Influence of Coxiella burnetii infection of male mice on their offspring.

The dramatic spread of Q fever in Poland among cattle kept in isolation from natural environment (ticks, wild animals) has suggested the possibility that the infection may also be transmitted sexually. To test this hypothesis series of experiments have been performed in controlled laboratory conditions. Male mice infected with C. burnetii were allowed to mate with healthy female mice. On day 18 of pregnancy serum IgM antibodies to C. burnetii antigens and bacteria in spleen, liver and placenta were detected. The influence of C. burnetii transmission between parents of their offspring was investigated. It has been found that C. burnetii infection in males diminish the number of fertilized females. Their litters are fewer in number and the number of dead embryos is increased.


DNA probes for detecting Coxiella burnetii strains.

Methods have been developed for the rapid detection of C. burnetii by specific hybridization of labelled DNA probes to rickettsial plasmid DNA sequences present in clinical samples. One DNA probe detects all C. burnetii strains, while additional probes differentiate, between organisms associated with chronic or acute disease. Using these probes, C. burnetii can be identified in blood, urine, and tissue samples. The plasmid-derived DNA probes detect as few as 10(4) organisms and less than 1 ng of Coxiella DNA. Host-cell DNA has no effect on the hybridization signal from C. burnetii DNA, and these probes do not cross-react with a variety of microorganisms, including both common laboratory contaminants and organisms that cause clinical symptoms similar to those of Q fever. The sensitivity of the assay is markedly enhanced when the procedure employs the polymerase chain reaction (PCR) to amplify C. burnetii DNA. This requires construction of oligonucleotide primers to DNA sequences flanking the target region of the DNA being amplified. For C. burnetii detection, several sets of primers have been prepared. One set is derived from the QpH1 H fragment, a region that is shared by all C. burnetii plasmids (homologous sequences are also present in the plasmidless strains of C. burnetii). The H primers detect all strains of C. burnetii. To differentiate between C. burnetii strains, additional primers, specific for DNA sequences that are unique either to chronic or acute disease-related strains of C. burnetii are employed. PCR amplifies target sequences up to 10(6)-fold. When DNA hybridization is used in conjunction with PCR, the test can detect less than 10 C. burnetii cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Cloning and expression in Escherichia coli of the 37-, 14-, and/or 16-kilodalton antigens genes from Rickettsia prowazekii strain E.

A gene bank of Rickettsia prowazekii strain E constructed in the phage vector lambda EMBL4 was screened for antigen production with anti-R. prowazekii serum. One of the immunoreactive clones, grown at 37 degrees C exhibited the expression of at least two antigens of molecular weight (M(r)) 37 kD and 14 kD. Subcloning and further analysis revealed that the antigens (polypeptides) of Mr 37, 14, and/or 16 kD apparently represent structural units of the 138 kD complex antigen. Assembly of the above mentioned polypeptides was found to be thermosensitive as it took place at 30 degrees C but not at 37 degrees C and resulted in an oligomeric structure of M(r) 138 kD. The nucleotide sequence of the gene coding for a precursor of the mature polypeptides of Mr 14 and/or 16 kD was determined.


Neuroleptic medication and prescription practices with sheltered-care residents: a 12-year perspective.

Most adult residents of sheltered-care facilities (board and care, family care, psychosocial rehabilitation, and other supported housing arrangements) for the chronically mentally ill receive neuroleptics. These facilities house over 300,000 mentally ill residents, but neuroleptic prescription practices with this population have not been studied. A probability sample (n = 393) of all adult former psychiatric patients in sheltered care in California was surveyed in 1973; 94% of the located survivors (n = 243) were reinterviewed 12 years later. In 1973, 79% received neuroleptics; in 1985, 76%. Polypharmacy decreased, and the elderly remained less medicated than adults. Yet, mean daily neuroleptic doses doubled, more persons received higher doses, and 62% reported adverse effects. Furthermore, high dosing was attributed to psychiatrists rather than other physicians, even when controlling for residents clinical and sociodemographic characteristics. Neuroleptic drugs became the staple pharmacological treatment for mentally ill sheltered-care residents. While physicians more cautiously medicated the elderly, they had not reduced doses by 1985, even after a decade of treatment. The specialty of the prescriber was an important factor in preference for high-dose treatment.


Serotype, hemolysin production, and adherence characteristics of strains of Escherichia coli causing urinary tract infection in dogs.

Virulence factors were studied in 82 strains of Escherichia coli isolated from the urine of dogs with urinary tract infections. The most frequently expressed O antigens were 2, 4, 6, 25, and 22/83. Most strains were K nontypeable. Mannose-sensitive hemagglutination (MSH) with canine erythrocytes was observed in 71 strains and mannose-resistant hemagglutination (MRH) was observed in 32 strains. Strains that caused MSH of erythrocytes from dogs also caused MSH of erythrocytes from guinea pigs. Most strains that caused MRH of human A1P1 erythrocytes also reacted with erythrocytes of dogs. Of 22 strains (27%) that agglutinated human A1P1 erythrocytes, but not A1p erythrocytes, 17 (77%) had specificity for globo A, but did not react with the galactose alpha 1----4galactose beta disaccharide receptor. The remaining 5 strains and 2 others that simultaneously expressed an X adhesin agglutinated galactose alpha 1----4galactose beta-coated latex beads. Bacterial adherence to canine uroepithelial cells from the bladder was most often observed in strains expressing MSH, less often observed in strains expressing MRH, and least often observed in strains that failed to induce hemagglutination. Adherence of MSH strains to canine uroepithelial cells was inhibited by alpha-methyl-D-mannoside. As a group, MRH strains expressing globo-A- and galactose alpha 1----4galactose beta-specific adhesins did not have strong adherence. Strains of E coli isolated from dogs with urinary tract infections most commonly expressed type-1 fimbriae, and the main mechanism of in vitro adherence to canine uroepithelial cells involved a mannose-sensitive mechanism. Overrepresentation of globo-A-specific adhesins did not appear to be related to adherence of canine uroepithelial cells.


Efficacy of AHR-13268, an antiallergenic compound, in the management of pruritus caused by atopic disease in dogs.

Twenty-nine pruritic, atopic dogs were entered into a double-blind, placebo-controlled, crossover study to evaluate the efficacy of an investigational antiallergenic compound, AHR-13268. Fourteen dogs were evaluated by a veterinary dermatologist (at intervals) and the owner (daily). Fifteen dogs were evaluated only by the owner. The mean (+/- SE) owner scores for pruritus, erythema, and lesions with placebo treatment (higher score = worse signs) were 3.24 (+/- 0.12), 2.73 (+/- 0.12), and 2.61 (+/- 0.09), respectively. With drug treatment, the corresponding scores were 2.89 (+/- 0.12), 2.50 (+/- 0.12), and 2.25 (+/- 0.09). Scores for pruritus and lesions (but not erythema) were significantly better with drug treatment than with placebo treatment. Investigator scores showed similar trends, but the differences were not great enough to be statistically significant. Overall, 11/29 (38%) owners reported their dogs had moderate or better improvement from drug capsules, and 4/29 dogs (14%) improved on placebo capsules. A variety of adverse effects were reported following both drug (9/29 dogs) and placebo (8/29 dogs) capsule administration, but were mild and well tolerated. Results of this study indicate that AHR-13268 has potential for empiric treatment of allergic inhalant dermatitis in some dogs.


Apparent heterozygote deficiencies observed in DNA typing data and their implications in forensic applications.

Restriction fragment length polymorphisms (RFLP) analysis using the Southern blot technique can be used to recognize copy number variation of variable number of tandem repeats (VNTR) of conserved core sequences at several regions of the human genome. This new class of polymorphisms reveals a high degree of genetic variation, useful for individual identification purposes. Criticisms against forensic applications of such DNA typing data include the limitation of employing Hardy-Weinberg expectation of genotype frequencies, since several surveys indicate apparent deficiency of heterozygosity (or excess homozygosity) in comparison with Hardy-Weinberg expectations. This research postulates an alternative explanation of deficiency of apparent heterozygosity which is caused by the inability to detect extremely small-sized alleles (called 'non-detectable' alleles) due to the sensitivity of Southern gel electrophoresis. We show that the presence of 'non-detectable' alleles can produce pseudo-homozygosity and their frequencies can be predicted from the observed proportional heterozygote deficiency. Furthermore, in the covert presence of such 'non-detectable' alleles, we show that the gene-count method provides over-estimates of allele frequencies in the sample population, and hence the Hardy-Weinberg predictions of genotype frequencies avoid wrongful bias against suspects in forensic applications of DNA typing data. Applications of this theory to population data on six VNTR loci in US Caucasians and US Blacks suggest that the presence of 'non-detectable' alleles could be the major cause of apparent heterozygote deficiency, and the current approaches of predicting the population frequency of specific DNA phenotypes are practically free of the possible wrongful bias in courtroom applications of DNA typing data.


