A thromboxane A2 synthetase inhibitor retards hypertensive rat diabetic nephropathy. Spontaneously hypertensive rats (SHR) were injected with streptozotocin (STZ-SHR) to induce diabetes. The effect of DP-1904, a thromboxane A2 synthetase inhibitor, on diabetic nephropathy was then studied by administering it for 5 months (1 or 10 mg/kg). DP-1904 did not affect renal 6-keto prostaglandin (PG)F1 alpha production in STZ-SHR, but markedly inhibited renal thromboxane (TX) B2 production, so that the 6-keto PGF1 alpha/TXB2 ratio was significantly increased (P less than 0.05). STZ-SHR showed significant uraemia and proteinuria, plus increases in urinary gamma-glutamyl-transpeptidase and urinary N-acetyl-beta-glucosaminidase. DP-1904 significantly decreased (P less than 0.01) the urinary changes. STZ-SHR also showed an increase in mesangial periodic acid-Schiff-positive substance and in relative renal weight, both of which were significantly inhibited by DP-1904 (P less than 0.05). Thus, DP-1904 inhibited both TXB2 production and the progression of renal damage in STZ-SHR.


Variable alpha 2-adrenoceptor-mediated inhibition of bronchoconstriction and peptide release upon activation of pulmonary afferents. In the present study, we evaluated the possible regulation by alpha 2-receptor agonists (SKF 35886 and UK 14304) of peptide release and functional responses upon sensory nerve activation in the guinea-pig lung. The peptide release and bronchoconstriction caused by antidromic vagal nerve stimulation (low frequency, 1 Hz), and a low concentration of capsaicin (10(-8) M) and resiniferatoxin (3 x 10(-10) M) were attenuated by alpha 2-adrenoceptor agonists. The effects of capsaicin and nicotine in high concentrations and high frequency nerve stimulation (10 Hz) were influenced to a much smaller extent by alpha 2-adrenoceptor stimulation. The calcitonin gene-related peptide release evoked by bradykinin but not the functional effects was inhibited by alpha 2-adrenoceptor activation. It is concluded that alpha 2-adrenoceptor stimulation mainly inhibits the release of mediator and/or the bronchoconstriction caused by moderate activation of sensory nerves. It is necessary to measure mediator release directly to reveal prejunctional effects and not to rely only on indirect functional evidence.


Nitric oxide is the endogenous neurotransmitter of bronchodilator nerves in humans. In human airways, there is a prominent neural bronchodilator mechanism which is non-adrenergic. In human tracheal segments, we have demonstrated that this response is mediated entirely by nitric oxide (NO), since an inhibitor of NO synthesis, L-NG-nitroarginine methyl ester (L-NAME) (10(-4) M) abolishes this neural response. Identification of the neurotransmitter of this bronchodilator pathway may now make it possible to study its role in physiological control of airway calibre and in airway disease.


Cardiovascular responses of sinoaortic-denervated rats to intracerebroventricular injection of alpha 1- and alpha 2-adrenoceptor agonists. The aim of the present work was to analyse the cardiovascular responses induced by i.c.v. administration of the alpha 1- and alpha 2-adrenoceptor agonists, phenylephrine and clonidine, respectively, in conscious normal and sinoaortic-denervated rats. Sinoaortic denervation involves changes in central and peripheral catecholaminergic pathways. Clonidine (1-10 micrograms) produced a dose-dependent rise in blood pressure and a bradycardiac response in sham-operated animals, whereas in sinoaortic-denervated rats it provoked a brief rise in blood pressure followed by a marked fall as well as bradycardia. The responses involved mostly activation of central alpha 2-adrenoceptors, but the blood pressure responses induced by clonidine in sinoaortic-denervated rats may also have involved alpha 1-adrenoceptors. The bradycardia induced by the alpha 2-agonist in both groups of rats involved preferentially central alpha 2-adrenoceptors but also partially stimulated alpha 1-adrenoceptors. Phenylephrine, at a dose of 10-60 micrograms, induced a rise in blood pressure and a bradycardiac response while 90 micrograms produced a biphasic pressure response (early transient rise followed by a fall) as well as bradycardia in both sham-operated and sinoaortic-denervated animals. Phenylephrine activated alpha 1-adrenoceptors in every case, but the fall in blood pressure and the bradycardia also involved alpha 2-adrenoceptors. The responses were significantly higher in the sinoaortic-denervated rats than in the sham-operated. Our findings suggest that arterial baroreceptor reflexes can modify the effects of alpha-agonists initiated in the central nervous system. Sinoaortic denervation preparations enable one to unmask the depressor response to clonidine and also demonstrate the true magnitude of the phenylephrine response.


Classification of the beta-adrenoceptor subtype in the rat portal vein: effect of altered thyroid hormone levels. The potencies of the beta 1-adrenoceptor agonist, noradrenaline, and the beta 2-adrenoceptor agonist, fenoterol, at beta-adrenoceptors in portal vein were examined using preparations isolated from control, methimazole-treated or l-thyroxine-treated rats. Tissues were preincubated with phenoxybenzamine (1 mumol/l) to block alpha-adrenoceptors and neuronal and extraneuronal uptake. Fenoterol was approximately 400 times more potent than noradrenaline (-log IC50 7.85 vs. 5.26) in inhibiting the spontaneous contractions of the portal vein. The beta 2-adrenoceptor antagonist, ICI 118,551, was approximately 3000 fold more potent than the beta 1-adrenoceptor antagonist, atenolol, in blocking the effects of fenoterol (pA2 9.32 and 5.88 respectively) and 400 times more potent in antagonising noradrenaline (pA2 8.96 vs. 6.23). Treatment of rats with methimazole led to decreased myogenic tone, and treatment with thyroxine to increased tone. beta-Adrenoceptor binding densities and the relative potencies of the agonists and antagonists used were unaffected by methimazole treatment. Thyroxine administration was also without effect on the relative potencies of these compounds. Our data indicate that although the portal vein is a target tissue for thyroxine, as indicated by its influence on myogenic tone, the beta-adrenoceptor population in this preparation, confirmed to be of the beta 2-subtype, is unaffected.


Terguride stimulates locomotor activity at 2 months but not 10 months after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment of common marmosets. The mixed dopamine (DA) agonist/antagonist terguride acts as a DA antagonist on normosensitive receptors but shows DA agonistic properties at supersensitive DA receptors. Such a compound could offer an alternative to the treatment of Parkinson's disease with indirect or direct DA agonists. The present study compares the actions of terguride, 4-12 mg/kg i.p., in naive common marmosets with its effects in animals rendered parkinsonian by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 2 months or 10 months previously, in order to test its antiparkinsonian efficacy. Terguride reduced locomotor activity in naive common marmosets, similar to its effects in rodents and in line with the DA antagonistic activity of the compound. In marmosets treated with MPTP 2 months previously and exhibiting pronounced behavioural motor deficits, terguride stimulated locomotor activity, showing DA agonistic properties under these conditions. In contrast, the locomotor activity of animals that had recovered from MPTP treatment 10 months previously was not altered by terguride. It is concluded that terguride has anti-akinetic efficacy in this primate model of Parkinson's disease. In addition, terguride offers a unique opportunity to differentiate, pharmacologically, the extent of dopaminergic recovery from MPTP treatment in this primate species.


The importance of P and type 1 fimbriae for the persistence of Escherichia coli in the human gut. The faecal Escherichia coli flora was studied in 89 infants. Each infant was followed with a mean of 12 faecal samples (range 5-21) between 0 and 18 months of age. All isolates were assayed for P fimbriae and biochemically phenotyped and the persistence of each strain (phenotype) in the infant's gut was determined. In a subset of strains the occurrence of type 1 fimbriae and adherence to HeLa cells was studied. Thirty-one per cent of isolates belonging to strains colonizing for longer than 6 months expressed P fimbriae compared to 19% of the isolates from strains colonizing 1-6 months or transient strains colonizing less than 1 month. Type 1 fimbriae and adherence to HeLa cells occurred similarly often in all groups of strains. We conclude that P fimbriae, but not type 1 fimbriae or HeLa cell adherence seemed to contribute to the ability of the E. coli strain to colonize the human intestine.


Amino acids and N-acetyl-aspartyl-glutamate as neurotransmitter candidates in the monkey retinogeniculate pathways. The identity of the neurotransmitter(s) in the mammalian retinogeniculate pathway is unclear. To investigate the possibility that some amino acids and certain dipeptides, such as N-acetyl-aspartyl-glutamate (NAAG), fulfill this function, changes in their concentration were measured in the optic tract, and the parvocellular and magnocellular segments of the LGNd of six monkeys (Macaca fascicularis), seven days after right optic tractotomy. The LGNd was studied also in two additional macaques, three months after occipital lobectomy. Tissue was frozen within five minutes of death, regions were dissected with the micropunch technique, and substances were analyzed by HPLC. Optic tractotomy induced significant, large reductions in NAAG, glutamate and aspartate in the optic tract distal to the lesion. Significant decreases in NAAG were also measured in the LGNd, and these changes were apparent in both the parvocellular and magnocellular segments. A small reduction in glutamate reached significance in the parvocellular laminae, and that of aspartate only approached significance in the magnocellular division. Occipital lobectomy produced large declines in aspartate and glutamate in the LGNd. The results of optic tractotomy support the role of NAAG as a neurotransmitter candidate in the monkey retinogeniculate pathways; its significant decrease in both geniculate segments suggests that both P- and M- retinal axons utilize this substance. Although at times the reductions in glutamate or aspartate failed to reach significance, their role cannot be excluded. The findings after occipital lobectomy strongly favor these latter substances as corticogeniculate and/or geniculocortical transmitters.


Non-cholinergic effects of acetylcholinesterase in the substantia nigra: a possible role for an ATP-sensitive potassium channel. Within the substantia nigra acetylcholinesterase has non-cholinergic actions that can be demonstrated at both behavioural and cellular levels: the aim of this study was, thus, to explore, in the in vitro guinea pig substantia nigra, the ionic mechanisms which mediate these non-classical phenomena. Acetylcholinesterase had a reversible hyperpolarizing action, via an opening of potassium channels, on a selective population of nigral neurons. These neurons could be identified by an ability to generate bursts of action potentials and by a sensitivity to either amphetamine or to a reduction of glucose in the perfusing medium. The acetylcholinesterase-induced hyperpolarization could not be attributed to a contaminant in the exogenous solution, since a highly purified preparation was even more potent. Furthermore, enzymatic action of any kind could be eliminated as boiled acetylcholinesterase was equally efficacious. The effect of acetylcholinesterase was not subject to tachyphylaxis and was resistant to blockade of potassium channels with tetraethylammonium: since both these phenomena are features of the D2 autoreceptor for dopamine within the substantia nigra, it seems unlikely that acetylcholinesterase is operating on the same target as dendritically released local dopamine. On the other hand, the actions of acetylcholinesterase were enhanced by low glucose and blocked by the sulfonylurea, tolbutamide. These results strongly suggest that acetylcholinesterase can exert a nonenzymatic action and that this action, in the substantia nigra, is mediated by an ATP-sensitive potassium channel.


