulation with the selective agonist B-HT 933 under euvolemic conditions, and the other to test whether B-HT 933 would alter the response to exogenous AII under conditions of plasma volume expansion. In euvolemic rats, B-HT 933 caused SNGFR to decline as the result of a decrease in glomerular ultrafiltration coefficient (LpA), an effect that was blocked by saralasin. After plasma volume expansion, B-HT 933 showed no primary effect on LpA but heightened the response of arterial blood pressure, glomerular transcapillary pressure gradient, and LpA to AII. The parallel results of these converse experiments suggest a complementary interaction between renal alpha 2-adrenergic and AII systems in the control of LpA.


Both immunity and hyperresponsiveness to Pneumocystis carinii result from transfer of CD4+ but not CD8+ T cells into severe combined immunodeficiency mice.

The opportunistic pathogen Pneumocystis carinii (Pc) is considered to be the leading cause of morbidity in patients with AIDS. It is important, therefore, to determine the immunological mechanisms of resistance to Pc. We have taken advantage of the lack of both T and B lymphocytes in severe combined immunodeficiency (scid) mice to determine the critical factors in resistance to spontaneously acquired Pc pneumonia. Using adoptive transfer of unfractionated or fractionated lymphocyte subsets or hyperimmune serum from congenic normal donors, we have demonstrated that effective immunity to Pc results from the action of CD4+ but not CD8+ T cells (in the absence of antibody) or from humoral immunity (in the absence of T cells). However, responses of CD4+ T cells (but not antibody) to already well-established burdens of Pc are often accompanied by a fatal hyperinflammatory reaction. The activity of CD4+ T cells against Pc thus illustrates a broadly applicable principle that T cell immunity represents a critical balance between consequences beneficial and harmful to the host.


Round table conference on ventilatory failure, Brussels, Belgium, March 16-18, 1991.

It was possible to reach agreement on several important issues relating to VF. First, the phenomenon of CO2 retention may have both pathophysiologic and compensatory components. There is increased awareness of the nature, intensity, and significance of the cross-talk between the ventilatory control center and the pump itself, as expressed in breathing pattern and indices of ventilatory drive. We are learning to interpret that information more effectively to assess functional reserve. Second, knowledge concerning the relative importance of various muscle groups is still incomplete, and the impact of disease on muscle function, lung mechanics, and ventilatory control is not fully understood. Dynamic hyperinflation and sleep disturbances provide two clear examples of conditions whose wide-ranging influence on drive, workload, and muscle function was, until quite recently, under appreciated. Finally, there was a general consensus that our therapeutic approaches to VF should be modified to reflect improved understanding of the pathogenesis of CO2 retention and iatrogenic lung injury. In the acute setting, measures to limit alveolar distention, such as controlling airway pressure, revising blood gas targets, and/or using adjunctive methods for blood gas exchange may avoid barotraumatic edema and rupture. The potential for non-invasive ventilation to avert intubation, facilitate ventilator withdrawal, and help patients with chronic VF to achieve compensation without machine dependence is now being actively investigated. This two day conference proved a stimulating forum for interchange of ideas regarding the state of the field, and allowed many opportunities for scientific interaction, both during outside the formal program.(ABSTRACT TRUNCATED AT 250 WORDS)


Do interactional capacities based on observed behaviour interfere with improvement in severely depressed patients? A longitudinal study.

This study examined whether the interaction of severely depressed patients and a psychiatrist was related to the course of depression during hospitalization. Interactional processes were defined on the basis of directly observed behaviour displayed during an interview and by the use of ethological methods. The behavioural structure of the interaction could be described by five factors. The severity of depression and the level of the behaviour factors were assessed just after admission (at baseline) and 10 weeks later. Patients were divided a posteriori into non-improved patients (N = 13) and improved patients (N = 18). Patients who did not improve displayed more 'speaking-effort' (looking, gesticulating, head movements during speech) and less 'active-listening' (head movements and intensive body touching during listening to the psychiatrist) than those who did improve. These factors increased over time in the improved patients but not in the non-improved patients suggesting that they may play a role in the maintenance of depression. The study illustrates the possible value of an ethological approach in the study of interactional processes.