Chronic neuroleptic treatment: D2 dopamine receptor supersensitivity and striatal glutamatergic transmission.

We studied the in vitro electrical activity of rat neostriatal neurons following chronic neuroleptic treatment. In haloperidol-treated rats, unlike naive animals, activation of neostriatal D2 dopamine receptors induced a potent presynaptic inhibition of glutamate-mediated excitatory synaptic potentials. Haloperidol treatment did not affect the intrinsic membrane properties of the neostriatal neurons. Pre- and postsynaptic physiological responses to direct and indirect gamma-aminobutyric acid (GABA)-ergic and cholinergic agonists were not affected by chronic haloperidol treatment. These findings suggest that movement disorders induced by chronic neuroleptic treatment may result, at least in part, from a hypersensitivity of presynaptic D2 dopamine receptors regulating the release of glutamate.


Prospective trial comparing the use of sulphasalazine and auranofin as second line drugs in patients with rheumatoid arthritis.

Two hundred patients with rheumatoid arthritis were studied in a prospective open trial comparing treatment with sulphasalazine and auranofin in patients with active disease over 12 months. The two drugs improved many parameters of disease activity at 12, 24, and 48 weeks. At 12 weeks, the group treated with sulphasalazine had a lower platelet count (Mann-Whitney U test), erythrocyte sedimentation rate, and articular index, with a greater decrease in erythrocyte sedimentation rate (Students t test) and C reactive protein between 0 and 12 weeks. There were no significant differences between sulphasalazine and auranofin treatment after 24 and 48 weeks. Life table analysis showed no significant differences in the rate of side effects which caused treatment to be stopped. Sulphasalazine works more rapidly, may be a more effective disease modifying antirheumatic drug, and is as well tolerated as auranofin.


Dipeptidyl peptidase II- and IV-like activities in gingival tissue and crevicular fluid from human periodontitis lesions.

Gingival tissue and gingival crevicular fluid were collected from patients with chronic periodontitis. Gel filtration chromatography of crude tissue extracts yielded separate fractions active against Lys-Ala-7-amino-4-trifluoromethyl-coumarylamide (AFC) at acid pH and Gly-Pro-AFC at alkaline pH. The molecular weights, pH optima and inhibitor responses of these activities were consistent with those of dipeptidyl peptidases (DPP) II and IV, respectively. When tested with the same substrates, crevicular fluid was also found to contain DPP II- and IV-like activities with very similar pH profiles and inhibitor responses to those in tissue. The close resemblance suggested that the crevicular fluid enzymes were derived mainly from inflamed gingival tissues. Slight differences in the DPP II-like activities might be explained by the additional presence in crevicular fluid of enzymes from subgingival bacteria. With use of appropriate buffers, a third substrate, Ala-Pro-AFC, gave selective detection of both DPP II- and IV-like activities in tissue and crevicular fluid. Assays with Ala-Pro-AFC had the advantage of greater sensitivity, especially with DPP II-like activity. Raised levels of this enzyme have previously been found in the gingiva of periodontitis patients and thus DPP II-like activity in crevicular fluid might prove of value in monitoring disease activity.


Inflammatory bowel disease presenting in pregnancy.

A case of inflammatory bowel disease (IBD) presenting in pregnancy is described. Despite previous reports of severe fulminating disease in this type of patient, this woman did well with an uncomplicated course; she responded to standard medical therapy and there were no fetal complications. IBD should not be a contraindication to pregnancy unless the disease is poorly controlled. Pregnancy does not increase the risk of relapse of IBD, but should this occur it is more likely in the first trimester or in the postpartum period. Treatment of IBD in pregnancy should be much the same as in the nonpregnant woman. Corticosteroids and sulphasalazine are safe in pregnancy and are the mainstays of medical treatment. Surgery should proceed for the usual indications of toxic megacolon and perforation. In the group requiring surgery fetal mortality is considerable but the maternal outcome is improving. Patients presenting with IBD in pregnancy may have more severe disease but recent reports suggest that the outcome for mother and infant in this group is improving.


The homeodomain: a new face for the helix-turn-helix?

The discovery of conserved protein domains found in many Drosophila and mammalian developmental gene products suggests that fundamental developmental processes are conserved throughout evolution. Our understanding of development has been enhanced by the discovery of the widespread role of the homeodomain (HD). The action of HD-containing proteins as transcriptional regulators is mediated through a helix-turn-helix motif which confers sequence specific DNA binding. Unexpectedly, the well conserved structural homology between the HD and the prokaryotic helix-turn-helix proteins contrasts with their divergent types of physical interaction with DNA. A C-terminal extension of the HD recognition helix has assumed the role that the N-terminus of the prokaryotic helix plays for specification of DNA binding preference. However, the HD appears also capable of recognizing DNA in an alternative way and its specificity in vivo may be modified by regions outside the helix-turn-helix motif. We propose that this intrinsic complexity of the HD, as well as its frequent association with other DNA binding domains, explains the functional specificity achieved by genes encoding highly related HDs.


Neuropeptides and classical transmitters. Localization and interaction.

The present article briefly reviews some aspects on the localization and possible functional roles of neuropeptides. It is emphasized that a large number of peptides can be found in the nervous system and that they in many instances occur together with classical transmitters such as acetylcholine and catecholamines in the same neurons. In agreement, functional studies have revealed that they interact in different ways, both synergistically and antagonistically, with the transmitters. In some instances peptides may also have trophic effects. The recent cloning of neuronal peptide receptors has further substantiated a physiological role for these compounds in the nervous system. Moreover, the recent development of peptide antagonists, which pass the blood brain barrier, now opens up new possibilities to elucidate the functional role of neuropeptides and thus of the coexistence phenomenon.


Glutamatergic-dopaminergic balance in the brain. Its importance in motor disorders and schizophrenia.

Dopamine appears to be of less importance in the regulation of psychomotor functions than was previously thought. A central dopaminergic-glutamatergic balance may be important for both akinetic motor disorders and psychosis. In Parkinson's disease glutamate antagonists may counteract central glutamatergic hyperactivity and may be of value as anti-parkinsonian drugs. An increase of dopaminergic activity and/or a reduction of glutamatergic activity may contribute to the development of paranoid hallucinatory psychosis in schizophrenic patients and of pharmacotoxic psychosis in Parkinson's disease. Because of possibly severe side-effects of glutamatergic antagonists and agonists in the treatment of akinesia and psychosis, the development of partial glutamate agonists/antagonists could be an alternative strategy capable of producing antipsychotic or anti-kinetic effects with only mild adverse reaction.


Overexpression of the c-erbB-2/neu-encoded p185 protein in primary lung cancer.

The c-erbB-2/neu gene encodes a transmembrane protein of 185 kDa (p185) with tyrosine kinase activity and extensive sequence homology to epidermal growth factor receptor. Amplification and overexpression of the c-erbB-2/neu gene has been shown in certain human tumors and is postulated to be important in human carcinogenesis. High levels of expression of the c-erbB-2/neu gene have been reported in non-small-cell lung cancer (NSCLC) cell lines and primary tumors from the United States. Since geographical and cultural factors may contribute to the development of certain types of cancer, we examined p185 examined p185 expression in 120 tumors from Chinese patients with lung cancers of different cell types and used immunohistochemical staining to determine the extent and general significance of p185 expression in human primary lung cancer. Our results demonstrate that 58.8% of the NSCLCs expressed p185 and that expression of p185 was observed only in NSCLC and not in small-cell lung cancers. Thirty-three of 41 adenocarcinomas and 24 of 55 squamous cell carcinomas among the NSCLCs examined were found to express p185 at levels different from those of normal lung. For the squamous cell carcinomas, p185 expression was correlated with lymph node metastasis (P less than 0.01), but for the adenocarcinomas, it was not (P greater than 0.05). In addition, expression of p185 in NSCLC was significantly more frequent in patients in advanced clinical stages. Our findings indicate that p185 expression is a frequent event and a general phenomenon in NSCLC and is correlated with poor clinical prognostic indicators, suggesting that expression of p185 may be of potential prognostic importance in NSCLC.