Evidence that non-NMDA receptors are involved in the excitatory pathway from the pedunculopontine region to nigrostriatal dopaminergic neurons. Extracellular single-neuron recordings were obtained from electrophysiologically identified nigrostriatal neurons in chloral hydrate anesthetized rats, in order to test the hypothesis that excitatory amino acid receptors are involved in responses of these neurons to electrical stimulation of the pontine region where the pedunculopontine nucleus (PPN) is located. The effects of iontophoretic application of excitatory amino acids and their antagonists as well as of cholinergic antagonists were tested on the fast orthodromic excitation of nigrostriatal neurons evoked by stimulation of the PPN region. The N-methyl-D-aspartate (NMDA) receptor antagonist D-alpha-aminoadipic acid as well as the cholinergic receptor antagonists mecamylamine and atropine failed to suppress the synaptic excitation of nigral neurons. The NMDA receptor antagonist DL-2-amino-5-phosphonovalerate exerted a weak depressant action on the synaptic response in a few neurons only. On the contrary, the broad spectrum antagonists of excitatory amino acid receptors kynurenic acid and gamma-D-glutamyl-amino-methyl-sulphonate were found to block simultaneously both the synaptic excitation and the neuronal responses to iontophoretic pulses of glutamate while leaving unaffected the neuronal responses to local application of acetylcholine or carbachol. The competitive antagonist of non-NMDA receptors 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline suppressed the synaptic excitation at ejection currents which antagonized neuronal responses to quisqualate and kainate. These results suggest that PPN excitatory fibers synapsing onto pars compacta nigrostriatal neurons utilize an excitatory amino acid as a synaptic transmitter acting preferentially on non-NMDA receptors.


Selection for class II Mhc heterozygosity by parasites in subterranean mole rats. Mhc organization and polymorphism have previously been studied in the four chromosomal species of the Spalax ehrenbergi superspecies in Israel, serologically, and at the DNA, RFLP and sequence levels of class I and class II genes. Here we demonstrate that the observed heterozygosity of Mhc class II genes P alpha 1 with 11 alleles, and Q beta, with at least 14 alleles, is positively and significantly correlated with infectivities of ectoparasites (gamasid mites) and endoparasites (helminths). Mhc heterozygosity is highest in the most infected area, which is in the most humid-warm region of the superspecies range, or where two zoogeographic regions overlap. We conclude that the evolutionary forces responsible for the Mhc class II two-gene polymorphisms include selection for increased heterozygosity as a defense strategy against ecto- and endoparasite infections.


The obstetric performance of sickle cell disease patients and homozygous hemoglobin C disease patients in Ile-Ife, Nigeria. In a 9-year period (1980-1988), 46 delivered hemoglobinopathy patients were studied. There was a high incidence of complications during pregnancy. The perinatal and maternal mortalities were 13.4% and 9.2% respectively. The complications could be minimized by adequate supervision in pregnancy, labor and puerperium.


Maternal height and the outcome of labor in rural Tanzania. The influence of maternal height (standardized for parity and birthweight) on obstetrical outcome is studied in 1095 women giving birth in Lugarawa hospital and 3869 women delivering in Mbozi hospital, both rural hospitals in the South Western Highlands of Tanzania. Short stature was found to increase the need for augmentation of labor in primiparae, the need for operative delivery (cesarean section/symphyseotomy) in all parity groups and the need for vacuum extraction in multiparae. The absence of such an effect of height on perinatal mortality is interpreted as the result of obstetric intervention. It is concluded that maternal height, which is easy to measure, remains a useful tool to predict difficult childbirth and cephalopelvic disproportion.


Sonographic assessment of the incompetent cervix in pregnancy. Ultrasonography (US) was done in 40 women with a history of recurrent midtrimester abortion. The results were compared with those of a control group who consisted of 53 women with no previous history of abortion and had had at least one full term pregnancy with normal vaginal delivery. Mean internal cervical os diameters of 16.0 mm and 22.5 mm at 10 and 27 weeks gestation respectively were recorded in the cervical incompetent patients while mean values of 7.7 mm and 14.5 mm at 13 and 28 weeks gestation were observed in the normal control subjects. Full analysis of covariance showed statistically significant difference in the internal os diameter between the control group and the cervical incompetence cases (t90 = 9.33, P less than 0.001).


Vaginal microbial flora as a cofactor in the pathogenesis of uterine cervical intraepithelial neoplasia. The vaginal microbial flora of 106 women with histopathologically confirmed cervical intraepithelial neoplasia and 79 women without disease, was evaluated for Gardnerella vaginalis, Trichomonas vaginalis, Candida albicans and other yeasts. Flora morphology was assessed by gram staining of secretions. Cervical cultures were examined for Herpes Simplex virus, Cytomegalovirus and Neisseria gonorrhoeae. Chlamydia trachomatis antigens in cervical secretions were detected by enzyme immunoassay. Human Papillomavirus was identified by koilocytosis in cytologic or histopathologic specimens. Human Papillomavirus infection (P less than 0.00001), vaginal infection with Mycoplasma hominis (P = 0.012) and abnormal vaginal flora (P = 0.006) were significantly associated with CIN, suggesting that CIN may be promoted by vaginal microorganisms in conjunction with human papillomavirus cervical infection.


Health and economic consequences of septic induced abortion. Over a period of 7 years, 230 cases of illegally induced abortions complicated by sepsis were treated at the University College Hospital, Ibadan, Nigeria. The number of terminations complicated by sepsis doubled from 25.4 (between 1981 and 1985) to 51.0 (between 1986 and 1987) cases per year. Peritonitis was the commonest associated complication while maternal mortality was 8.3%. The average cost of treatment was US$223.11, while the average monthly earnings was US$45.00. Legalization of abortion would have resulted in a saving of US$50,022.28. Provision of legal abortion would reduce the incidence of sepsis after termination while reproductive health education and information dissemination and provision of easily accessible family planning services would greatly reduce the number of unwanted pregnancies.


Cytomegalovirus hepatitis in late pregnancy. A case of pregnancy complicated by cytomegalovirus hepatitis is represented. The mother had a fulminant disease, but she delivered spontaneously at 31 weeks of gestation. The baby was unaffected.


Growth and rupture of an ovarian endometrioma in pregnancy. Pregnancy has long been considered to have beneficial effects on endometriosis. We describe a patient who underwent emergency exploratory laparotomy at gestation week 35 for rupture of an ovarian endometrioma.


Giant broad ligament leiomyoma.

Giant broad ligament leiomyoma. Giant fibroids are known to arise from the uterus, but occasionally from the broad ligament also. A case of giant broad ligament fibroid is reported for its rarity, and the diagnostic difficulties it posed are discussed.


An immunocytochemical study of endocrine cell development in the early fetal guinea pig pancreas. The presence of insulin, glucagon, pancreatic polypeptide, and somatostatin containing cells and their ontogenic changes were investigated immunocytochemically in the early fetal pancreas of the guinea pig (Days 25-40). In the earliest tissues examined (Day 25 and Day 30) brightly staining glucagon cells were the most predominant endocrine cell population, followed by slightly fewer and weaker staining pancreatic polypeptide cells. Insulin and somatostatin immunoreactive cells were less numerous. At Day 25 all endocrine cells were located within the pancreatic tubules where some glucagon cells also coexpressed insulin. Similar dual immunoreactivity was present at Day 30. At Day 25 some of the pancreatic polypeptide cells also showed coexpression of somatostatin which persisted until Days 35-40. At these later stages insulin and somatostatin cells were increasingly frequent. Glucagon and pancreatic polypeptide cells were also conspicuous. The four endocrine cell types were found either in the pancreatic tubules or in the developing islets where they began to acquire an adult-like topographic distribution. These studies in the fetal guinea pig show that the four islet hormonal cells cytodifferentiate from an early stage. A small proportion of endocrine cells coexpress either insulin and glucagon or pancreatic polypeptide and somatostatin.


Presence and subcellular localization of tyrosine aminotransferase and p-hydroxyphenyllactate dehydrogenase in epimastigotes of Trypanosoma cruzi. Cell-free extracts of epimastigotes of Trypanosoma cruzi contain tyrosine aminotransferase (TAT) and p-hydroxyphenyllactate dehydrogenase (pHPLDH). The TAT activity could be separated from aspartate aminotransferase (ASAT) by polyacrylamide gel electrophoresis or DEAE-cellulose chromatography; the latter procedure also allowed complete separation of pHPLDH. The subcellular localization of both T. cruzi enzymes, as determined by digitonin extraction, subcellular fractionation by differential centrifugation, and isopycnic ultracentrifugation in sucrose gradients, was mainly cytosolic, with low mitochondrial activities.


Comparison of coagulase-negative staphylococci by pulsed-field electrophoresis. Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.


Structures of the genes encoding the alpha and beta subunits of the Neurospora crassa mitochondrial ATP synthase. We have isolated and sequenced cDNA and genomic clones encoding the alpha and beta subunits of the Neurospora crassa ATP synthase. The genes are not linked to each other: atp-1(alpha) maps to either linkage group I or V, and atp-2(beta) lies on linkage group II. The two genes resemble each other in having a large number of introns, five in atp-1 and seven in atp-2, mostly positioned near their 5' ends and varying in length from 60-332 bp. The coding regions of both genes have a high G+C content (59%) and use a low number of codons, 46 (atp-1) and 44 (atp-2), a feature associated with highly expressed genes. Northern-blot analysis shows both genes are expressed at high levels during mycelial growth. Comparison of the exon-intron structures of the beta-subunit-encoding gene with those from human and tobacco showed a similar number of introns, several closely positioned, but no exact conservation in position, size or sequence of introns.


Homeobox-containing genes in the newt are organized in clusters similar to other vertebrates. In vertebrates, the majority of homeobox (HBox) genes are found in four clusters and this structural organization is believed to be of functional importance. Many HBox genes sustain their expression in the appendages of the adult newt. To further understand their regulation, the genomic loci of four newt HBox genes (two from the human HBox (HOX)-2 complex and two from the HOX-3 complex) were analysed and compared with homologous loci in other vertebrates. Notophthalmus viridescens HBox (NvHBox) genes were selected from a lambda EMBL3 library and analysed by restriction mapping and nucleotide (nt) sequencing. The nt sequences of the NvHBox genes have a very high degree of homology (more than 90%) with the human and mouse HBox genes, HOX-3.3, HOX-3.4, HOX-2.7 and HOX-2.8. The sequences flanking the HBox are also very homologous to their human and mouse counterparts. Moreover, the size of the DNA spacer separating NvHBox-3.3 from NvHBox-3.4, and NvHBox-2.7 from NvHBox-2.8 in the newt is similar in the homologous regions of the mouse and human, despite there being a C value ten times greater in the newt genome. Finally, three of these NvHBox genes are expressed in the limbs of the adult newt.