Repair of ionizing radiation damage in primate alpha DNA transfected into rat cells.

The time-course of repair of irradiated primate alpha DNA was studied after transfection and recovery from rat NRK cells. Rat cells were chosen for transfection because they have no alpha DNA. Plasmid pBUC4 alpha 10, containing 10 tandem 172 bp alpha DNA subunits in its 5 kbp DNA, was irradiated and introduced into the rat cells by electroporation. The transfected alpha DNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected alpha DNA in time-course experiments. alpha DNA continuously entered nuclei for processing in the first 2.5 h after transfection. The pool of damaged bases in alpha DNA in NRK nuclei was detectable 2.5 h after transfection. Radiation-induced alpha DNA fragments of electrophoretic mobility intermediate between those of unit nucleotide length were prominent in sequencing gel analyses of alpha DNA for 5-150 min after transfection. These intermediate mobility fragments initially disappeared with T 1/2 of 6-20 min. The alpha DNAs of intermediate mobility are presumed to be intermediates in DNA repair. Residual DNA base damage which had not been processed in the transfected cells could later be unmasked in vitro by conversion to strand breaks by beta-elimination using heat and piperidine or endonuclease III of E. coli. Irradiation of the recipient NRK cells with 5 Gy 4 hours before transfection prolonged the time during which intermediate mobility species could be found, consistent with the increased frequency of intermediate mobility species observed in DNA of monkey CV-1 cells pretreated with small doses of radiation before 300 Gy (Bases et al. 1990).


Radical effects on mutation spectra in lambda phage.

Mutations in the lambda repressor gene cI (710 bp) were induced by 60Co-gamma radiation in dissolved lambda phage DNA. After in vitro DNA packaging to lambda phage particles (pack phage) and phenotypic expression of the mutants, DNA was sequenced directly. Two-thirds of mutations were located in the amino terminus region of the gene without any signs of hotspots. Changes consisted of (+1) insertions (25%) and base substitutions (75%). Transitions were exclusively G/C to A/T. Transversions were mostly G/C to C/G and few G/C to T/A. We did not find A/T to T/A transversions, A/T to G/C transitions, deletions and gross rearrangements. In most of the base substitutions a pre-existing base pair had been replaced by an A/T pair; this might come from 'non-instructional sites' like abasic sites. Several mechanisms for base substitutions are considered.


Dermatofibrosarcoma protuberans: 4 years after local trauma.

Dermatofibrosarcoma protuberans (DFSP) is a low grade, fibrohistiocytic malignant soft tissue tumor that arises infrequently in the foot and ankle region. It most frequently demonstrates a "storiform" histologic pattern, and if incompletely excised, may be recurrent, more anaplastic and metastatic. Soft tissue plain film radiography, as well as magnetic resonance imaging (MRI) help outline the tumor and involved tissues, which is beneficial in staging, determining surgical approach and evaluation of therapy. Definitive diagnosis is made by biopsy. A dermatofibrosarcoma protuberans is reported in the foot of a 14-year-old female patient, 4 years after trauma to the site.


Detection of single-strand breaks and base damage in DNA of blood cells from leukaemia patients receiving chemo- and radiotherapy.

Chemotherapy combined with total-body irradiation (TBI), a conditioning regimen for bone-marrow transplantation (BMT), causes lesions in the cellular DNA of the patients treated. To understand possible consequences of the DNA damage induced during such treatment, information is required about the nature of the damage, the level of induction and its persistence, and about the importance of the various lesions for cell-lethality and/or mutation induction. Recently, we developed a sensitive immunochemical method to quantify single-strand breaks (SSB) in the DNA of mammalian cells. In addition, a modification of the so-called alkaline elution technique was introduced which allows quantification of SSB together with base damage (SSB+BD). These methods have now been applied successfully to study the in vivo induction and repair of DNA damage in WBC of leukaemia patients who prior to BMT were treated with cyclophosphamide (CY) and received TBI. SSB and SSB+BD were determined after two treatments with CY (60 mg kg-1) followed by TBI (4.5-8.6Gy). The CY treatments gave rise to rather persistent SSB. In addition to these, radiation-induced SSB and SSB+BD could be detected shortly after TBI. However, 105 min after TBI, these SSB could be observed no longer, as a result of rapid repair.