Inhibition of human, ovine, and baboon neutrophil elastase with Eglin c and secretory leukocyte proteinase inhibitor.

The association rate constants (kon) of human, ovine, and baboon neutrophil elastase with two recombinant serine proteinase inhibitors (Eglin c, secretory leukocyte proteinase inhibitor) were compared. The association rate constant of sheep leukocyte elastase (SLE) with Eglin c is about 100 times lower (kon = 2.2 x 10(5) M-1s-1) than that of human elastase (kon = 2.4 x 10(7) M-1s-1). Baboon elastase, however, is as effectively blocked with Eglin c (kon = 2.5 x 10(7) M-1s-1) as human elastase. Secretory leukocyte proteinase inhibitor (SLPI) blocks the elastase of all three species with high efficiency; baboon elastase shows the highest association rate constant (kon = 5.6 x 10(7) M-1s-1) followed by human elastase (kon = 4.1 x 10(7) M-1s-1) and finally sheep elastase (kon = 1.2 x 10(7) M-1s-1). These findings demonstrate marked differences in the inhibition kinetic properties of ovine and human elastase. Concerning a future clinical application of proteinase inhibitors, the baboon seems a more suitable model than sheep to evaluate the effects of Eglin c and SLPI, since both inhibitors block baboon and human elastase with comparable efficiency.


Molecular cloning and expression of a high affinity L-proline transporter expressed in putative glutamatergic pathways of rat brain.

We have used the polymerase chain reaction (PCR) with degenerate oligonucleotides derived from two conserved regions of the norepinephrine and gamma-aminobutyric acid transporters to identify novel Na(+)-dependent transporters in rat brain. One PCR product hybridized to a 4.0 kb RNA concentrated in subpopulations of putative glutamatergic neurons including mitral cells of the olfactory bulb, pyramidal cells of layer V of the cerebral cortex, pyramidal cells of the piriform cortex, and pyramidal cells of field CA3 of the hippocampus. Transient expression of the cognate cDNA conferred Na(+)-dependent L-proline uptake in HeLa cells that was saturable (Km = 9.7 microM) and exhibited a pharmacological profile similar to that for high affinity L-proline transport in rat brain slices. The cloned transporter cDNA predicts a 637 aa protein with 12 putative transmembrane domains and exhibits 44%-45% amino acid sequence identity with other members of the emerging family of neurotransmitter transporters. These findings support a synaptic role for L-proline in specific excitatory pathways in the CNS.


Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene family, in the developing rat brain.

We have previously characterized a novel transcription factor family in mammals, the HNF-3 family, by the members' homology to one another and to the Drosophila homeotic gene fork head. The expression of fork head is restricted to the anterior and posterior termini of the early fly embryo. Brain factor 1 (BF-1) is a new member of this family isolated from rat brain with an expression pattern and DNA binding specificity distinct from the HNF-3 genes. Expression is highly restricted in the developing neural tube to its rostral end, which gives rise to the telencephalon. These results suggest that BF-1 plays an important role in the establishment of the regional subdivision of the developing brain and in the development of the telencephalon.


Oxygen or glucose deprivation-induced neuronal injury in cortical cell cultures is reduced by tetanus toxin.

We examined glutamate-mediated neurotoxicity in cortical cell cultures pretreated with 1-5 micrograms/ml tetanus toxin to attenuate the Ca(2+)-dependent release of neurotransmitters. Efficacy of the tetanus toxin pretreatment was suggested by blockade of electrical burst activity induced by Mg2+ removal and by reduction of glutamate efflux induced by high K+. Tetanus toxin reduced neuronal injury produced by brief exposure to elevated extracellular K+ or to glutamate, situations in which release of endogenous excitatory neurotransmitter is likely to play a role. Furthermore, although glutamate efflux evoked by anoxic conditions may occur largely via Ca(2+)-independent transport, tetanus toxin attenuated both glutamate efflux and neuronal injury following combined oxygen and glucose deprivation. With prolonged exposure periods, the neuroprotective efficacy of tetanus toxin was comparable to that of NMDA receptor antagonists. Presynaptic inhibition of Ca(2+)-dependent glutamate release may be a valuable approach to attenuating hypoxic-ischemic brain injury.


Light stimulation of the hypothalamic neuroendocrine system.

This paper demonstrates that, in the mediation of light, the suprachiasmatic nucleus (SCN) functionally associates with the anterior periventricular and parvocellular paraventricular neuron systems in rats. Intact rats (group 1) and rats undergoing a hemicomplete cutting of the SCN (group 2) were housed in a dark room (2-3 weeks) and killed after an exposure to light for 10, 30 or 60 min. Other intact animals (group 3) kept in a dark room (2 weeks) were exposed to light for 10 min, then stored 60 min in the dark room, and killed in darkness. The SCN, anterior periventricular nucleus, and parvocellular paraventricular nucleus were examined immunohistochemically using antisera for vasoactive intestinal polypeptide (VIP), arginine vasopressin, somatostatin, rat corticotropin releasing factor (rCRF), and c-fos protein. In comparison with animals kept in darkness, animals exposed for 10 and 30 min to light indicated a remarkable reduction of VIP immunoreactivity in the SCN and some increase of CRF immunoreactivity in the parvocellular paraventricular nucleus. The diminution of VIP immunoreactivity did not occur in the isolated SCN of group 2 animals. In group 3, a 10 min-light exposure induced a remarkable enhancement of nuclear c-fos immunoreactivity in neurons in the ventrolateral region of the SCN, in the anterior periventricular nucleus, and in the parvocellular paraventricular nucleus, most strongly in the SCN. Double immunolabeling methods have shown that VIP, somatostatin, and CRF neurons in the respective nuclei were c-fos positive.


Enantioselective pharmacokinetics of ethotoin in humans following single oral doses of the racemate.

Racemic ethotoin (1000 mg) was administered orally as a single dose to six healthy adult volunteers. Blood samples were collected at appropriate times for 120 h following the dose. Ethotoin was quantified enantio-selectively in plasma using a novel chiral column HPLC procedure. One of the enantiomers of the chiral metabolite, 5-phenylhydantoin, was also quantified in the HPLC method. The Cmax and AUC0-infinity values for (+)-(S)-ethotoin were significantly greater than those for (-)-(R)-ethotoin (ratio of mean AUC0-infinity values 0.88), but the elimination half-lives of the isomers were virtually identical [12.35 +/- 5.15 h for (-)-(R)-ethotoin; 12.28 +/- 5.34 h for (+)-(S)-ethotoin]. Parameters derived from AUC0-infinity (Cl0/F and V(area)/F) also differed slightly between the isomers. The data were interpreted as indicating a small difference in the absorption of the two isomers; it seemed unlikely, in terms of the identical elimination rates, that their metabolic profiles would differ greatly. The 5-phenylhydantoin was eliminated with a significantly longer half-life (18.69 +/- 6.11 h) than that of ethotoin. Enantioselectivity in the pharmacokinetics of ethotoin is therefore a minor issue.


Long-term cholinergic denervation caused by early postnatal AF64A lesion prevents development of muscarinic receptors in rat hippocampus.

The effect of early postnatal (day 8) intracerebroventricular injections of the putative cholinotoxin ethylcholine aziridinium mustard (AF64A) on development of cholinergic innervation and postsynaptic muscarinic acetylcholine receptors in the rat hippocampus was examined. The cholinotoxin applied at this stage of development leads to a permanent denervation of cholinergic fibres in the hippocampus in adulthood demonstrated by (immuno)histochemical methods and biochemical assays. Muscarinic receptor expression in the principal neurons of dentate gyrus and cornu ammonis was strongly reduced as studied by immunostaining with antibodies against muscarinic receptor proteins and binding assays with the muscarinic antagonist quinuclidinyl benzilate. Cholinoceptive interneurons and somatostatinergic interneurons are not affected by the developmental cholinergic lesion. Immunoreactivity to protein kinase C type I as a marker for inositolphosphate-related cellular activation systems slightly decreased in the apical dendrites of the hippocampal principal neurons. These findings indicate that damage to ingrowing cholinergic terminals in the hippocampus in the early postnatal period is a critical hazard for development of the muscarinic receptor system in the hippocampal principal neurons. These results are discussed for their significance to the neural mechanisms that underlie perinatal brain damage and associated cognitive dysfunction.