Expression of a single lignin peroxidase-encoding gene in Phanerochaete chrysosporium strain ME446. A previously described linked set of lignin peroxidase-encoding genes (Lpo) from Phanerochaete chrysosporium (P.c.) ME446 is not expressed under standard growth conditions for ligninolytic activity. However, a single unlinked Lpo gene, not previously described in P.c. strain ME446, is expressed. The transcription start points of this gene are mapped and the gene is assigned to a genetic linkage group by the use of restriction-site polymorphism segregation analysis. No transcripts from Lpo-related genes, including that normally expressed in ME446, could be detected within RNA extracted from three nonligninolytic mutant strains, but a hyper-ligninolytic strain showed an increased level of Lpo expression. This increase is due to expression of additional Lpo genes, rather than to an increased level of transcription from the normally expressed sequence.


Sequence of the bovine CD18-encoding cDNA: comparison with the human and murine glycoproteins. The bovine cDNA (CD18) encoding CD18, a cell-surface glycoprotein involved in multiple leukocyte functions, was sequenced and compared with the human and murine sequences. Portions of the 5'- and 3'-untranslated regions of the nucleotide sequences are conserved among the three species, including a 3' A+T-rich region believed to regulate mRNA stability and translational efficiency. The 2833-bp bovine sequence coded for a protein of 769 amino acids (aa). Overall, the deduced aa sequences were greater than 80% identical among the three species. The aa 96-389 and those in the cytoplasmic domain were very highly conserved with approx. 95% aa identity. All Cys residues and potential Asn-glycosylation sites present in the bovine sequence were also present in the human and murine sequences. The aa identity was also found in those regions where mutations were found to cause the genetic disease, leukocyte adhesion deficiency. These data identify functionally important regions of the CD18 mRNA and protein.


Statistical analysis of the measurement errors in the determination of fragment length in DNA-RFLP analysis. DNA from human whole blood samples was digested with the restriction enzyme HinfI and RFLP analysis performed using the single locus probes MS1, MS31, MS43a and YNH24. The intergel variation of 3291 duplicate measurements of fragment lengths in terms of basepairs was investigated. The difference between two measurements of the same fragment on different gels increased approximately exponentially with increasing fragment length. After transformation of the fragment length into a normalized migration distance it was found that the difference between two transformed measurements was normally distributed with a S.D. (0.70 mm) which was independent of the fragment length. The errors of band 1 and band 2 on the same lane were correlated (r2 = 0.8). It is useful in the calculation of frequencies and in retrieval procedures and also in the calculation of likelihood ratios to be able to use a S.D. which is independent of the fragment length.


[Prevention of migraine using bisoprolol. Results of a double-blind study versus metoprolol]. Comparison of bisoprolol, a beta-1 selective beta blocker with no intrinsic sympathomimetic activity (ISA) and metoprolol, which numerous studies have shown to be an effective migraine prophylactic. Multicentric, cross-over study. 125 patients suffering at least from 3 attacks of classic or common migraine a month for at least two years. Bisoprolol 5 mg given once a day, or metoprolol 50 mg given twice a day, for two periods of 12 weeks. 125 patients were admitted to the 4-week run-in phase. A comparison of the main target: frequency of migraine attacks was thus carried out in 78 patients (f. = 63, m. = 15). Both substances reduced the average frequency of migraine per 28-day period by about 50%. There was no statistically significant difference between the two beta-blockers (p greater than 0.05). The results of the study show that 5 mg of bisoprolol and 100 mg of metoprolol a day have comparable efficacy for migraine prophylaxis, and show comparable tolerability.


[Interactions of theophylline and H2 receptor antagonists. Interaction modes--clinical relevance]. Basic considerations: peptic ulcers and obstructive diseases occur jointly, since cigarette smoking is an important causal factor in both diseases. Major topics: The interaction possibilities of theophylline and H2-receptor antagonists on the various levels from absorption to elimination of the two drugs are discussed, with emphasis being attached to oxidative breakdown in the liver. The clinical relevance of such interactions arises from the limited therapeutic spectrum of theophylline. Treatment with both drugs should take account of the varying tendency of the individual H2-receptor antagonists to interact.


[Neuroleptic therapy and suicide--review of the literature and personal results]. The majority of schizophrenic patients receive neuroleptics (NL) and a relatively high number of them commit suicide. Is there a relationship between the two variables? Long-term observations failed to demonstrate an interdependence between a large-scale introduction of NL in the therapeutic practice and suicide rates. There is a relationship between depression and suicide and depressive syndroms are frequent in schizophrenic patients. Depressions due to an exclusive use of NL probably do occur, but quite seldom so. Also, possible relationships between other NL side effects (akathisia, dysphoric reaction to NL) and suicidal behavior are not sufficiently supported by the clinical data. Direct comparisons in controlled studies (the own study included) between NL therapy of suicide and control subjects yielded no consistent results. Suicide promoting effect of NL cannot be postulated on the basis of the available data, however, it also cannot be fully excluded in individual cases.


[Clinical symptoms and therapy of status epilepticus]. The term "status epilepticus" was first coined in 1824 by Calmeil as this condition had such a poor prognosis. Although still commonly misused today, from the beginning this term actually included all kinds of epileptic seizures, since there are as many types of status epileptici as there are seizure types. Status epileptici are usually triggered by a combination of factors including sleep deprivation, alcohol withdrawal, failure to take medication regularly and fever. In status epilepticus epileptic seizures and EEG discharges initially appear to be no different from isolated seizures. The longer the status epilepticus continues, however, the more atypical the seizures and EEG discharges become. Usually status epilepticus ends gradually. Irreversible damage or fatalities may occur especially in infants or under certain conditions (e.g. long status duration, protracted interval between seizure onset and medical treatment and symptomatic etiology). In most cases benzodiazepines and diphenylhydantoine are the preferred drugs used for treatment.


Phenotype of disease in three patients with identical mutations in methylmalonyl CoA mutase. We have previously identified a mutation in the gene for methylmalonyl CoA mutase in a patient with the mut- phenotype of methylmalonic aciduria. This mutation (G717V) interferes with the binding of the deoxyadenosylcobalamin cofactor to the apoenzyme producing a mutant holoenzyme that is defective, but not completely inactive, in vitro. This report describes the clinical phenotype associated with this mutation in the original patient and two additional patients who are homozygous for this allele. All three patients presented in the first years of life with multiple episodes of life-threatening organic acidosis and hyperammonemia. None had evidence of disease in the perinatal period, and all three have low-normal intelligence. These three children exhibit a distinctive phenotype of disease that is intermediate between the fulminant and benign forms of methylmalonic aciduria. These data suggest that this phenotype is the specific consequence of the G717V mutation, and that the degree of residual enzyme activity associated with the G717V mutation is close to the threshold required in vivo for maintaining metabolic homeostasis.


Huntington disease in Finland: a molecular and genealogical study. Huntington disease (HD) is found at exceptionally low frequency in the Finnish population. In this population, linkage disequilibrium was earlier established with markers from the D4S10 and D4S43 loci. We now report a continuation to the restriction fragment length polymorphism haplotype analysis, in combination with a genealogical study of all the Finnish HD families. When the HD pedigrees were systematically traced to the 18th century, only one consanguinity was found, and a high percentage (28%) of the families had foreign ancestors. The majority of the Finnish ancestors were localized to border regions or trade centers of the country following the old postal routes. The observed high risk haplotypes formed with markers from the D4S10 and D4S43 loci were evenly distributed among the HD families in different geographical locations. Consequently, the HD gene(s) has most probably arrived in Finland on several occasions via foreign immigrants during the last few centuries.


Genetics of neurofibromatosis 1 in Japan: mutation rate and paternal age effect. We have performed formal genetic studies on 26 patients (14 males, 12 females) with neurofibromatosis 1 (von Recklinghausen's disease, NF1) in Japan. Family studies of 74 members of 18 kindreds revealed that 50% of the cases were caused by a new mutation; the mutation rate was assumed to be 7.3-10.5 x 10(-5). A tendency of paternal age effect, which was not accounted for by the maternal age effect, was observed, but live-birth order had no significant effect. Genetic linkage of neurofibromatosis 1 to the NF1 gene or the genetic marker in the pericentric region of chromosome 17 was established in 3 informative families.


Linkage disequilibrium detected between myotonic dystrophy and the anonymous marker D19S63 in the Spanish population. We used the following polymorphic markers: APOC2 (BanI, BglI, TaqI), CKMM (NcoI, TaqI), and D19S63 (PstI) to haplotype 33 Spanish myotonic dystrophy (DM) families. We analysed the allele and haplotype frequencies of our sample, and the possible association of alleles or haplotypes with the disease. We found a slight linkage disequilibrium between APOC2 (BanI) and DM, but no disequilibrium when using all other APOC2 and CKMM RFLPs; this agrees with data previously reported. In addition, we found a very strong linkage disequilibrium when using D19S63 (PstI), the + allele being associated with the DM locus. This disequilibrium in the Spanish population indicates that D19S63 is very close to the DM locus.


Loss of normal allele of the APC gene in an adrenocortical carcinoma from a patient with familial adenomatous polyposis. Endocrine neoplasms have been reported occasionally in patients with familial adenomatous polyposis (FAP). An adrenocorotical carcinoma was studied in a patient with a family history of FAP. Loss of heterozygosity (LOH) in the region close to the adenomatous polyposis coli (APC) gene was detected in this carcinoma, and evidence was obtained that there was a loss of the normal allele of the APC gene. This is the first demonstration of LOH at the APC locus in adrenocortical tumors. The present results and our previous data on LOH in a recurring desmoid tumor suggest that the heterozygous mutant/wild-type condition of the APC gene may give rise to benign tumors, and that functional loss of this gene leads to development of tumors not only in the colon but also in other various parts of the body in FAP patients.


RNA analysis from newborn screening dried blood specimens. We have previously demonstrated that DNA can be extracted from the dried blood specimen of the type used for newborn screening. The technique presented here allows us to extract RNA from newborn screening specimens for cDNA synthesis by reverse transcriptase and amplification by the polymerase chain reaction (PCR). Products of the PCR reaction are then analyzed by restriction enzymes. This method successfully distinguishes beta A and beta S transcripts in unaffected (AA), carrier (AS), and affected (SS) individuals. The value of this approach for identification of a compound heterozygous patient with S/beta-thalassemia, using the original newborn screening specimen, is also demonstrated. This work shows that mRNA is stable in dried blood specimens and that analysis of the mRNA phenotype can be a useful adjunct in the application of molecular genetic technology to newborn screening.