Oncogenes in X-ray-transformed C3H 10T1/2 mouse cells and in X-ray-induced mouse fibrosarcoma (RIF-1) cells.

In order to better understand the molecular basis of X-ray induced carcinogenesis we have investigated RNA levels of oncogenes in an X-ray transformed C3H 10T1/2 fibroblast line (XTD) and RIF-1 cells isolated from an X-ray-induced fibrosarcoma in a C3H mouse. Steady-state levels of K-ras, H-ras, N-ras, abl, sis, src, and fos were unchanged in the X-ray-transformed cells compared with non-transformed C3H 10T1/2 cells. However, myc and raf mRNA levels were increased dramatically in the transformed cells. Data further suggests a possible alteration in processing of raf RNA in the XTD cells. Southern blot analysis of secondary transfectants induced with XTD DNA indicated that the oncogenic phenotype did not segregate with the myc or raf loci; nor with nine other oncogenes analysed.


Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation.

This paper presents an analysis of the utility of fluorescence in situ hybridization (FISH) with whole-chromosome probes for measurement of the genomic frequency of translocations found in the peripheral blood of individuals exposed to ionizing radiation. First, we derive the equation: Fp = 2.05fp(1-fp)FG, relating the translocation frequency, Fp, measured using FISH to the genomic translocation frequency, FG, where fp is the fraction of the genome covered by the composite probe. We demonstrate the validity of this equation by showing that: (a) translocation detection efficiency predicted by the equation is consistent with experimental data as fp is changed; (b) translocation frequency dose-response curves measured in vitro using FISH agree well with dicentric frequency dose-response curves measured in vitro using conventional cytogenetic procedures; and (c) the genomic translocation frequencies estimated from FISH measurements for 20 Hiroshima A-bomb survivors and four workers exposed to ionizing radiation during the Y-12 criticality accident are approximately the same as the translocation frequencies measured using G-banding. We also show that translocation frequency dose response curves estimated using FISH are similar for Hiroshima A-bomb survivors and for first division lymphocytes irradiated in vitro. We conclude with a discussion of the potential utility of translocation frequency analysis for assessment of the level of acute radiation exposure independent of the time between analysis and exposure.


Radioprotection mediated by the haemopoietic stimulation conferred by AM5: a protein-associated polysaccharide.

The haemopoietic and radioprotective effects of a protein-associated polysaccharide named AM5, have been studied following i.v. injection in mice. A dose-related accumulation of the splenic granulocyte-macrophage colony-forming units (CFU-GM) and colony-forming units in the spleen (CFU-S) was observed in mice treated with doses ranging from 0.1 to 0.4 mg/kg of AM5. The accumulation of splenic CFU-S, CFU-GM and BFU-e (erythroid burst-forming units) was always maximal 5 days after treatment with 0.4 mg/kg of AM5, with increases over control values between 300% and 500%. When the number of haemopoietic progenitors was quantified in the bone marrow, only slight increases of CFU-S were obtained, corresponding to the administration of low doses of AM5 (0.1 mg/kg). However, significant increases of circulating CFU-S were observed following administration of higher doses of AM5, suggesting a mobilization of haemopoietic progenitors from this organ. A faster recovery of spleen CFU-GM was observed in mice treated with 0.4 mg/kg of AM5 3 days or 1 day prior to a sublethal irradiation, and at this later time AM5 produced a significant survival enhancement from 10% to 90% in mice irradiated with 7.6 Gy X-rays. This effect was correlated with an increase in the nadir of leucocytes, characteristic of the radiation syndrome.


Radioreductive effect of bestatin (Ubenimex) in BALB/c mice.