Chromatographic resolution of the chiral isomers of several beta-blockers over cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase.

The optical resolution of seven beta-blockers which have in common the N-isopropyl-3-aryloxy-2-hydroxypropylamine moiety was carried out by HPLC using the cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase to quantitatively characterize the enantioselectivity of these compounds. The capacity factors and separation factors at different column temperature were determined with some qualitative trends derived. A compensation effect was observed for these compounds where there exists an approximately linear relationship between the enantiomeric differences in enthalpic and entropic energies.


Follow-up of retinoblastoma patients having prenatal and perinatal predictions for mutant gene carrier status using intragenic polymorphic probes from the RB1 gene.

We have carried out presymptomatic prediction of mutant gene carrier status in ten individuals with a family history of retinoblastoma. In all cases standard linkage studies were employed using intragenic DNA probes which recognise restriction fragment length polymorphisms. In four cases foetal DNA samples were obtained by chorionic villus sampling, the remaining six were derived from either cord blood samples or venipuncture of neonates. We demonstrated that the mutant gene was inherited by only one of these patients who has subsequently developed bilateral tumours. Six of the other cases have now reached the age beyond which it might have been expected that tumours would develop and are all disease free. It must be concluded that repeated ophthalmological examination of these and future patients shown not to have inherited the mutant gene, is unnecessary.


Retinogeniculate EPSPs recorded intracellularly in the ferret lateral geniculate nucleus in vitro: role of NMDA receptors.

We used an in vitro preparation of the ferret lateral geniculate nucleus (LGN) to examine the role of the NMDA class of excitatory amino acid (EAA) receptors in retinogeniculate transmission. Intracellular recordings revealed that blockade of NMDA receptors both shortened the time course and reduced the amplitude of fast and slow components of excitatory postsynaptic potentials (EPSPs) evoked by optic tract stimulation. The amplitude and width of the EPSPs mediated by NMDA receptors increased as membrane potential was depolarized towards spike threshold. Individual LGN cells were influenced to varying extents by blockade of NMDA receptors; NMDA and non-NMDA receptor blockade together attenuated severely the entire retinogeniculate EPSP. The dependence of all components of retinogeniculate EPSPs (and action potentials) on NMDA receptor activation supports the hypothesis that the NMDA receptor participates in fast (less than 10 ms) synaptic events underlying conventional retinogeniculate transmission. The voltage dependence of the NMDA receptor-gated conductance suggests strongly that the transmission of retinal information through the LGN is subject to modulation by extraretinal inputs that affect the membrane potential of LGN neurons.


Different types of non-P-glycoprotein mediated multiple drug resistance in children with relapsed acute lymphoblastic leukaemia.

Although cellular drug resistance is considered to be an important cause of the poor prognosis of children with relapsed acute lymphoblastic leukaemia (ALL), the knowledge of drug resistance in these patients is very limited. Different aspects of drug resistance were studied in 17 children with relapsed ALL. The in vitro sensitivity profile was determined using the MTT assay. Cells from relapsed children were significantly more resistant to 6-thioguanine, prednisolone, cytosine arabinoside, daunorubicin (DNR), mustine-HCl and mafosfamide but not to L-asparaginase and vincristine (VCR) than cells from 41 children with ALL at initial diagnosis. Some relapsed patients showed a general drug resistance while others were resistant to only 1-3 drugs. The relevance of the multidrug resistance (MDR) model was analysed: In all DNR- and VCR resistant cases a co-resistance to drugs not involved in the MDR model was found. P-glycoprotein was not detected in any of 28 untreated and 14 relapsed samples tested. VCR- and DNR accumulation in the most resistant cells were not lower than in sensitive cells. Resistance modifiers did not potentiate the cytotoxicity of VCR and DNR. We conclude that resistance to anthracyclines and vinca alkaloids in childhood relapsed ALL is not due to P-glycoprotein mediated MDR. Different types of drug resistance varying from a resistance to only one drug to a general chemoresistance, can be detected in children with relapsed ALL. VCR and L-asparaginase seemed to be only infrequently involved in drug resistance. Knowledge of drug resistance might lead to more effective and less toxic therapies for children with relapsed ALL.


Lower respiratory tract infections after abdominal operations: epidemiology and risk factors.

To find out the incidence and aetiology of lower respiratory tract infections after abdominal operations and identify predisposing factors. Prospective open study. Department of General Surgery, Hospital Germans Trias i Pujol, Barcelona, Spain. 2,083 patients having abdominal operations between March 1985 and June 1987 excluding splenectomies, and those requiring thoracoabdominal incisions. The presence of three or more of the following: temperature of 38 degrees C or more, cough with mucopurulent sputum, raised white cell count, or changes on the chest radiograph or bronchogram. 50 patients (2.4%) developed lower respiratory tract infections. Micro-organisms were isolated from 15 (30%), the most common being Haemophilus influenzae. The most important risk factors were American Society of Anesthesiologist's grade, duration of anaesthesia, age, and operations on the upper gastrointestinal tract. Further work is needed to investigate other possible predisposing factors and develop a predictive score.


Wound infection after cholecystectomy. Correlation between bacteria in bile and wound infection after operation on the gallbladder for acute and chronic gallstone disease.

To see if there was a difference in the wound infection rates after operation for acute and chronic cholecystitis, and to see if the presence of bacteria in the bile had any influence on those rates. Prospective open study. University hospital. 213 Patients undergoing cholecystectomy for acute or chronic gallstone disease. Incidence of postoperative wound infection, and of bile cultures growing pathogenic organism. There was no difference in wound infection rates between patients operated on for acute and those operated on for chronic cholecystitis. The presence of bacteria in the bile did not seem to influence the wound infection rate in either group. Early cholecystectomy and appropriate antibiotic prophylaxis result in an acceptably low wound infection rate, and the growth of bacteria from bile is not predictive of the development of wound infection.


Advantages and disadvantages of using the hydrogen clearance technique to measure pancreatic blood flow.

This study was undertaken to identify the advantages and disadvantages of using the hydrogen clearance technique (HCT) to measure pancreatic blood flow (PBF) in the opossum over short and long periods. An open study was performed on 8 opossums. A total of 72 platinum electrodes were implanted into the pancreas. On the fifth day 41 additional electrodes were implanted. All implantation sites were examined histologically after five days. Measurements of pancreatic blood flow were taken every other day for five days. Three measurements were taken from each electrode and the mean of the three was used for analysis. In the anaesthetized animal mean PBF (SD) was 63.5 (12.8) ml min-1 100 g-1 immediately after electrode implantation and 34.3 (11.4) ml min-1 100 g-1 on the 5th postoperative day. Of the implanted electrodes, 35% failed during the experimental course, mainly as the result of proliferation of scar tissue around the electrodes. Mild to moderate fibroproliferation had taken place around the platinum tips of the remaining 65%. Primary electrode implantation caused no or negligible trauma to the pancreas. We recommend the HCT only for short-term measurements of PBF. Implantation of electrodes for any length of time leads to inflammatory changes around the platinum tips that cause proliferation of scar tissue and lead to failure of the electrodes, misleadingly low PBF recordings, and fewer monoexponential clearance curves.


Pancreatico-duodenectomy: 13-years' experience.

This paper reports a retrospective study of 51 consecutive pancreatico-duodenectomies from a Swedish university hospital concerning postoperative morbidity, number of reoperations and mortality. Resection of the distal common bile duct, duodenum, and head of the pancreas with hemigastrectomy and cholecystectomy was done in 48 patients and modified pancreaticoduodenectomy with preservation of the pylorus was done in three patients. Three patients (6%) died within 30 days. Nine patients required reoperation, three more than once. The most common major complications were anastomotic breakdown (n = 7), severe hemorrhage (n = 3) and abscess formation (n = 3). We conclude that a low postoperative morbidity and mortality can be achieved outside large, specialist centres, but the operation should be done by experienced surgeons. The results may justify carrying out the operation when the chances of cure are small, but it has yet to be evaluated as a palliative procedure.