The molecular basis of beta-thalassemia in Turkey. By using oligonucleotide hybridization, restriction endonuclease analysis and direct sequencing of amplified genomic DNA, we have been able to characterize 18 different mutations in the beta-globin genes of 161 beta-thalassemia homozygotes and 107 beta-thalassemia heterozygotes from Turkey (429 beta-thalassemia chromosomes). Previous studies dealing with beta-thalassemia in Mediterranean countries have shown that, in most Mediterranean populations, only a few mutations are prevalent. In contrast, beta-thalassemia in Turkey does not seem to be associated with a few predominant mutations. The six most frequent alleles, IVS-I-110 (G----A), IVS-I-6(T----C), FSC-8 (-AA), IVS-I-1(G----A), -30(T----A) and FSC-5 (-CT), account for only 69.3% of the disease genes; indeed, all 26 mutations assayed represent 85.8% of the disease genes, confirming the considerable molecular heterogeneity of beta-thalassemia among Turks, and indicating the possible presence of rare, previously undefined, mutations in the population. Two mutations observed in this study, IVS-I-116 (T----G) and Cd44(-C), have not been reported in the Turkish population to date. Since preventive medical services, such as genetic counseling and prenatal diagnosis, are greatly improved by detailed knowledge of the molecular pathology of beta-thalassemia, we strongly believe that the presented data will facilitate the intended establishment of a prenatal diagnosis center, based on DNA analysis, in Turkey.


Human alpha-globin gene expression is silenced by terminal truncation of chromosome 16p beginning immediately 3' of the zeta-globin gene. The high level expression of the human alpha-globin genes in erythroid tissue appears to require a set of DNaseI hypersensitive sites located upstream of the human alpha-globin gene cluster. These sequences, termed the locus control region (LCR), include two erythroid specific and a number of less restricted DNaseI hypersensitive sites. In this report we describe an individual with alpha-thalassemia associated with a truncation of the short arm of chromosome 16 that removes the LCR region and inactivates the adjacent intact alpha-globin genes. This genetic study supports the critical role of the LCR in the transcriptional activation of the human alpha-globin gene cluster and substantiates the importance of LCR deletions in the etiology of alpha-thalassemia.


Beta-thalassemia major resulting from a compound heterozygosity for the beta-globin gene mutation: further evidence for multiple origin and migration of the thalassemia gene. We describe in a Japanese family beta zero-thalassemia resulting from a compound heterozygosity for a beta-globin gene mutation. One mutation is a C-to-T transition at IVS-2 nucleotide position 654 on the background of Mediterranean haplotype IX. Another mutation is a G-to-A transition at IVS-2 nucleotide position 1, associated with a novel haplotype XI. The occurrence of these mutations on various chromosomal backgrounds provides strong evidence for an interplay of gene migration, interallelic gene conversion, and multiple origins of the same mutation.


Transthyretin Pro55, a variant associated with early-onset, aggressive, diffuse amyloidosis with cardiac and neurologic involvement. Mutations in the protein transthyretin cause amyloidosis involving the heart, peripheral nerves, and other organs. A family from West Virginia developed an unusually aggressive form of widespread transthyretin amyloidosis. Single-strand conformation polymorphism analysis revealed a variant in the transthyretin gene, which was found on sequencing to be a T----C transversion at position 2 of codon 55, corresponding to a Leu----Pro substitution. The variant sequence was confirmed by restriction analysis and polymerase chain reaction (PCR)-primer introduced restriction analysis.


A rare FokI RFLP in the human dopamine D2 receptor gene (DRD2). A new biallelic polymorphism for FokI restriction enzyme due to C----T transition in the fourth intron of human DRD2 is described. It must be a usefull marker of this candidate gene for several mental disorders.


Method for storing Lagenidium (Oomycetes: Lagenidiales). Techniques for storing the mosquito pathogenic fungus, Lagenidium, were evaluated. A technique, which involves storage of fungal mycelia in sterile distilled water of pH 6-7 with 0.0025 M glucose at 30-35 degrees C, was found to be useful. When stored in this manner the fungus retained it's larvicidal activity for 190 days.


An attempt to define sets of cooperating genetic alterations in human breast cancer. The extent and the variation of losses of genetic material were examined in a series of 191 human breast cancers by means of a set of 18 polymorphic DNA probes, specific of 7 chromosomal arms (1p, 1q, 3p, 11p, 13q, 17p and 18q) known to be frequently affected by allele losses. Frequencies of losses of heterozygosity ranged from a low of 3.5% (chromosome 13q) to a high of 27% (chromosome 3p). The number of sites involved in breast cancer added to the frequent occurrence of concomitant losses at several chromosomal arms within the same tumor suggest cooperative effects of these LOHs. We were therefore interested in assessing the existence of preferential associations between sets of LOHs in our panel of tumors. Statistically significant associations were found between LOHs at chromosomes 1p and 17p, and between LOHs at chromosomes 11p and 17p. Furthermore, since all the tumors presently studied had previously been analyzed for proto-oncogene amplification at 5 distinct chromosomal sites, we tested for associations between LOH and DNA amplification. Such associations were indeed observed as exemplified by the correlations observed between the LOH at 11p and amplification of the erbB2 gene and LOH at 17p and the amplification of the flg gene. The only correlation with clinico-pathological parameters that could be observed linked the occurrence of LOHs on 11p with recurrent breast cancer (p = 0.015). Sets of several LOHs or LOHs and gene amplifications could not be significantly related to any marker of tumor aggressiveness.


Frequent expression of the tumor antigen CAK1 in squamous-cell carcinomas. K1 is a murine monoclonal antibody (MAb) derived from a hybridoma generated by the fusion of splenocytes of BALB/c mice immunized with a human ovarian tumor cell line, OVCAR-3. This antibody reacts strongly with epithelial ovarian tumors and mesotheliomas. The antigen recognized by MAb K1, designated CAK1, has recently been characterized as a 40-kDa protein probably anchored to the cell surface by glycosyl-phosphatidylinositol. Using immunoperoxidase histochemical methods, we examined 37 squamous-cell carcinoma (SqCC) samples from cervix, lung, esophagus and other origins, and 12 normal squamous epithelia of the cervix and esophagus for their reactivity with MAb K1. Of the SqCC specimens, 81% showed K1 reactivity with variable intensity, but none of 12 normal tissue samples of squamous epithelia did so. Two patterns of CAK1 expression in tumor samples were found, i.e., a heterogeneous pattern with strong intensity, and a homogeneous pattern with weak intensity. Three carcinomas in situ of the larynx, vulva and esophagus were moderately positive with K1, suggesting that CAK1 antigen may occur in the early stage of carcinogenesis of SqCC. The expression of CAK1 was also compared with expression of CA125, HER-2/neu, p53 and P-glycoprotein, and MAb K1 was found to react most consistently with SqCC. Since K1 reacts with a majority of cervical and esophageal carcinomas but has no detectable reactivity in normal epithelia of the cervix uteri and esophagus, MAb K1 could be of value as a reagent to help distinguish between normal and neoplastic cells on sections as well as in cytological samples.


Heatstroke and hyperthermias.

Heatstroke and hyperthermias. We describe the pathogenesis and the typical clinical and laboratory features of the hyperthermia syndromes that may develop during neuroleptic drug treatment, environmental exposure or following the use of inhalational anesthetics. Since the features are similar and confusion is possible we emphasize the need for prompt diagnosis so that appropriate treatment, can be started without delay. Hyperthermic reactions have common pathophysiological mechanisms and they may be seen as an abnormal individual response to organic and functional stressors.


Levamisole augments roxatidine's immune modulatory effects in the graft-versus-host reaction (GVHR). Levamisole is known to have anti-anergic properties in immune compromised, but no or only marginal effects in immunologically competent subjects. In this study the possibility that levamisole would act as an 'immunoadjuvant' with roxatidine, a histamine H2 receptor blocker, is explored in healthy animals. Sixteen female, inbred Sprague-Dawley rats acted as lymphocyte donors and were treated for 8 days with either a roxatidine-levamisole combination, or levamisole alone, or roxatidine alone or placebo. Five randomly bred guinea pigs and eight inbred BD IX rats acted as lymphocyte acceptors. The in vivo effects of the four treatment modalities on transferred lymphocytes were expressed as the sizes of the allogeneic and xenogeneic graft-versus-host reactions as well as the radioactivity in each AGVHR. The combination of levamisole and roxatidine significantly augmented the cell mediated immune response in vivo and resulted in the most reactive lymphocytes as indicated by the AGVHR's and XGVHR's. These cells mediated reactions with significantly larger wheals as well as more lymphocyte proliferation in each reaction site than those caused by either levamisole or placebo treatment.


Neurogenic inflammation and asthma.

Neurogenic inflammation and asthma. The release of neurotransmitters may exacerbate the inflammatory response. Such neurogenic inflammation has been documented in a number of inflammatory diseases. Neurogenic inflammation due to release of neuropeptides from sensory nerves has been demonstrated in airways of several species, particularly rodents, and may contribute to the inflammatory response in asthmatic airways. Tachykinins (substance P and neurokinin A) released from airway sensory nerves may cause bronchoconstriction, vasodilatation, plasma exudation, and mucus secretion, whereas another sensory neuropeptide, calcitonin generelated peptide, may contribute to hyperemia of inflammation. Airway epithelial damage in asthma exposes sensory nerves which may become sensitized by inflammatory products (including prostaglandins and cytokines) so that neuropeptides are released via a local reflex trigger such as bradykinin, resulting in exaggerated inflammation. The effects of tachykinins may be amplified further by loss of the major degrading enzyme, neutral endopeptidase, from epithelial cells. Direct evidence for neurogenic inflammation in asthma is still awaited, however. Several strategies for reducing neurogenic inflammation are possible, particularly inhibition of neuropeptide release from sensory nerves by stimulating prejunctional receptors such as mu-opioid receptors.