The radioreductive effect of Bestatin (Ubenimex), a biological response modifier, was examined in BALB/c mice. Consecutive daily administrations from day 0 to day 3 after lethal irradiation resulted in a dose reduction factor (DRF) of 1.03. In haematological studies Bestatin enhanced the recovery of peripheral leucocytes in sublethally irradiated mice by activating colony-stimulating factors (CSF) in serum, leading to enhanced recovery of GM-CFC. There was no enhancement of CSF in BALB/c nude mice. On the other hand, Bestatin showed no effect on day 12 CFU-S and day 7 CFU-S after the sublethal irradiation, though there was a trend that the drug stimulated numbers of day 7 CFU-S after irradiation. These results strongly suggest that Bestatin enhanced the CSF activity by modulating T cell function, resulting in the augmentation of peripheral leucocytes and prolongation of survival of irradiated mice. This drug now used in the clinic proved to be modestly effective.


Cytotoxicity, uptake and dissolution of 241AmO2 particles in dog alveolar macrophages in vitro.

Because alveolar macrophages play a role in the pulmonary clearance of inhaled particles, experiments were conducted to investigate the toxicity of 241AmO2 particles to alveolar macrophages and the role of these macrophages in the dissolution of 241AmO2 particles. Beagle dog pulmonary alveolar macrophages obtained by bronchopulmonary lavage were exposed in vitro to selected concentrations of 241AmO2. Macrophage viability determined by trypan-blue dye exclusion technique and the ability of the alveolar macrophages to phagocytose opsonized sheep red blood cells were the measures of 241AmO2 toxicity. The uptake of 241AmO2 particles was studied as a function of time. In addition, the dissolution of 241AmO2 by macrophages was determined for periods up to 72 h. After 20 h exposure to concentrations of 241Am higher than 4.63 kBq/ml, the phagocytic ability of macrophages was reduced, whereas no significant change in cell viability was observed at this concentration. Significant cell killing occurred at concentrations higher than 18.5 kBq/ml. After 72 h in the cultures, 10% of the 241AmO2 was dissolved by the alveolar macrophages. These findings imply that the inhalation of radioactive particles such as 241AmO2 particles might cause a reduction in the alveolar macrophage population in the lungs. In addition, the dissolution of 241AmO2 particles by alveolar macrophages might play a role in the short-term pulmonary clearance of inhaled 241AmO2 particles in the beagle dog.


Model of DNA damage induced by radiations of various qualities.

A theoretical model permitting estimation of yields of various DNA damages induced by radiations of varying qualities is described. It is based on the Monte-Carlo track structure simulation and DNA structure, and links physical, physicochemical and chemical stages of radiation action. Direct and indirect effects are not strictly distinguished but treated cooperatively. Good agreement between calculated and measured initial yields of double-strand breaks was observed. Other multiple and single damages of DNA are studied. When radiation quality is changed there are quantitative and qualitative transitions in the damage spectrum. The proportion of multiple damage in the damage spectrum is about 30% for low-LET radiations and increases considerably with increasing ionization density.


An interspecies comparison of the phagocytosis and dissolution of 241AmO2 particles by rat, dog and monkey alveolar macrophages in vitro.

Because of the role that alveolar macrophages (PAM) play in the pulmonary clearance of inhaled particles via mechanical transport and dissolution, understanding the uptake and dissolution of particles by these cells might provide insight into the mechanisms of particle dissolution in lungs of various species and hence facilitate the extrapolation of animal data to humans. Therefore, experiments were conducted to study the phagocytosis and dissolution of 241AmO2 particles by rat, dog and monkey PAM in vitro. Rat, dog and monkey PAM were exposed for up to 72 h to 0.19, 0.93 or 4.6 kBq/ml 241Am, after which cell viability was determined. The 241Am concentration, 4.63 kBq/ml, was used for the phagocytosis and dissolution experiments. The phagocytosis and dissolution of 241AmO2 particles were followed up to 20 and 72 h, respectively. Dog and monkey PAM took up 241AmO2 particles at similar rates, whereas rat PAM phagocytosed only 60% of the amount phagocytosed by dog and monkey PAM at 20 h. The PAM of the three species dissolved 241AmO2 particles at similar rates; 8-10% was dissolved by 72 h. The results of the 241AmO2 uptake in vitro may reflect in vivo situations, where the differences in uptake seen in vitro would probably diminish at later times after exposure. The dissolution results imply that the dissolution of 241AmO2 particles by alveolar macrophages of the three species might be species-independent. This, at least, might be true for dog and monkey, where in vivo data have shown that 241AmO2 was translocated similarly in both species. Finally, the alveolar macrophage culture system provides a useful simulation to investigate uptake and dissolution of inhaled particles.