Acute mesenteric ischaemia.

To identify any differences in presentation among the four types of acute mesenteric ischaemia, and to correlate time between presentation and treatment with outcome, we retrospectively analysed 100 cases of acute mesenteric ischaemia at a University hospital diagnosed by radiography (n = 21), at laparotomy (n = 61), or at necropsy (n = 18). A total of 68 patients died. Mortality was 50% when the aetiology was embolic occlusion of the superior mesenteric artery and 95% when the occlusion was thrombotic; 67% when the disease was "non"-occlusive; and 30% in cases of splanchnic vein thrombosis. We conclude that early diagnosis is critical for successful management of acute mesenteric ischaemia, but outcome is also influenced by the aetiology.


Spontaneous rupture of the spleen.

A 40-year-old woman with bronchial asthma was admitted as a medical emergency with suspected myocardial infarction. Spontaneous splenic rupture was diagnosed during hospitalization and splenectomy successfully performed. No histopathologic changes or signs of previous injury or adhesions were found.


Subcutaneous avulsion of the breast caused by a seat belt injury. Report on a case requiring emergency mastectomy.

A 60-year-old female car driver wearing a combined three-point lap-shoulder seat belt was involved in a head-on-collision. The entire right breast was separated from the chest wall, whereas the overlying skin remained intact. The injury resulted in a rapid development of a monstrous haematoma in the retromammary space, and required emergency mastectomy. Seat belt lesions of the female breast are seldom referred in the literature.


Importance of dehydration in anastomotic and subcutaneous wound healing: an experimental study in rats.

To find out if preoperative and postoperative dehydration adversely affect anastomotic and subcutaneous healing. Randomized study. 18 Wistar rats. Dehydration established in nine rats by withdrawal of food and water for 24 hours before operation, and by injections of frusemide twice daily. Laparotomy and division of intestine 5-8 cm from the ileocaecal valve. Implantation of expanded polytetrafluoroethylene (ePTFE) tubes in the backs of the necks. All rats killed after five days. Measurements of weight loss and of hydroxyproline per centimetre in the ePTFE tubes and standardized biopsy specimens of the intestine. Presence of anastomotic dehiscence at necropsy. Preoperative and postoperative dehydration caused a 24% weight loss in the experimental group on day 5 compared with 8% in the control group. Dehydrated animals accumulated less collagen in the ePTFE tubes than control animals (p less than 0.05). There were three anastomotic breakdowns in the dehydrated group compared with one in the control group. There was a loose but significant correlation between collagen accumulation in the anastomoses and the weight of the animal (r = 0.5, p less than 0.05). Preoperative and postoperative dehydration has a deleterious effect on subcutaneous, and to a lesser extent on anastomotic healing in rats.


Efficacy of recombinant erythropoietin for stimulating erythropoiesis after blood loss and surgery. An experimental study in rats.

Perioperative administration of recombinant human erythropoietin (rEpo) may reduce the need for allogeneic blood transfusions by diminishing the time lag between blood loss and erythropoiesis and by generating more adequate Epo levels. The efficacy of pre- and postoperative rEpo was studied in rats subjected to blood loss (20% of the blood volume) and surgery (ileal resection). After 200 U rEpo/kg daily for 5 days postoperatively, hemoglobin had increased by 15.7 g/l in these rats but by 36.9 g/l in rEpo-treated controls without surgery (p less than 0.05), indicating an inhibitory effect of surgery on erythropoiesis. A course of 200 U rEpo/kg/day for 5 days, starting 4 or 2 days before operation and blood loss, resulted in significantly higher postoperative hemoglobin levels than in untreated controls. Such difference did not occur if rEpo treatment was begun on the day of operation. Prolonged (10-day) postoperative rEpo treatment was of minor benefit, inducing significant increase in hemoglobin and hematocrit only from day 8 onwards. The study indicates that rEpo is a promising agent to obviate need for perioperative blood transfusions, provided that the treatment is begun before operation.


Septic shock in rats treated with terbutaline alone and in combination with chemotherapeutics, dexamethasone, and infusion of 3% albumin.

The effects of a beta 2-receptor agonist, terbutaline, on haematocrit and survival were studied in rats in which septic shock had been induced by intraperitoneal injection of a mean (SD) dose of 6.0 (4.5) x 10(8) live E. coli. Untreated septic animals developed haemoconcentration, the mean (SD) haematocrit increasing from 47.5 (1.4) to 53.1 (2.2). Mean (SD) survival time was 8.9 (0.6) hours, and no animal survived for 24 hours. Terbutaline given as the only treatment in doses of 0.1, 0.5, and 2.5 mg/kg before injection of E. coli significantly reduced the haemoconcentration, with haematocrit of 51.9, 46.6 and 47.9, respectively, at 4 hours. Survival was not significantly prolonged. When terbutaline was started 5.5 hours after injection of E. coli and given in addition to a chemotherapeutic drug (trimethoprim + sulphamethoxazole) and dexamethasone, haematocrit were reduced, 24 hour survival improved from 44% to 68%, and 7 day survival improved from 20% to 48%. We conclude that terbutaline given alone counteracts the loss of plasma volume during septicaemia and, when combined with a chemotherapeutic and dexamethasone, significantly improves long term survival.


On the value of giving a combination of drugs for the treatment of endotoxaemia in pigs.

To see if treatment with a combination of drugs each directed at a different mediator was successful in preventing activation of those mediators in experimental endotoxic shock. Controlled study. 40 juvenile pigs. 33 animals received 0.01 mg/kg endotoxin infusion, the rest being given the same volume of saline; 10 of the 33 received no treatment. Of the remaining 23, 5 were given combination treatment with methylprednisolone, naloxone, ketanserin, promethazine, C1 esterase inhibitor, antithrombin III and aprotinin; 7 were given methylprednisolone only; 6 were given the three protease inhibitors (C1 esterase inhibitor, antithrombin III and aprotinin); and 5 were given naloxone, ketanserin and promethazine. Assessment of haemodynamic, proteolytic, and cellular effects of endotoxaemia. Only the combination treatment totally blocked all the effects of the infusion of endotoxin. As endotoxin affects several mediators, combination treatment is necessary to block all its deleterious effects in pigs.


Cloning and analysis of the CD18 promoter.

CD18, the common beta chain of the leukocyte integrin adhesion proteins, is expressed exclusively by myeloid cells and lymphocytes. During myeloid differentiation, the increase in CD18 cell surface expression is paralleled by increased CD18 messenger RNA levels. Nuclear run-on studies show that CD18 expression is transcriptionally regulated during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL-60 monocytic differentiation. The CD18 transcriptional start site was defined by primer extension and RNAse protection. The gene encoding CD18 was cloned and a fragment that overlaps the transcriptional start site was isolated. DNA sequence analysis of this promoter fragment identified potential AP-1 elements that may mediate the TPA transcriptional response. The CD18 promoter also contains a putative binding site for PU.1, a leukocyte-specific transcription factor. DNA elements resembling those found in other myeloid and integrin promoters were identified. The CD18 promoter fragment was linked to the luciferase reporter gene, electroporated into the U937 monocytic cell line, and its expression increased after exposure to TPA. Thus, CD18 may serve as a model for identifying the cis elements and trans-acting factors that regulate gene expression during myeloid differentiation.


Prognostic value of proliferating cell nuclear antigen expression in chronic lymphoid leukemia.

Chronic lymphocytic leukemia (CLL) is a usually indolent disease that can assume an aggressive clinical course in some patients. To develop assays that would be predictive of how a particular patient's disease would evolve, we studied the expression of proliferating cell nuclear antigen (PCNA) by Western blotting in 40 patients with CLL. The concentration of PCNA, a cofactor for delta DNA-dependent DNA polymerase, is indicative of the proliferative state of the cell. Significantly lower PCNA levels were observed in earlier stage CLL when compared with more advanced disease. The leukemic cell proliferative rate, assessed by lymphocyte doubling time and flow cytometry, also correlated significantly with the level of PCNA expression. These results suggest that a high level of PCNA in the cells of CLL patients at presentation identifies a subgroup of patients whose CLL cells have a higher proliferative activity and who may, therefore, have a potentially shorter survival.


Human erythrocyte protein 4.2: isoform expression, differential splicing, and chromosomal assignment.