The effect of repeat action albuterol sulfate (Proventil Repetabs) in nocturnal symptoms of asthma. At four medical centers, 98 patients with stable asthma, histories of nighttime awakening at least three times weekly and nighttime declines of pulmonary function of at least 15%, who were not taking oral adrenergic agonists, were randomly treated with either oral repeat-action albuterol sulfate (Proventil Repetabs), 4 mg in the morning and 4-16 mg at bedtime, or a placebo for 2 weeks. All patients were required to have nocturnal symptoms of asthma, with prior use of bronchodilators other than oral adrenergic agonists to be eligible for the study. The patients maintained a diary of asthma symptom scores and recorded peak flow rates at home at bedtime and in the morning. They had spirometry (FEV1, FVC, and PEFR) after a 1-week baseline stabilization period, and after 1 and 2 weeks of double-blind oral therapy as noted above. Efficacy was evaluated by changes in the bedtime and morning peak flow rates, changes in the number of nighttime awakenings, results of office spirometry testing, and by physician and patient global evaluations of response to therapy. Of the 98 patients in the study, 47 received oral albuterol, and 51 received placebo. The patients on albuterol had a statistically significant reduction in the number of nighttime awakenings (p less than or equal to 0.01), as compared with the patients on placebo; this included both the average number of awakenings per week (p = 0.04), and the mean number of nights with awakenings per week (p = 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)


N-[9H-(2,7-dimethylfluorenyl-9-methoxy)carbonyl]-L-leucine, NPC 15669, prevents neutrophil adherence to endothelium and inhibits CD11b/CD18 upregulation. NPC 15669, a member of a new class of antiinflammatory agents termed leumedins, blocks inflammation in several animal models, including contact dermatitis and Arthus reaction, by inhibiting recruitment of neutrophils and lymphocytes into inflammatory lesions. These compounds do not block lipid metabolic enzymes, nor do they antagonize receptors for various chemoattractants, including LTB4, PAF, C5a, and fMLP. This report demonstrates that in vitro, pretreatment of stimulated neutrophils with NPC 15669 results in the dose-dependent inhibition of adherence to cultured human umbilical vein endothelial cells or to the protein substrate keyhole limpet hemocyanin. Adherence of HL-60 cells (a promyelocytic line) is unaffected when stimulated endothelial cells are pretreated with NPC 15669. Flow cytometric analysis of adhesion molecules expressed on neutrophils revealed that pretreatment of neutrophils with NPC 15669 prior to activation inhibits the increase in expression of the CD11b and CD18 adhesion molecule subunits. We conclude that (1) NPC 15669 acts on neutrophils to block activation-induced adherence, and (2) NPC 15669 inhibits the upregulation of the CD11b/CD18 (Mac-1) adhesion receptor.


Effects of caudal elevation on testicular function in rats. Separation of effects on spermatogenesis and steroidogenesis. A variety of biologic processes are perturbed when exposed to microgravity (space flight) for more than 7 days, including testicular function. Suspension of rats in a special harness (caudal elevation) to induce thoracic pooling of blood fluids and remove the support function of the hind limbs is used to mimic, on earth, the effects of microgravity encountered during space flight. Typically, this induces cryptorchidism in male rats. Three experiments were conducted to differentiate the effects of caudal elevation (30 degrees angle) and anatomic location of testes on spermatogenesis and steroidogenesis. Rats were subjected to caudal elevation for 7 days using either a tail harness (experiments 1 and 2) or a whole-body harness (experiment 3). Testes of rats fell into the abdominal cavity when a tail harness was used, but ligation of the inguinal canal prevented this repositioning. For rats with abdominal testes, testicular weight was reduced (P less than 0.05) and histology of testes was abnormal; the number of spermatids per gram parenchyma was lower (P less than 0.05) in tail-suspended rats compared with control rats. In contrast, spermatogenesis was not affected by caudal elevation in most rats in which the inguinal canal was ligated or in rats elevated by whole-body harness. Concentrations of testosterone in serum and testicular interstitial fluid were lower (P less than 0.05) in suspended rats, regardless of the method used for caudal elevation or anatomic location of testes. Concentrations of luteinizing hormone in serum were elevated (P less than 0.05) in rats with intra-abdominal testes.(ABSTRACT TRUNCATED AT 250 WORDS)


Dynamics of atrial natriuretic factor-guanylate cyclase receptors and receptor-ligand complexes in cultured glomerular mesangial and renomedullary interstitial cells. The dynamics of the guanylate cyclase receptor of atrial natriuretic factor (GCA-ANF receptor) were investigated in cultured glomerular mesangial and renomedullary interstitial cells from the rat. In these cells, the GCA-ANF receptor did not mediate internalization and lysosomal hydrolysis of 125I-ANF1-28 and did not undergo ligand-induced endocytosis. Glomerular mesangial cells were able, however, to mediate internalization and lysosomal hydrolysis of 125I-ANF1-28 via clearance ANF (C-ANF) receptors and to promote rapid receptor-mediated internalization and lysosomal hydrolysis of 125I-(Sar1) angiotensin II. Radioligand specifically bound to surface GCA-ANF receptors was rapidly dissociated at 37 degrees C (k(off) greater than 0.8 min-1), with a Q10(30-37 degrees C) greater than 6. The dissociation was markedly slower at subphysiological temperatures (Q10(4-30 degrees C), 2-3) or in the presence of 0.5 mM amiloride. The results demonstrate that the GCA-ANF receptor, contrary to C-ANF receptors and most other polypeptide hormone receptors, is a membrane resident protein that does not mediate internalization and lysosomal hydrolysis of ligand. The termination of the interaction of ANF with GCA-ANF receptors results from a physiological process that leads to rapid dissociation of receptor-ligand complexes. The unique dynamics of GCA-ANF receptor-ligand complexes are likely to contribute importantly to stimulus-response homeostasis of ANF.


Intercellular adhesion molecule-1 gene expression in human endothelial cells. Differential regulation by tumor necrosis factor-alpha and phorbol myristate acetate. Intercellular adhesion molecule-1 (ICAM-1) is an inducible glycoprotein expressed on the surface of inflamed endothelium which mediates in part the extravasation of granulocytes into sites of infection or injury. ICAM-1 mRNA is not detected in unstimulated human umbilical vein endothelial cells (HUVECs), but accumulates transiently following tumor necrosis factor-alpha (TNF-alpha) or phorbol myristate acetate (PMA) treatment with maximal steady state levels occurring at 2 or 4 h, respectively. Pretreating HUVECs with PMA for 72 h down-regulates protein kinase C and inhibits the subsequent induction of ICAM-1 mRNA by PMA, but does not affect TNF-alpha-induced message accumulation. Nuclear run-on assays showed that the ICAM-1 gene is transcribed under basal conditions in HUVECs, and that TNF-alpha stimulates transcriptional activity 3- to 4-fold within 30 min of treatment. In contrast, PMA has little effect on ICAM-1 gene transcription up to 4 h following stimulation. Message stability studies established that ICAM-1 mRNA induced by PMA has a longer half-life than the TNF-alpha-induced message. These results suggest that PMA acts through protein kinase C to up-regulate ICAM-1 expression primarily at a post-transcriptional level by stabilizing ICAM-1 mRNA, whereas TNF-alpha transcriptionally regulates ICAM-1 gene expression through an undefined, protein kinase C-independent pathway.


Regulated coupling of the Neu receptor to phosphatidylinositol 3'-kinase and its release by oncogenic activation. The neu protooncogene encodes a tyrosine kinase receptor that is involved in the regulation of normal growth and malignant transformation. To circumvent the use of the incompletely characterized ligand of Neu, we constructed a chimeric protein composed of the ligand-binding domain of the epidermal growth factor receptor and the transmembrane and cytoplasmic portions of Neu. By expressing this Neu-epidermal growth factor receptor chimera (termed NEC), we found that following stimulation by the heterologous ligand, the tyrosine kinase of Neu became associated with a phosphatidylinositol (PI) kinase activity. The association was dependent on the concentration of the ligand and was almost maximal within 30 s after ligand binding. The lipid kinase was identified as a type I PI 3'-kinase on the basis of its inhibition by Nonidet P-40 and high pressure liquid chromatography of the phosphorylated product. To confirm the identification of PI 3'-kinase as an effector of Neu, we raised antibodies to the alpha-isoform of the regulatory subunit of PI 3'-kinase (p85). Using these antibodies, it was possible to directly demonstrate ligand-dependent formation of a tyrosine-phosphorylated complex of NEC and PI 3'-kinase. Apparently, both PI 3'-kinase and phospholipase C gamma, another substrate of the Neu kinase, simultaneously associated with the same activated NEC molecule. Nevertheless, immunofluorescence localization of PI 3'-kinase revealed no significant cellular redistribution of the enzyme after activation of the Neu kinase. Interestingly, PI 3'-kinase was localized primarily to the cell nucleus and to confined regions of the plasma membrane. Analysis of mutants of the Neu protein indicated that the oncogenic point-mutated Neu (Glu664) was permanently coupled to PI 3'-kinase; but two nontransforming versions of the oncoprotein, a kinase-defective protein and a carboxyl-terminally deleted Neu, were devoid of the constitutive association with PI 3'-kinase. Hence, we concluded that phosphatidylinositol 3'-kinase is a physiological substrate of the Neu receptor, but the regulation of this coupling is released upon oncogenic activation.


Overexpression of the alpha-thyroid hormone receptor in avian cell lines. Effects on expression of the malic enzyme gene are selective and cell-specific. The role of the alpha-thyroid hormone receptor (TR alpha) in regulation of transcription of the gene for chicken malic enzyme was analyzed in fibroblast cell lines normally unresponsive to triiodothyronine (T3). The gene for this transcription factor was introduced stably and overexpressed using a replication-competent retroviral vector. In chick embryo fibroblasts (CEF), overexpression of TR alpha decreased malic enzyme activity by 90% in the absence of T3. Addition of T3 almost completely restored malic enzyme activity to the level of similarly treated control CEF infected with virus lacking TR alpha. These TR alpha-induced changes in malic enzyme activity were mediated by alterations in transcription of the malic enzyme gene. Similar results were obtained when transcriptional activity of TR alpha was analyzed using a transient co-transfection system. Thus, the unliganded TR alpha is a transcriptional repressor of the malic enzyme gene; binding of T3 to the receptor abolishes this repression. In contrast, stable overexpression of TR alpha in QT6 cells had no effect on malic enzyme expression in the absence or presence of T3. Nuclear T3 binding was equally high in CEF and QT6 cells overexpressing TR alpha. These findings suggest that cell-specific factors control the ability of TR alpha to regulate the malic enzyme gene. Overexpression of TR alpha in CEF had no effect on the expression of fatty acid synthase and acetyl-CoA carboxylase, lipogenic enzymes that are stimulated by T3 in hepatocytes in culture. Thus, gene-specific factors also may control the transcriptional activity of TR alpha.


The glycophosphatidylinositol-anchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells. Stimulation of murine T cells by engagement of the multi-component T cell antigen receptor or by cross-linking the Thy-1 molecule leads to a similar response characterized by lymphocyte activation and lymphokine production. The early biochemical events induced by engaging these molecules also are similar and begin with activation of a tyrosine kinase pathway and tyrosine phosphorylation of a comparable set of substrates. Previous work demonstrates that the protein tyrosine kinase p60fyn is associated with the antigen receptor and therefore it may participate in the tyrosine phosphorylations that are observed with antigen receptor signaling. In this study we demonstrate that the Thy-1 molecule is also associated with p60fyn in a murine T cell hybridoma and in murine thymocytes. The interaction is independent of antigen receptor expression. Thy-1 is a member of the class of molecules anchored to the plasma membrane by a glycophosphatidylinositol (GPI) group. The association of Thy-1 with p60fyn is dependent on the GPI linkage, since cleavage of the GPI anchor disrupts the interaction. The association of Thy-1 and p60fyn suggests a means by which Thy-1 cross-linking leads to tyrosine phosphorylation and T cell activation.