Incidence of brain tumours in rats exposed to an aerosol of 239PuO2.

Incidence of brain tumours was investigated in 3390 female and male Wistar rats exposed to an aerosol of 239PuO2, or as sham-exposed controls. Lung doses ranged from 0.05 to 22 Gy. In females, six brain tumours were found in 1058 control rats (incidence, 0.6%) and 24 brain tumours in 2134 rats exposed to Pu (incidence, 1.1%); the survival-adjusted level of significance was p = 0.29 for comparing control with exposed females. In males, two brain tumours were found in 60 control rats (incidence, 3.3%) and seven brain tumours in 138 rats exposed to Pu (incidence, 5.1%); the survival-adjusted level of significance was p = 0.33. Brain tumour incidence was about five times greater in male than in female rats (p = 0.0001), demonstrating a highly significant sex difference in brain tumour incidence. Tumour types were distributed similarly among control and Pu-exposed groups of both sexes; most tumours were astrocytomas. Mean lifespans for rats with brain tumours were not significantly different between control and Pu-exposed rats. Plutonium was not detected on autoradiograms of the brain. These results, like those for plutonium workers, show an increase of brain tumours which cannot be demonstrated statistically to be related to radiation exposure.


Monoamines and neuropeptides as transmitters in the sedentary polychaete Sabellastarte magnifica: actions on the longitudinal muscle of the body wall.

Pharmacological studies on the body wall musculature of the sedentary polychaete Sabellastarte magnifica show a potential neurotransmitter role for monoamines and neuropeptides in this organism. All catecholamines induced contraction of longitudinal muscle strips, while serotonin and the neuropeptides FMRFamide and substance P caused a relaxation of both resting and active muscle. In addition, we demonstrate catecholaminergic and serotonergic pathways in the nervous system of this sabellid, using immunohistochemistry and catecholamine-induced fluorescence. The presence of neuropeptide-containing fibers in the nervous system of this polychaete has been previously reported. Together these results suggest that catecholamines act as excitatory transmitters on the longitudinal muscle cells of the body wall of S. magnifica, while serotonin and FMRFamide, and possible substance P, are inhibitory transmitters. The possibility of coexistence of serotonin and FMRFamide within the same neuronal cell bodies and fibers of this polychaete is also explored.


Isolation and characterization of a ntrC mutant of Bradyrhizobium (Parasponia) sp. ANU289.

A mutant of Bradyrhizobium (Parasponia) sp. ANU289 affected in the regulation of nitrogen metabolism was isolated. The mutant, designated ANU293, was unable to induce ammonium transport (Amt), nitrate reductase (NR) or glutamine synthetase II (GSII) activities under conditions that induce these activities in the wild-type. However, glutamine synthetase I (GSI), which is expressed constitutively in the wild-type, was present at normal levels in the mutant. The mutant also retained the ability to fix nitrogen in vitro and in planta, although nodule development on siratro (Macroptilium atropurpureum) was retarded. Southern blot analysis showed that ntrC, the product of which is involved in regulation of nitrogen metabolism, is the site of pSUP1021 insertion in ANU293. These results indicate that the transcriptional activator NtrC is required for the expression of Amt, NR and GSII, but not GSI or nitrogenase in Bradyrhizobium (Parasponia) sp. ANU289.


Evaluation of a ribosomal RNA gene probe for the identification of species and subspecies within the genus Staphylococcus.