Human protein 4.2 (P4.2) is a major membrane skeletal protein in erythrocytes. Individuals with P4.2 deficiency exhibit spherocytosis and experience various degrees of hemolytic anemia, suggesting a role for this protein in maintaining stability and integrity of the membrane. Molecular cloning of P4.2 cDNAs showed that P4.2 is a transglutaminaselike molecule in erythrocytes but lacks the essential cysteine for cross-linking activity. Two cDNA isoforms have been identified from a human reticulocyte cDNA library, with the long isoform containing a 90-base pair (bp) in-frame insertion encoding an extra 30 amino acids near the N-terminus. Characterization of the P4.2 gene suggests differential splicing as the mechanism for generating these two cDNA isoforms. The donor site for the short isoform (P4.2S) agrees better with the consensus than the donor site for the long isoform (P4.2L) does. Expression of P4.2L was detected by a long-isoform-specific antibody raised against a peptide within the 30-amino acid insert. Western blot analyses showed P4.2L to be a minor membrane skeletal protein in human erythrocytes with an apparent molecular weight (mol wt) of approximately 3 Kd larger than the major protein 4.2, P4.2S. By in situ hybridization of a full-length 2.4-kilobase (kb) cDNA to human metaphase chromosomes, the gene for P4.2 was mapped to bands q15-q21 of chromosome 15, and it is not linked to the gene for coagulation factor XIIIa (plasma transglutaminase, TGase).


Double-intensive therapy in high-risk multiple myeloma.

A high remission rate is achieved with high-dose melphalan (HDM) in multiple myeloma (MM), and autologous transplantation of hematopoietic stem cells allows a prompt hematologic recovery after high-dose therapy. We treated 97 patients with high-risk MM (group 1:44 advanced MM including 14 primary resistances and 30 relapses; group 2: 53 newly diagnosed MM) with a first course of HDM. For responding patients a second course of high-dose therapy with hematopoietic stem cell support was proposed. After the first HDM, the overall response and complete remission rates were 71% and 25% with no significant difference between the two groups. The median durations of neutropenia and thrombocytopenia were significantly longer in group 1 (29.5 days and 32 days, respectively) than in group 2 (23 days and 17 days, respectively). This severe myelosuppression led to eight toxic deaths and the fact that only 38 of the 69 responders could proceed to the second course (three allogenic and 35 autologous transplantations). Among the 35 patients undergoing autologous transplantation (10 in group 1, 25 in group 2), 31 received their marrow unpurged collected after the first HDM, and four received peripheral blood stem cells. The median durations of neutropenia and thrombocytopenia after autologous transplantation were 24 days and 49 days, respectively. Two toxic deaths and nine prolonged thrombocytopenias were observed. The median survival for the 97 patients was 24 months (17 months in group 1, 37 months in group 2) and the median duration of response was 20 months. The only parameters that have a significant impact on the survival are the age (+/- 50 years) and the response to HDM. The median survival of the 35 patients undergoing autologous transplantation is 41 months, but the median duration of remission is 28 months with no plateau of the remission duration curve. Patients responding to HDM may have prolonged survival, but even a second course of high-dose therapy probably cannot eradicate the malignant clone.


Characterization of thymic cell subpopulations involved in IL-1- or GM-CSF-induced IL-6 production.

IL-6 has been demonstrated by in vitro studies to be a cytokine involved in thymocyte activation We show herein that thymocytes cultured at high concentrations in the absence of comitogen respond to IL-1 and, to a lesser degree, to GM-CSF, by producing IL-6. This phenomenon disappears rapidly with decreasing cell densities, suggesting the involvement of a minor cellular component of the thymus which may be solely responsible for or cooperate in IL-6 production. We have analysed several thymic subpopulations for IL-6 production and show that accessory cells, and eventually their precursors, are the major if not exclusive, producers of this cytokine. Mature steroid-resistant thymocytes do not secrete IL-6. Production of IL-6 by total CD4-CD8- thymic cells is largely reduced by the depletion of mature accessory cells which express I-A and Mac-1 antigens. As shown previously, accessory cell precursors within the CD4-CD8- compartment are induced to differentiate into M phi and DC in response to IL-1 and GM-CSF. We provide evidence that this maturation is associated with IL-6 production. Thymic DC and phagocytic cells of the thymic reticulum (P-TR) in vitro produce high levels of IL-6 which are enhanced by GM-CSF or IL-1. These factors have a synergistic effect on IL-6 production by total thymocytes, and on CD4-CD8- cells that are not depleted for mature I-A+ Mac-1+ accessory cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Mobilization of peripheral blood progenitor cells by chemotherapy and granulocyte-macrophage colony-stimulating factor for hematologic support after high-dose intensification for breast cancer.

High-dose therapy with autologous marrow support results in durable complete remissions in selected patients with relapsed lymphoma and leukemia who cannot be cured with conventional dose therapy. However, substantial morbidity and mortality result from the 3- to 6-week period of marrow aplasia until the reinfused marrow recovers adequate hematopoietic function. Hematopoietic growth factors, particularly used after chemotherapy, can increase the number of peripheral blood progenitor cells (PBPCs) present in systemic circulation. The reinfusion of PBPCs with marrow has recently been reported to reduce the time to recovery of adequate marrow function. This study was designed to determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF)-mobilized PBPCs alone (without marrow) would result in rapid and reliable hematopoietic reconstitution. Sixteen patients with metastatic breast cancer were treated with four cycles of doxorubicin, 5-fluorouracil, and methotrexate (AFM induction). Patients responding after the first two cycles were administered GM-CSF after the third and fourth cycles to recruit PBPCs for collection by two leukapheresis per cycle. These PBPCs were reinfused as the sole source of hematopoietic support after high doses of cyclophosphamide, thiotepa, and carboplatin. No marrow or hematopoietic cytokines were used after progenitor cell reinfusion. Granulocytes greater than or equal to 500/microL was observed on a median of day 14 (range, 8 to 57). Transfusion independence of platelets greater than or equal to 20,000/microL occurred on a median day of 12 (range, 8 to 134). However, three patients required the use of a reserve marrow for slow platelet engraftment. In retrospect, these patients were characterized by poor baseline bone marrow cellularity and poor platelet recovery after AFM induction therapy. When compared with 29 historical control patients who had received the same high-dose intensification chemotherapy using autologous marrow support, time to engraftment, antibiotic days, transfusion requirements, and lengths of hospital stay were all significantly improved for the patients receiving PBPCs. Thus, autologous PBPCs can be efficiently collected during mobilization by chemotherapy and GM-CSF and are an attractive alternative to marrow for hematopoietic support after high-dose therapy. The enhanced speed of recovery may reduce the morbidity, mortality, and cost of high-dose treatment. Furthermore, PBPC support may enhance the effectiveness of high-dose therapy by facilitating multiple courses of therapy.


Ex vivo incubation with growth factors enhances the engraftment of fetal hematopoietic cells transplanted in sheep fetuses.

Hematopoietic stem cells (HSC) transplanted in utero are in competition with endogenous HSC; thus, ultimately the graft constitutes a relatively small fraction of total HSC pool. To enhance the engraftment of donor cells in sheep fetuses, we preincubated these cells, ex vivo, for 16 hours at 37 degrees C with the conditioned medium from phytohemagglutinin-stimulated lymphocytes (PHA-LCM) before in utero transplantation. PHA-LCM is a rich source of hematopoietic growth factors in sheep. Subsequent engraftment was significantly higher in cells preincubated with PHA-LCM compared with fresh cells or those incubated with control medium only. This was reflected in all markers of the donor cells (hemoglobin type, karyotype, and progenitor cell assays). Brief ex vivo incubation with PHA-LCM also increased viability of all marrow cells as well as total numbers of progenitors. Similar enhancement of engraftment was also noted in monkeys after a brief preincubation of donor cells with interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We conclude that brief (16 hours) ex vivo incubation of donor cells with a source of such growth factors as IL-3 and GM-CSF enhances the subsequent engraftment of transplanted cells.


Effect of azelastine on the seasonal increase in non-specific bronchial responsiveness to methacholine in pollen allergic patients. A randomized, double-blind placebo-controlled, crossover study.