Evidence for the involvement of beta-adrenergic receptors in conditioned immunomodulation. This study investigates the role of beta-adrenergic receptors in the immunomodulatory effects of a conditioned aversive stimulus (CS). A CS is an environmental event that is not inherently aversive, but acquires aversive properties through pairings with a stimulus such as electric shock. This study evaluated the effects of administration of the beta 1-receptor selective antagonist atenolol, and the beta 2-receptor antagonist ICI 118,551 on conditioned immune alterations. Administration of either antagonist prior to presentation of the CS resulted in a dose-dependent attenuation of the CS-induced suppression of splenic T-cell proliferative response to concanavalin A, phytohemagglutinin, and the combination of ionomycin/phorbol-myristate-acetate. Furthermore, both antagonists dose-dependently attenuated the CS-induced suppression of gamma-interferon production by concanavalin-A (ConA)-stimulated splenocytes. In contrast, neither antagonist significantly attenuated the CS-induced suppression of the B-cell mitogenic response to LPS, interleukin-2 production, natural killer cell activity, or mitogenic responsiveness of blood lymphocytes. Thus it is likely that multiple mechanisms are involved in CS-induced immune alterations and these results clearly implicate beta 1 and beta 2-adrenergic receptors in a subset of immunomodulatory effects.


Immunomodulatory effects of therapeutic gold compounds. Gold sodium thiomalate inhibits the activity of T cell protein kinase C. Previous studies have shown that the gold compounds, gold sodium thiomalate (GST) and auranofin (AUR), which are effective in the treatment of rheumatoid arthritis, inhibit functional activities of a variety of cells, but the biochemical basis of their effect is unknown. In the current studies, human T cell proliferation and interleukin 2 production by Jurkat cells were inhibited by GST or AUR at pharmacologically relevant concentrations. Because it has been documented that protein kinase C (PKC) is involved in T cell activation, the capacity of gold compounds to inhibit PKC partially purified from Jurkat cells was assayed in vitro. GST was found to inhibit PKC in a dose-dependent manner, but AUR caused no significant inhibition of PKC at pharmacologically relevant concentrations. The inhibitory effect of GST on PKC was abolished by 2-mercaptoethanol. To investigate the effect of GST on the regulation of PKC in vivo, the levels of PKC activity in Jurkat cells were examined. Cytosolic PKC activity decreased slowly in a concentration- and time-dependent manner as a result of incubation of Jurkat cells with GST. To ascertain whether GST inhibited PKC translocation and down-regulation, PKC activities associated with the membrane and cystosolic fractions were evaluated after phorbol myristate acetate (PMA) stimulation of GST incubated Jurkat cells. Translocation of PKC was markedly inhibited by pretreatment of Jurkat cells with GST for 3 d, but the capacity of PMA to down-regulate PKC activity in Jurkat cells was not altered by GST preincubation. The functional impact of GST-mediated downregulation of PKC in Jurkat cells was examined by analyzing PMA-stimulated phosphorylation of CD3. Although GST preincubated Jurkat cells exhibited an increased density of CD3, PMA-stimulated phosphorylation of the gamma chain of CD3 was markedly inhibited. Specificity for the inhibitory effect of GST on PKC was suggested by the finding that GST did not alter the mitogen-induced increases in inositol trisphosphate levels in Jurkat cells. Finally, the mechanism of the GST-induced inhibition of PKC was examined in detail, using purified PKC subspecies from rat brain. GST inhibited type II PKC more effectively than type III PKC, and also inhibited the enzymatic activity of the isolated catalytic fragment of PKC. The inhibitory effect of GST on PKC activity could not be explained by competition with phospholipid or nonspecific interference with the substrate. These data suggest that the immunomodulatory effects of GST may result from its capacity to inhibit PKC activity.


Major histocompatibility complex class III genes and susceptibility to immunoglobulin A deficiency and common variable immunodeficiency. We have proposed that significant subsets of individuals with IgA deficiency (IgA-D) and common variable immunodeficiency (CVID) may represent polar ends of a clinical spectrum reflecting a single underlying genetic defect. This proposal was supported by our finding that individuals with these immunodeficiencies have in common a high incidence of C4A gene deletions and C2 rare gene alleles. Here we present our analysis of the MHC haplotypes of 12 IgA-D and 19 CVID individuals from 21 families and of 79 of their immediate relatives. MHC haplotypes were defined by analyzing polymorphic markers for 11 genes or their products between the HLA-DQB1 and the HLA-A genes. Five of the families investigated contained more than one immunodeficient individual and all of these included both IgA-D and CVID members. Analysis of the data indicated that a small number of MHC haplotypes were shared by the majority of immunodeficient individuals. At least one of two of these haplotypes was present in 24 of the 31 (77%) immunodeficient individuals. No differences in the distribution of these haplotypes were observed between IgA-D and CVID individuals. Detailed analysis of these haplotypes suggests that a susceptibility gene or genes for both immunodeficiencies are located within the class III region of the MHC, possibly between the C4B and C2 genes.


An immunohistochemical study of the telencephalon of the senegal bichir (Polypterus senegalus). The telencephalon in ray-finned fish (actinopterygians) is everted, in contrast to the evaginated telencephalic hemispheres in all other vertebrates. In the more derived ray-finned fish, the teleosts, proliferation of neurons and their migration from the ependymal zone of the pallium renders comparisons between telencephalic cell groups of the teleosts and members of other vertebrate groups extremely difficult. The telencephalon of Polypterus (a primitive living ray-finned fish), although everted, is cytoarchitecturally much simpler than that of teleosts. We have thus applied immunohistochemical techniques to the study of the telencephalon of Polypterus to help clarify the evolution of the telencephalon in teleosts and facilitate comparisons between the telencephalon in ray-finned fish and other vertebrates. Antisera against the following neuroactive substances were used: 1) serotonin (5HT), 2) tyrosine hydroxylase (TH), 3) substance P (SP), 4) leucine-enkephalin (ENK), 5) neuropeptide Y (NPY), and 6) the neurotensin-related hexapeptide LANT6. Several features of the labeling patterns obtained suggested that the dorsal and ventral subdivisions of the area ventralis are homologous as a field to the basal ganglia and septum plus other basal telencephalic regions of land vertebrates, sharks and lungfish: 1) an abundance of SP+, NPY+, and ENK+ fibers; 2) an abundance of TH+ fibers, possibly of posterior tubercle/tegmental origin; 3) the presence of an SP+ fiber bundle that appeared to descend from basal telencephalic levels and terminate in the posterior tubercle/tegmentum, which contain TH+ (possibly dopaminergic) neurons; and 4) an abundance of 5HT+ fibers, presumably of posterior tubercle/tegmental origin. It was not possible, however, to recognize distinct pallidal and striatal subdivisions within the area ventralis of Polypterus. The olfactory pallium (P1) was generally poor in most of the substances examined, except for the presence of LANT6+ fibers. The P3 pallial field was conspicuously rich in SP+ and ENK+ fibers throughout its extent, and the caudal and lateral parts of the P2 field were rich in SP+ fibers and ENK+ fibers. Since this is characteristic of the medial pallial and/or dorsomedial pallial walls of the telencephalon in lungfish, sharks, frogs, and reptiles, the P3 field and caudolateral part of the P2 field may be homologous to these portions of the telencephalon in other vertebrates. More rostromedial parts of P2 may correspond to those parts of the pallium in land vertebrates that are in receipt of specific sensory input from the thalamus, since low neuropeptide levels are characteristic of these regions.(ABSTRACT TRUNCATED AT 400 WORDS)


T cell receptor-alpha beta lacking the beta-chain V domain can be expressed at the cell surface but prohibits T cell maturation. A TCR-beta gene lacking V domain sequences (delta V-TCR-beta) was inserted into the germline of mice. Expression of the transgene inhibited endogenous TCR-beta, but not TCR-alpha gene rearrangement and expression. The mutated TCR-beta gene affected alpha beta T cell development: the common thymocyte pool was normal in cell number, with cells expressing CD4 and CD8, but the mature, "CD3bright" population expressing either CD4 or CD8 molecules was reduced by 90%. To help understand these effects on TCR-beta gene rearrangement and T cell development, biosynthesis of the delta V-TCR-beta protein was analyzed in a tumor cell line derived from a transgenic mouse. Despite absence of the V domain, the delta V-TCR-beta chain paired with endogenous TCR-alpha chains and assembled with CD3 gamma, -delta, -epsilon, and -zeta components in the endoplasmatic reticulum, followed by transport through the Golgi complex to the plasma membrane. Therefore, assembly of the complex, and even cell surface expression, may be relevant for allelic exclusion of the TCR-beta gene. In the common thymocyte population, the CD3 components, endogenous TCR-alpha, and the delta V-TCR-beta gene product were expressed at the RNA level, but endogenous TCR-beta was not. The TCR-alpha delta beta/CD3 complex was present at the cell surface at low levels and was functional in terms of anti-CD3-induced Ca2+ mobilization. The observed arrest of alpha beta T cell development at the CD4+8+ thymocyte stage indicates that ligand recognition by the TCR, with contribution of the beta-chain V domain, is not required for transition of CD4-8- thymocytes to the CD4+8+ phenotype, but necessary for entry into the "single positive," CD3bright differentiation stage.


Generation of B cell memory and affinity maturation. Induction with Th1 and Th2 T cell clones. We used an adoptive transfer system and CD4+ T cell clones with defined lymphokine profiles to examine the role of CD4+ T cells and the types of lymphokines involved in the development of B cell memory and affinity maturation. Keyhole limpet hemocyanin (KLH)-specific CD4+ Th2 clones (which produce IL-4 and IL-5 but not IL-2 or IFN-gamma) were capable of inducing B cell memory and affinity maturation, after transfer into nude mice or after transfer with unprimed B cells into irradiated recipients and immunization with TNP-KLH. In addition, KLH-specific Th1 clones, which produce IL-2 and IFN-gamma but not IL-4 or IL-5, were also effective in inducing B cell memory and high affinity anti-TNP-specific antibody. The induction of affinity maturation by Th1 clones occurred in the absence of IL-4, as anti-IL-4 mAb had no effect on the affinity of the response whereas anti-IFN-gamma mAb completely blocked the response. Th1 clones induced predominantly IgG2a and IgG3 antibody, although Th2 clones induced predominantly IgG1 and IgE antibody. We thus demonstrated that some Th1 as well as some Th2 clones can function in vivo to induce Ig synthesis. These results also suggest that a single type of T cell with a restricted lymphokine profile can induce both the terminal differentiation of B cells into antibody secreting cells as well as induce B cell memory and affinity maturation. Moreover, these results suggest that B cell memory and affinity maturation can occur either in the presence of Th2 clones secreting IL-4 but not IFN-gamma, or alternatively in the presence of Th1 clones secreting IFN-gamma but not IL-4.