To evaluate a 16S rRNA gene probe for the identification of staphylococcal species and subspecies, we have augmented previous studies involving 12 staphylococcal species by analysing the remaining 16 species currently classified in the genus Staphylococcus. HindIII- and EcoRI-restricted DNA of isolates from validly described species of Staphylococcus was probed with radiolabelled plasmid pBA2 containing 16S rDNA from Bacillus subtilis. The Dice coefficient was used to assess similarity between the 74 HindIII- and the 81 EcoRI-hybridization patterns obtained from a total of 271 isolates belonging to 31 staphylococcal taxa (28 species, of which three include two subspecies). The use of HindIII yielded a better discrimination of the staphylococci than the use of EcoRI. All of the isolates belonging to the same species or subspecies, except S. hyicus isolates, were recovered as homogeneous clusters using their HindIII hybridization patterns. The phenotypically close taxa were clearly distinguished. Thus, the method presented in this study constitutes a powerful tool for the identification of taxa within the genus Staphylococcus.


The molecular analysis of synonymy among medically important yeasts within the genus Candida.

Three sets of medically important yeasts, Candida albicans, C. tropicalis, and C. krusei, were compared with their putative synonyms (C. langeronii and C. claussenii, C. paratropicalis, and Itssatchenkia orientalis, respectively) to determine if these synonyms are genetically distinguishable from each other. Pulsed-field electrophoresis and hybridization to species-specific probes were used to accomplish this goal. The species-specific probes for C. albicans and C. tropicalis have been previously described (27A and CT13.8, respectively) whereas the probe for C. krusei (CK3) was cloned in this study. No distinguishing characteristics between synonyms were identified, thus supporting the current taxonomic treatment of these organisms.


Effect of pH on glutamine content derived from exogenous glutamate in astrocytes.

A shift in pH from 7.4 to 7.8 in the incubation solution caused a 3.4-fold increase in the free glutamine content of mouse cerebral astrocytes that were incubated with glutamate (100 microM) and ammonium (100 microM). This large and reversible steady-state increase in glutamine content was accompanied by smaller transient increases in the following: (a) net formation of glutamine; (b) clearance of glutamate from the incubation solution; and (c) glutamate content. The content of glutamine was reduced markedly by omission of either glutamate or ammonium from the incubation solution, or by inhibition of glutamine synthetase activity with methionine sulfoximine. The rate at which glutamine was exported from the astrocytes was unaffected by the pH change. The effects of pH on the concentration of free ammonia or on glutamate uptake do not appear to mediate the increase in glutamine content. Uptake of exogenous glutamine was little affected by the pH change. Therefore, possible mediation of the effect by an increase in intracellular pH must be considered. The response to altered pH described here may provide a cellular basis for the increased level of brain glutamine observed in hyperammonemia.


Distribution of protein phosphatase inhibitor-1 in brain and peripheral tissues of various species: comparison with DARPP-32.

The distribution of inhibitor-1, a cyclic AMP-regulated inhibitor of protein phosphatase-1, was analyzed in various brain regions and peripheral tissues of various species by immunolabeling of sodium dodecyl sulfate-poly-acrylamide gel transfers using specific antibodies. The distribution of inhibitor-1 was directly compared to that of DARPP-32, a structurally related cyclic AMP-regulated inhibitor of protein phosphatase-1. In rat CNS, a single immunoreactive protein of M(r) 30,000, identified as inhibitor-1, was widely distributed. In contrast, DARPP-32 was highly concentrated in the basal ganglia. Inhibitor-1 was detected in brain tissue from frog (M(r) 27,000), turtle (M(r) 29,000/33,000), canary (M(r) 26,000), pigeon (M(r) 28,000), mouse (M(r) 30,500), rabbit (M(r) 26,500), cow (M(r) 27,000), and monkey (M(r) 27,500), but not from goldfish. Inhibitor-1 was detected at various levels in most peripheral tissues of the species studied; however, it was not detectable in certain tissues of particular species (e.g., rat and cow liver). DARPP-32 was detected in brain tissue of all the species tested except frog and goldfish, but was not detectable in most peripheral tissues. Both inhibitor-1 and DARPP-32 were concentrated in the cytosol and synaptosomal cytosol of rat striatum. The developmental expressions of inhibitor-1 and DARPP-32 in rat striatum differed: the level of inhibitor-1 peaked in the first postnatal week and then declined by the third postnatal week, whereas the level of DARPP-32 increased to a peak level by the third postnatal week and remained elevated thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)


Striatal protection induced by lesioning the substantia nigra of rats subjected to focal ischemia.