Azelastine, a phthalazinone derivative, is a new potent, long acting, orally active anti-allergic compound with particularly strong H1-histamine receptor antagonistic effects which has been proven to possess in vitro and in vivo a number of anti-inflammatory properties. The aim of the present study was to investigate whether azelastine would be able to prevent and/or reverse the seasonal increase in non-specific bronchial responsiveness to methacholine in pollen allergic patients. Twelve atopic patients (5 males, mean age 31 years), skin positive exclusively to grass and/or Parietaria pollen extract, with rhinitis and mild asthma occurring in the spring for at least two years previously, were studied. After a 2 week run-in period, oral azelastine, 4 mg twice daily, or placebo, was given for 2 weeks from the start of the pollen season, according to a randomized, double-blind design. After 2 weeks, the treatments were crossed over. During both the run-in and study periods, patients recorded rhinitis and asthma symptoms, additional antihistamine and bronchodilator drugs taken and peak expiratory flow measurements. A methacholine inhalation test was carried out on four occasions in each patient: before the run-in period, before the start of the treatment, and at the end of the two 2 week treatment periods. Azelastine significantly reduced rhinitis symptoms and the need for antihistamine drugs, whereas asthmatic symptoms, use of bronchodilator drugs, peak flow recordings and bronchial responsiveness to methacholine were unaffected by the treatment. Compliance level and adverse side-effects were not significantly different between active treatment and placebo. In the final subjective evaluation of the two treatments, eight out of 12 patients preferred azelastine.(ABSTRACT TRUNCATED AT 250 WORDS)


Pharmacology of antiarthritic drugs.

The clinical use of corticosteroids and second-line antirheumatic drugs provides relief in many patients but is associated with short-term and long-term toxicity. The beneficial effects are evident but are not well understood, particularly for the second-line agents. Rheumatoid arthritis is associated with abnormalities in macrophage, lymphocyte, and fibroblast functions. Corticosteroids and second-line agents appear to alter many of these responses (Table 2). Effects on macrophage and other antigen processing and phagocytic cells are common, but T- and B-lymphocytes also may be affected. Some of these agents have direct anti-inflammatory properties by inhibiting prostaglandin or leukotriene synthesis. A few are able to inhibit fibroblast proliferation and secretion of inflammatory mediators. Many other activities are possible. Understanding pharmacokinetics also should assist in determining dosing, possible consequences of other disorders, and predicting duration of acute effects. A better understanding of the disease process in rheumatoid arthritis and other disorders treated with these agents will lead to the better therapeutic approaches and, it is hoped, the discovery of more specific and less toxic agents.


Modification of DENA-induced hepatocarcinogenesis by CCl4 cirrhosis. Comparison of the marker enzyme patterns.

The modifying action of chronic liver injury on the process of hepatocarcinogenesis was investigated. To induce cirrhosis or fibrosis F344 rats received CCl4 alone or in combination with phenobarbital, either before (model 1) or after (model 2) the application of initiator, diethylnitrosamine (DENA). In these models, morphology, tumor incidence as well as polysubstrate monooxygenase system, gamma-glutamyltransferase (GGT) and glucose-6-phosphatase (G-6-Pase) were studied. The data presented show that in model 1 the tumor incidence was much lower than in rats treated with DENA alone. This reduction appeared to be associated with the decrease in cytochrome P450 content occurring in model 1 after DENA administration. Promotion of the hepatocarcinogenic process was observed when CCl4 injury followed the application of DENA (model 2). Comparison of marker enzymes in cirrhotic livers and in tumors either with or without cirrhosis indicated that changes in cytochrome P450 and G-6-Pase were rather the results of parenchymal damage, while GGT was elevated only in tumorous livers. In tumorous livers none of the xenobiotic metabolizing activities decreased as much as the cytochrome P450 content of the same samples. Thus conceivably the cytochrome P450 operates more rapidly in tumors than in normal livers.


Neuroendocrine and side effect profile of pramipexole, a new dopamine receptor agonist, in humans.

The effects and tolerability of pramipexole, a new dopamine D2-receptor agonist, on prolactin, human growth hormone, thyrotropin, cortisol, and corticotropin levels were investigated in a randomized, double-blind, crossover study in 12 healthy volunteers. Single oral doses of 0.1, 0.2, and 0.3 mg pramipexole and placebo were studied over a period of 24 hours. Pramipexole decreased serum prolactin levels in a dose-dependent manner, with a maximum effect after 2 to 4 hours. Serum levels of human growth hormone were dose-dependently increased; however, this effect was only significant 2 hours after drug administration. Furthermore, a slight increase in serum cortisol levels and a slight decrease in serum thyrotropin levels was observed. Our findings show for the first time pharmacodynamic effects of pramipexole after single oral doses in healthy volunteers. The compound was well tolerated and showed an endocrine profile similar to other dopamine D2-agonists.


The hemodynamic effects of adrenergic blocking agents.

The various antihypertensive agents reduce blood pressure by different mechanisms. Alpha-1 receptor blockers reduce vascular resistance and maintain cardiac output. Chronic treatment with beta blockers without intrinsic sympathomimetic activity produces a fall in blood pressure which is associated with a fall in cardiac index and heart rate. Beta blockers with strong intrinsic sympathomimetic activity showed reduced heart rate during exercise. Labetalol reduces cardiac output and peripheral vascular resistance with little or no reduction in peripheral blood flow. Alpha-1 blockers are suitable for patients with active life-styles, with peripheral vascular disorders, or with high blood-cholesterol levels. Beta blockers are useful in patients who have tachycardia, palpitation problems, or angina pectoris, or who have survived a heart attack. They should not be used in patients with bronchial asthma, reduced peripheral blood flow, or heart failure. Labetalol reduces blood pressure in a somewhat larger fraction of patients than the pure alpha- or beta-blocking agents. It is hoped that its long-term results will include regression of cardiovascular damage, improved quality of life, and increased life expectancy.


[The mechanism of action of benzodiazepine and its consequences for therapy].

Kinetics and mechanism of action of benzodiazepines are at the base of their proper use in clinical management of anxiety, tension, panics, and insomnia. These important agents have specific receptors within the complex GABA-ergic system: according to the most recent studies, GABAA receptors are more important than GABAB receptors. The author illustrates the management of anxiety states, as well as the treatment of anxiety in children, in old people and of panic attacks. Since the use of benzodiazepines has become excessive, the risk of dependence is discussed as well as possible alternatives (buspirone, antidepressants). Finally, the usefulness of anxiolytic drug treatment is stressed which is one of the pivots of psychopharmacology and the study of the GABA-ergic system.


The structure, role, and regulation of type 1 protein phosphatases.

Type 1 protein phosphatases (PP-1) comprise a group of widely distributed enzymes that specifically dephosphorylate serine and threonine residues of certain phosphoproteins. They all contain an isoform of the same catalytic subunit, which has an extremely conserved primary structure. One of the properties of PP-1 that allows one to distinguish them from other serine/threonine protein phosphatases is their sensitivity to inhibition by two proteins, termed inhibitor 1 and inhibitor 2, or modulator. The latter protein can also form a 1:1 complex with the catalytic subunit that slowly inactivates upon incubation. This complex is reactivated in vitro by incubation with MgATP and protein kinase FA/GSK-3. In the cell the type 1 catalytic subunit is associated with noncatalytic subunits that determine the activity, the substrate specificity, and the subcellular location of the phosphatase. PP-1 plays an essential role in glycogen metabolism, calcium transport, muscle contraction, intracellular transport, protein synthesis, and cell division. The activity of PP-1 is regulated by hormones like insulin, glucagon, alpha- and beta-adrenergic agonists, glucocorticoids, and thyroid hormones.


Retinal microvessels express less gamma-glutamyl transpeptidase than brain microvessels.