Alloantigenicity of human endothelial cells. 1. Frequency and phenotype of human T helper lymphocytes that can react to allogeneic endothelial cells. To determine the relative ability of allogeneic endothelial cells to stimulate helper T lymphocytes (HTL), human PBMC or purified T cells were incubated in conventional lymphocyte microcultures or in limiting dilution microcultures with allogeneic human umbilical vein endothelia (HUVE), with cytokine-treated allogeneic HUVE, or with allogeneic peripheral blood monocytes. These cultures were tested for IL-2 production as an index of HTL stimulation. Dose-response studies in conventional lymphocyte cultures indicated that allogeneic monocytes were better than allogeneic HUVE at stimulating IL-2 production. Limiting dilution analyses revealed that untreated HUVE and TNF-treated HUVE stimulated small numbers of HTL (approximately 1 HTL/30,000 PBMC), whereas 5 to 10 times more HTL were stimulated by IFN-gamma-treated HUVE and 10 to 20 times more HTL were stimulated by allogeneic monocytes. Serologic deletion studies revealed that most of the high frequency HTL responding to IFN-gamma-treated HUVE were CD4+, whereas most of the low frequency HTL responding to nontreated HUVE or to TNF-treated HUVE were CD8+. Interestingly, mAb to MHC class I and class II molecules, which significantly impaired HUVE-induced proliferation, caused little interference with HUVE-induced IL-2 production. Finally, polymerase chain reaction analysis demonstrated that untreated allogeneic HUVE cells could stimulate PBMC to produce mRNA for IFN-gamma, as well as for IL-2. These data demonstrate the following hierarchy of allogeneic stimulatory capacity for human HTL: monocytes greater than IFN-gamma-treated HUVE much greater than TNF-treated HUVE = nontreated HUVE. Further, these data suggest that non-activated allogeneic endothelial cells can initiate immune responses by inducing IL-2 and IFN-gamma. Because IFN-gamma can induce MHC class II expression by the endothelial cells, this could recruit large numbers of CD4+ T cells for IL-2 production.


Production of transmembrane and secreted forms of tumor necrosis factor (TNF)-alpha by HIV-1-specific CD4+ cytolytic T lymphocyte clones. Evidence for a TNF-alpha-independent cytolytic mechanism. Candidate AIDS vaccines consisting of recombinant forms of the HIV-1 envelope glycoprotein induce, in seronegative human volunteers, an env-specific T cell response that includes CD4+, MHC class II-restricted CTL capable of lysing HIV-1-infected target cells. In this study, we have analyzed the production of the cytokines TNF-alpha and lymphotoxin (LT) by a set of env-specific CD4+ human CTL clones. TNF-alpha and LT are of interest because of their potential role in target cell destruction by CD4+ CTL. Our studies focused on the possibility that a cell surface form of TNF-alpha expressed by CTL after physiologic activation with target APC might participate in the cytolytic reactions mediated by these clones. We found that, upon interaction with target cells expressing env epitopes in the context of the appropriate MHC class II molecules, CD4+ CTL released TNF-alpha with kinetics that were rapid, compared with other cytokines, and that were generally similar to the kinetics of target cell destruction. LT secretion was not detected during the time course of the cytolytic reactions. A novel flow cytometric assay was used to show that physiologic activation of CD4+ CTL with target APC induced expression by the CTL of cell surface forms of TNF-alpha. Immunoprecipitations from activated, surface-iodinated CTL clones revealed two forms of surface TNF-alpha, a 26-kDa form, representing the transmembrane precursor of secreted TNF-alpha, as well as the 17-kDa secreted form bound to the cell surface. For a subset of CD4+ CTL, we found that treatment of CTL with cyclosporin A inhibited Ag-induced production of both transmembrane and secreted forms of TNF-alpha but had no effect on cytolysis. Thus, although transmembrane and secreted TNF-alpha produced by HIV-1-specific CD4+ CTL may have important effects in vivo, the rapid destruction of target APC by the set of CD4+ CTL clones described here occurs through a TNF-alpha-independent mechanism.


The lymphocyte-specific tyrosine protein kinase p56lck is endocytosed in Jurkat cells stimulated via CD2. Incubation of the human T cells, Jurkat, with two sets of activating anti-CD2 mAb (T11(2) + T11(3), D66 + T11(1)) induced delocalization of p56lck and CD2 receptors from the plasma membrane and increased the tyrosine kinase activity of p56lck. The anti-CD2 mAb combination (T11(2) + T11(3)) that produced the most rapid increase in p56lck kinase activity also induced the most rapid delocalization of the kinase. In stimulated cells, both p56lck and CD2 receptors are detected in cytoplasmic vesicles. The internalization of p56lck in endocytic vesicles was established by confocal microscopy. By double staining it was shown that only part of the p56lck colocalized with the internalized CD2 receptor suggesting distinct sorting processes. Internalization of p56lck appeared to be specific of CD2 stimulation as: 1) in Jurkat cells triggered with an anti-CD3 mAb, p56lck was not internalized whereas CD3 receptors were completely endocytosed; 2) when cells were stimulated via CD4, the kinase and CD4 receptors remained associated with the plasma membrane. In addition, internalization of p56lck upon stimulation of CD2 receptors was not modified in CD2+/CD3-Jurkat cells indicating that CD3 is not involved in this process. The identification of different subcellular localizations of p56lck in resting and stimulated T cells should represent an important step in the definition of its functional activity.


Reduced susceptibility to HIV-1 infection of ethyl-methanesulfonate-treated CEM subclones correlates with a blockade in their protein kinase C signaling pathway. We have described the isolation of chemically induced CEM subclones that express CD4 receptors and bind soluble gp120, yet show a markedly reduced susceptibility to infection with HIV-1. Two subclones were found to have an abnormal response to the protein kinase C (PKC) activator PMA. PMA treatment induced CD3 and CD25 (IL-2R) receptors on the parental line and on other ethyl-methanesulfonate-derived subclones, but not on these two mutants. Direct assays of PKC activity were conducted. Total cellular PKC enzymatic activity was found to be normal in these subclones. PMA-induced CD4 down-modulation occurred normally. In addition, activation of c-raf kinase was normal. Since HIV-1 long terminal repeat contains two functional nuclear factor kB (NF-kB) regulatory elements, we studied the ability of PMA to induce NF-kB binding activity by different assays. Chloramphenicol acetyl transferase (CAT) assays using the HIV-1 (-139)long terminal repeat-CAT construct showed no PMA induction of CAT activity in these subclones (unlike the parental line and other subclones). Okadaic acid, an inhibitor of phosphatases 1 and 2A, did not overcome the defect in these subclones. Gel retardation assays, using a 32P-probe containing the HIV-1 NF-kB probe and nuclear extracts from PMA-treated cells, showed significantly reduced induction of nuclear NF-kB binding proteins in these two subclones compared with wild type CEM and a control subclone. Deoxycholate treatment of cytoplasmic extracts from these subclones released much reduced NF-kB binding proteins from their cytoplasmic pools. Thus, reduced levels of PKC-induced nuclear NF-kB activity in two T cell subclones did not affect their normal cell growth, but correlated with a pronounced reduction in their susceptibility to HIV-1 infection.


Inhibition of S-phase progression in macrophages is linked to G1/S-phase suppression of DNA synthesis genes. Some of the important controlling events regulating eukaryotic S-phase progression are considered to occur late in the G1 stage of the cell cycle. We show here that stimulation of DNA synthesis in bone marrow-derived macrophages (BMM) by macrophage CSF-1 is preceded by G1 expression of three genes which encode proteins associated with the DNA synthesis machinery--the M1 and M2 subunits of ribonucleotide reductase and proliferating cell nuclear Ag (PCNA). Increased expression for these genes correlated well with the mitogenic response and sustained expression required de novo RNA and protein synthesis and also the presence of CSF-1 for at least most of G1. Inhibitors of BMM proliferation (LPS, TNF-alpha, IFN-gamma, and cAMP elevating agents) suppressed CSF-1-induced expression of M1, M2, and PCNA mRNA measured at 22 h. This suppression occurred even when added up to 12 h after the CSF-1, a period coinciding with the G1/S-phase boundary. The delayed kinetics of this effect parallels the ability of these agents to maximally inhibit CSF-1-induced BMM DNA synthesis when added at similar times. Decreased expression of M1, M2, and PCNA was not merely a consequence of DNA synthesis inhibition because the S-phase inhibitor, hydroxyurea, did not suppress CSF-1-induced gene expression. These results suggest that inhibition of DNA synthesis by antiproliferative agents involves inhibition of expression of several genes associated with the DNA synthesis machinery.


CD45 alternative exon expression in murine and human CD4+ T cell subsets. Leukocytes express a family of high m.w. glycoproteins called leukocyte common Ag (CD45), which are involved in phosphotyrosine signal transduction. Antibodies to different CD45 isoforms distinguish functionally different CD4+ T cell subsets in humans, rats, and mice. Selected protein isoforms are expressed through a process of exon splicing that is cell-type and differentiation-state specific. Splicing of the three variable exons, A, B, and C, which encode amino acids located near the extracellular amino terminus of the protein, potentially results in generation of eight different mRNA transcripts. The purpose of the present study was to determine the relative levels of all eight different CD45 transcripts present in a panel of murine CD4+ T cell lines and normal murine and human CD4+ T cell subsets separated with antibodies to CD45 variable exons. We show, as expected, that the broad features of CD45 surface isoform expression in these cells can be accounted for by the relative amounts of the eight differentially spliced transcripts. Unexpectedly, all the differences in CD45 isoform expression among the CD4+ T cell subpopulations that we measured could be accounted for by differences in the overall level of variable exon expression. We did not see differences among T cell populations in the relative expression of particular variable exons. Exon B was always found in greater abundance than exons C or A. Of the dual exon species, only AB and BC were found in CD4+ T cells. The AC species was undetectable. Human CD4+ T cells, especially those in the naive subset, express higher levels of CD45 variable exons than murine CD4+ T cells.


Multiple endocrine neoplasia syndrome--type 2b. Case report and review. The multiple endocrine neoplasia syndromes are an association of tumours of 2 or more endocrine glands. Multiple endocrine neoplasia type 2b (MEN 2b) patients develop medullary thyroid carcinoma and pheochromocytomas as well as unique physical characteristics. Most commonly, MEN2b is inherited with an autosomal dominant pattern although sporadic cases are not uncommon. If untreated the disease may be lethal. The facial, oral and ocular characteristics are reliable markers of the disease. These patients give a history most commonly of slipped capital femoral epiphysis, hypertension and life-long diarrhoea and/or constipation. MEN2b is most commonly characterised by nodules on the anterior aspect of the tongue, thickened lips with nodules, thickened upper eyelids, broadened nasal bridge, thickened corneal nerves and dilated, symmetrical, pedunculated nodules on the cheek mucosa. The patient described has most of these characteristics. Radiographic features of the jaws which have not been previously described are reported. These include a markedly enlarged and bifurcated inferior alveolar canal and shortened roots of the lower incisor teeth. Due to the lethality of the disease, patients who present with the above physical characteristics must be further investigated to exclude MEN2b.