Unilateral 6-hydroxydopamine lesion of the substantia nigra reduced the volume of striatal necrosis and suppressed the increase in extracellular glutamate concentration in the striatum induced by middle cerebral artery occlusion in rats. These results indicate that the dopaminergic nigrostriatal pathway is highly involved in the vulnerability of the striatum to ischemia and suggest that glutamate-dopamine interactions may play a key role in the striatal ischemic insult.


Glutamate inhibits adenylate cyclase activity in dispersed rat hippocampal cells directly via an N-methyl-D-aspartate-like metabotropic receptor.

Three major subtypes of glutamate receptors that are coupled to cation channels--N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors--are known as ionotropic receptors in the mammalian CNS. Recently, an additional subtype that is coupled to GTP binding proteins and stimulates (or inhibits) metabolism of phosphoinositides has been proposed as a metabotropic receptor. Incubation of dispersed hippocampal cells from adult rats with glutamate or NMDA decreased forskolin-stimulated cyclic AMP (cAMP) accumulation; half-maximal effects were obtained with 5.6 +/- 2.2 and 6.4 +/- 2.3 microM, respectively. Kainate and quisqualate were less potent. The effect of glutamate was antagonized by 2,3-diaminopropionate and 2-amino-5-phosphonovalerate, NMDA/glutamate receptor antagonists, but not by 0.5 microM Joro spider toxin, a specific blocker of the AMPA receptor. The inhibitory effect of glutamate on cAMP formation was not blocked by 2 microM tetrodotoxin or by the absence of Ca2+. In hippocampal membranes, glutamate, similar to carbachol, inhibited adenylate cyclase activity in a GTP-dependent manner. These findings suggest that the glutamate inhibition of adenylate cyclase is direct and is not due to a result of the release of other neurotransmitters. The effect of glutamate on cAMP accumulation was observed in an assay medium containing 0.7 mM MgCl2, which is known to inhibit both ionotropic NMDA receptor/channels in the hippocampus and metabotropic NMDA receptors in the cerebellum. The inhibitory effect of glutamate was abolished by pertussis toxin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Activation by nitric oxide of guanylate cyclase in endothelial cells from brain capillaries.

Endothelial cells (ECs) from brain microvessels respond to exogenous nitric oxide (NO) donor molecules (N-ethoxycarbonyl-3-morpholinosydnonimine and sodium nitroprusside) with large (greater than 15-fold) increases in cyclic GMP (cGMP) levels. Comparable actions of sodium nitroprusside were observed in vascular smooth muscle cells and in neuroblastoma cells. Coculturing brain capillary ECs in the presence of N1E-115 neuroblastoma cells increased their cGMP levels fourfold. A further increase was observed in the presence of 50 nM neurotensin, although brain capillary ECs lack receptor sites for neurotensin. The neuroblastoma cell-dependent formation of cGMP was suppressed by 0.1 mM L-NG-monomethylarginine, indicating that NO, produced by N1E-115 cells in response to neurotensin, activated guanylate cyclase in brain capillary ECs. Similarly, culturing brain capillary ECs in the presence of aortic ECs increased their cGMP content in a manner that was amplified by bradykinin and that was inhibited by L-NG-monomethylarginine. Bradykinin had no action in pure cultures of brain capillary ECs. It is concluded that brain capillary ECs express high levels of guanylate cyclase activity that could be activated by exogenous NO donor molecules and by NO produced by neuroblastoma cells and by aortic ECs in response to specific agonists. Brain capillary ECs are thus potential target cells for brain-derived NO.