Acute foot compartment syndromes.

Acute foot compartment syndromes. Twelve cases of compartment syndrome of the foot in 10 patients were retrospectively reviewed. All were high-energy injuries sustained in a fall from a height (six), crush (three), or motor vehicle accident (three). Bone injuries of the foot included five calcaneal fractures, three multiple metatarsal and/or phalangeal fractures, and two Lisfranc fracture-dislocations with multiple metatarsal neck fractures. The most consistent physical finding was tense swelling of the foot. The diagnosis was confirmed with compartmental pressure measurements in all cases. Decompressive fasciotomies were adequately performed by the medial approach of Henry or a combined approach with medial and dorsal incisions. An additional lateral incision was used in two instances.


Characterization of prejunctional alpha-2 adrenergic receptors involved in modulation of adrenergic transmitter release in the isolated perfused rat kidney. The subclassification of alpha-2 adrenergic receptors into A and B subtypes is based on radioligand binding and functional studies. Radioligand binding studies also have suggested the existence of C and D subtypes, which have only been described as binding sites. This study was designed to determine the subtype of prejunctional alpha-2 adrenergic receptor involved in inhibition of norepinephrine release from sympathetic nerves in the rat kidney. Electrically induced (0.25 Hz, 50 V, 0.5 msec, 10 pulses) fractional tritium overflow was measured in kidneys isolated from male Sprague-Dawley rats and prelabeled with [3H]norepinephrine. The alpha-2 receptor antagonists rauwolscine and yohimbine did not enhance fractional tritium overflow, suggesting the lack of autoinhibition at this frequency of stimulation. The alpha-2 receptor agonist, UK-14,304, inhibited electrically stimulated fractional tritium overflow with an ED50 of 4.85 +/- 0.35 nM. pA2 values for various alpha receptor antagonists against UK-14,304-induced inhibition of fractional tritium overflow were: rauwolscine, 8.8; yohimbine, 8.1; prazosin, 7.4; BAM 1303, 7.5; SKF 104078, 6.4; phentolamine, 8.7; WB 4101, 8.1; corynanthine, 6.1; and ARC-239, 7.2. The correlation coefficients and slopes of the regression lines between pA2 values of alpha receptor antagonists at the prejunctional alpha-2 adrenergic receptor and the reported pKi values obtained from radioligand binding studies were: 0.48 and 0.82; 0.61 and 0.58; 0.53 and 0.85; and 0.89 and 1.05 for the alpha-2A, B, C and D adrenergic receptors, respectively. These data suggest that the prejunctional alpha-2 adrenergic receptor modulating [3H]norepinephrine release in the rat kidney most closely resembles the alpha-2D adrenergic receptor characterized by radioligand binding studies.


Opposite rank order of potency for alpha-2 adrenoceptor agonists on water and solute excretion in the rat: two sites and/or receptors? Preliminary studies determined that, unlike other purported alpha-2 adrenoceptor agonists, 2,6-dimethyl clonidine (2,6-DMC) increased urine flow rate independent of vasopressin. We therefore compared the dose-response curves of three alpha-2 adrenoceptor agonists, clonidine, UK 14,304 and 2,6-DMC. Unilaterally nephrectomized Sprague-Dawley rats were anesthetized and the left kidney was exposed and the ureter cannulated. A 31-gauge needle was advanced into the renal artery to permit direct intrarenal infusion of the study drugs. All three agonists produced a dose-related increase in urine flow rate and sodium excretion. A clear opposite rank order of potency was observed when the urine flow rate was analyzed as free water and osmolar clearance. For free water clearance, clonidine much greater than UK 14,304 much greater than 2,6-DMC, with 2,6-DMC producing little change. The effect on osmolar clearance was opposite with 2,6-DMC much greater than clonidine = UK 14,304. The V2 antagonist [1-(beta-mercapto-beta,beta-pentamethylene-proprionic acid), 2-d-isoleucine,4-isoleucine]arginine-vasopressin blocked the effects of clonidine but not 2,6-DMC. In a water-loaded rat model, 2,6-DMC but not clonidine increased the delivery of filtrate out of the proximal segments of the nephron. These results are consistent with the postulate that lower doses of 2,6-DMC increase solute excretion independent of vasopressin, possibly in proximal segments of the nephron. Clonidine on the other hand increases free water clearance and this effect is mediated through an interaction with the renal actions of vasopressin. Whether these disparate effects represent two distinct receptors or two sites of alpha-2 adrenoceptors in the kidney is not known.


Postnatal development of organic cation transport and mdr gene expression in mouse kidney. The apical surface of the proximal tubular epithelium is the site of both P-glycoprotein localization and postulated active secretion of organic cations in the mammalian kidney. P-glycoprotein has been shown to act as a pleiotropic drug efflux pump across the cell membrane of tumor cells expressing the multidrug resistance phenotype, whereas the renal organic anion and organic cation secretory systems serve the function of pleiotropic drug transport across the proximal tubule epithelium. Because most known substrates for P-glycoprotein are organic cations, we tested the hypothesis that the physiological function of this protein in the kidney is to mediate renal organic cation secretion. In one approach, we compared the postnatal development of organic cation transport with that of kidney mdr gene expression. Cimetidine-sensitive uptake of classical substrates for renal secretion (N-methyl nicotinamide and tetraethylammonium) into kidney slices developed gradually in neonate mice, reaching adult capacity in 4 to 6 weeks. P-glycoprotein and its mRNA, as estimated by immunohistochemical methods and RNAse protection analysis, were undetectable at birth and were expressed abruptly at the adult level between 2 and 3 weeks of age. In another approach, classical inhibitors of renal organic cation secretion (cimetidine and cyanine 863) failed to reverse resistance to adriamycin in Chinese hamster ovary and P388 cell lines, which possess the phenotypic traits of multidrug resistance. These results suggest that the cimetidine-sensitive component of organic cation secretion is mediated by a protein other than the P-glycoprotein in the mammalian kidney.


Possible location and function of neuropeptide Y receptor subtypes in the rat mesenteric arterial bed. Earlier investigation of the vascular actions of Neuropeptide Y (NPY) led us to propose that distinct receptors mediated the prejunctional inhibition of periarterial nerve-stimulated norepinephrine (NE) release and the postjunctional potentiation of the increase in perfusion pressure elicited by vasoconstrictors. These receptors were designated Y2 and Y1, respectively, based on the ability of C-terminal fragments to mimic the former action. The present study investigates further the involvement of these putative receptor subtypes in the isolated and perfused mesenteric arterial bed. [Leu31Pro34]NPY, a novel analog with specificity at the Y1 receptors, potentiated the increase in perfusion pressure elicited by exogenously administered NE and arginine vasopressin, confirming the existence of this NPY subtype postjunctionally. This immediate and prolonged potentiation was abolished by phentolamine, attenuated by benextramine and the reputed NPY antagonist, PYX1. [11-36]NPY also produced a concentration-dependent potentiation of NE-stimulated increase in perfusion pressure suggesting that the Y2 receptor subtype may also be present postjunctionally in this model of the vascular neuroeffector junction. The finding that the profile of this potentiation differed from that elicited by [Leu31Pro34]NPY and, in contrast to the latter, was not attenuated by PYX1, intimates the existence of both distinct subtypes postjunctionally. [Leu31Pro34]NPY also reduced periarterial nerve-stimulated release of NE with a concomitant reduction in perfusion pressure indicating, in addition to the Y2 subtype, the presence of the Y1 receptor prejunctionally in the rat mesenteric arterial bed.


Epidural narcotics: mechanism of action and nursing implications. Patients who receive narcotics by epidural injection or infusion have the potential to develop pharmacological side effects. Theories of epidural pain control and a brief review of spinal anatomy and pain physiology are presented in this article. Narcotic medications used for epidural infusion are pharmacologically distinguished by their hydrophilic or lipophilic character. Important side effects of epidurally administered narcotics, clinical presentation, manifestations, and appropriate nursing interventions are discussed.


Effects of petrochemicals and ultraviolet radiation on epidermal IA expression in vitro. We previously demonstrated that combined treatment of mice with crude oil and longwave ultraviolet radiation (UVA) led to the depletion of IA-positive cells from the epidermis. In the present study, we have developed an in vitro screening assay for combined effects of purified petrochemicals and UVA on epidermal IA and Thy-1 expression. This method involves removal of skin from donor mice prior to treatment with chemicals and UVA (20,000 J/m2), followed by in vitro culture and subsequent immunoperoxidase staining. In this study, a complete correlation was observed in terms of IA-positive cell density among similarly treated cultured skin and live mice. In vivo and in vitro studies both indicated that anthracene but not phenanthrene or benzo[a]pyrene led to significant depletion of both epidermal Langerhans cells and Thy-1-positive dendritic cells when followed by UVA treatment. The in vitro assay developed for this study should prove to be a valuable tool for the screening of a wide variety of chemicals for contact photosensitizing activity.


Transcellular activation of the human immunodeficiency virus type 1 long terminal repeat in T lymphocytes requires CD4-gp120 binding. Cells expressing human immunodeficiency virus type 1 (HIV-1) tat can transactivate the HIV-1 long terminal repeat (LTR) in cocultured T lymphocytes. In this report, we describe the molecular requirements for transcellular activation of the LTR in Jurkat cells. An analysis with deletion mutants and blocking antibodies demonstrated a requirement for env expression in addition to tat expression for transcellular activation to occur. The results suggest that the transient association of CD4 and gp120 in cocultured cells is required for tat-mediated transcellular activation. The events that follow CD4-gp120 binding in transactivation, however, do not require the gp120-neutralizing domain, in contrast to HIV-mediated fusion and infection. The consequences of this interaction on cellular function are currently under investigation.


Role of human T-cell leukemia virus type 1 X region proteins in immortalization of primary human lymphocytes in culture. Human T-cell leukemia virus type 1 (HTLV-1) immortalizes human CD4+ T lymphocytes in culture. Previous studies show that in the context of a herpesvirus saimiri vector, the sequence of the X region at the 3' end of the HTLV-1 genome is also capable of immortalizing CD4+ lymphocytes in the absence of HTLV-1 structural proteins. The X region of HTLV-1 encodes two trans-acting viral proteins, the 42-kDa Tax protein and the 27-kDa Rex protein. Infection of human cord blood cells with herpesvirus saimiri recombinants which contain HTLV-1 X region sequences defective for expression of tax, rex, or both tax and rex demonstrates that tax function is necessary and sufficient for immortalization of primary human CD4+ cord blood lymphocytes in culture in the context of the herpesvirus saimiri vector.