In this investigation we localized and compared the level of gamma-glutamyl transpeptidase (GGTP) activity in retinal and brain preparations using histochemical, enzymatic and in situ hybridization assays. We compared GGTP distribution to another microvessel specific enzyme, alkaline phosphatase (AP). In the rat brain, GGTP activity was observed in microvessels and choroid plexus by a histochemical method. Similar studies in the rat retina revealed activity in the pigment epithelium but only a very weak reaction in microvessels. Histochemical staining for alkaline phosphatase was observed in both retinal and brain microvessels choroid plexus and pigment epithelium. Biochemical analysis verified that GGTP activity was significantly lower in retinal than brain microvessels, while alkaline phosphatase activity was similar in both types of microvessels. GGTP specific activity of bovine brain and retinal microvessels was 185 +/- 39 mUnits and 8.5 +/- 1.5 mUnits (p less than 0.001), respectively. By contrast, alkaline phosphatase specific activity in brain and retinal microvessels was 732 +/- 139 and 471 +/- 114 (p greater than 0.1), respectively. Choroid plexus and retinal pigment epithelium exhibited similar levels of GGTP and alkaline phosphatase. Differences in GGTP expression between retinal and brain microvessels were also observed on the mRNA level. In situ hybridization studies revealed that brain microvessels expressed four times more GGTP specific mRNA than retinal microvessels. We conclude that retinal microvessels do not express high levels of GGTP which may make them more vulnerable than brain microvessels to injuries mediated by leukotrienes and oxidative stress.


Laser lithotripsy of pancreatic and biliary stones via 3.4 mm and 3.7 mm miniscopes: first clinical results.

The pulsed dye laser is safe and effective in lithotripsy of biliary and pancreatic ductal stones, however delivery of the laser energy to the site of the calculi is technically difficult. A 3.4 mm miniscope inserted through a standard duodenoscope was used for transpapillary laser application under direct vision in one patient with an impacted pancreatic stone and in three patients with bile duct stones not amenable to treatment by routine endoscopy. Lithotripsy and ductal clearance was achieved in 3 of the 4 patients within a single treatment session. Percutaneous transhepatic laser lithotripsy via a 3.7 mm miniscope succeeded in the patient in whom the peroral approach had failed and in two further patients with bile duct stones not accessible by a retrograde approach. There were no major complications. The miniscopes provide an appropriate direct visual control for laser lithotripsy of pancreatobiliary calculi. This approach using miniscopes seems to be an effective, minimally invasive and time-saving alternative to conventional endoscopic laser lithotripsy.


An anti-peptide antibody that recognizes the dopamine D2 receptor from bovine striatum.

The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.


Synergistic effect of IL-3 and IL-6 on highly enriched murine hemopoietic progenitors.

It has been reported that interleukin 6 (IL-6) acts on hemopoietic stem cells in synergism with interleukin 3 (IL-3), but it has not yet been clarified whether IL-6 acts directly on the stem cells or not. To investigate the mechanism of the synergism between IL-3 and IL-6, we sorted hemopoietic stem cells from untreated murine bone marrow cells using a two-laser fluorescence-activated cell sorter (FACS). Cells negative for the lymphohemopoietic lineage (lineage-negative, Lin-), with a high affinity to wheat germ agglutinin (WGA+), and showing a low expression of Thy-1 antigen (Thy-1low) were sorted and analyzed by in vitro and in vivo colony formation. This fraction was 0.4% of the total mononuclear bone marrow cells. Approximately 25% of these Lin-WGA+Thy-1low cells showed in vitro colony formation, whereas approximately 1% of them formed day-8 and day-12 spleen colonies. Thus, it appears that the Lin-WGA+Thy-1low cells were a highly enriched stem cell population. By FACS clone sorting, single cells were isolated from the enriched stem cell fraction and cultured in semisolid or liquid culture systems. Addition of IL-6 to methylcellulose medium containing IL-3 did not significantly increase the number of colonies. It is thus suggested that the target cells of IL-3 and IL-6 are the same as those of IL-3. The secondary colony-forming ability of primary colonies that developed in the presence of IL-6 and IL-3 was higher than that of colonies formed in the presence of IL-3 alone. In correspondence with this finding, the numbers of myeloid colonies and spleen colony-forming units (CFU-S) were increased by the incubation of these sorted cells for 7 days with IL-6 and IL-3 when compared with the effect of IL-3 alone. Therefore, it is concluded that IL-6 acts directly on hemopoietic stem cells to enhance their proliferation.


In vitro effect of P-glycoprotein (P-gp) modulators on drug sensitivity of leukemic progenitors (CFU-L) in acute myelogenous leukemia (AML).

Cyclosporine A (CyA), a potent reversant of multidrug resistance (mdr), was studied for its effects on the sensitivity of leukemic progenitors (leukemia colony-forming units, CFU-L) to daunorubicin (DNR) and mitoxantrone. CyA was first compared to verapamil and cefoperazone for reversion of mdr in the mdr1+ cell line K562/DOX. A dramatic increase of sensitivity to 10(-6) M DNR was noted with CyA (0.5 and 1 microgram/ml) and verapamil (1 and 5 micrograms/ml), but not for cefoperazone (0.3 and 0.6 mg/ml). The sensitivity of K562/DOX to 10(-6) M mitoxantrone was also slightly enhanced by CyA. The change in CFU-L drug sensitivity in the presence of CyA was then tested in 12 relapsing/resisting patients and in 3 untreated patients with acute myelogenous leukemia (AML). No change in CFU-L sensitivity to DNR was noted, despite the presence of a subset of P-glycoprotein positive (P-gp+) cells in three out of the ten evaluable cases. Among the six evaluable cases tested with mitoxantrone and CyA, an increase of 50% in CFU-L sensitivity to mitoxantrone was noted in one (of three) P-gp+ patient. These data suggest that the CFU-L in AML rarely expressed the P-gp and that other mechanisms of drug resistance could be involved in AML.


Glutamate-induced action potentials are preceded by regenerative prepotentials in rat hippocampal pyramidal cells in vitro.

(1) The responses of CA1 pyramidal cells to short glutamate pulses (10-50 ms) delivered at sensitive spots in the apical dendrites have been analysed by intracellular recording. (2) The glutamate pulses elicited stable depolarizing responses in a dose- and frequency-dependent manner. (3) When a single action potential with a firing probability around 0.5 was elicited, a subtraction procedure showed that a slow depolarizing ramp preceded each spike. We call this ramp the glutamate-induced prepotential (GluPP). (4) In contrast to the upward convex subthreshold depolarization the GluPP was upward concave. (5) The GluPP amplitude and time course increased with depolarization of the membrane, a phenomenon which appears to be connected to the elevation of action potential threshold. (6) The GluPP was regenerative since once started, it ended in an action potential. (7) A specific N-methyl-D-aspartate receptor antagonist, DL-2-amino-5-phosphonovaleric acid (50 microM) reduced the glutamate-induced depolarization, but did not affect the form or amplitude of GluPP, once the latter was induced. (8) It is concluded that short glutamate pulses elicited action potentials through a prepotential mechanism, similar to the slow prepotentials induced by long depolarizing current pulses across the soma membrane. (9) A possible physiological role for the GluPP is discussed.


In vitro release of glutamate and aspartate from zebra finch song control nuclei.

The release of glutamate and aspartate from a song control nucleus, the robust nucleus of the archistriatum (RA) of the zebra finch was examined in slice preparations. The RA received inputs from two other song control nuclei, the high vocal center (HVc) and the lateral magnocellular nucleus of the anterior neostriatum (1MAN). The fibers that innervated the RA from either the HVc or the 1MAN caused calcium-dependent release of glutamate and aspartate after electrical stimulation. It is likely that synapses of the RA from the HVc and the 1MAN utilize glutamate or aspartate as a neurotransmitter.


Reaction of astrocytes in the gerbil hippocampus following transient ischemia: immunohistochemical observations with antibodies against glial fibrillary acidic protein, glutamine synthetase, and S-100 protein.

An immunohistochemical method was used to study the distribution and changes with time of the astrocytic reaction in the gerbil hippocampus following transient ischemia. Three markers were investigated with specific antibodies to glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and S-100 protein. On Day 2 after ischemia, and more prominently on Day 3, reactive astrocytes were intensely stained for GFAP in the hippocampal formation, especially in the CA1 region and dentate gyrus. This response by astrocytes preceded CA1 pyramidal cell degeneration, which became apparent on Day 5. On Day 5, immunoreactive cells were not stained as intensely as on Day 3, but cells in the CA1 region and dentate gyrus were still more intensely stained than those in normal animals. GS and S-100 showed similar changes in distribution after ischemia, although the change in GS was less prominent: the hilus of the dentate gyrus was most intensely stained. Both immunoreactivities seemed to increase rather transiently on Day 2 or 3 and to decrease to the initial level on Day 5. The fact that reactive astrocytes appeared in CA1 before the onset of visible neural degeneration indicates that signals from indisposed neurons may be transmitted to astrocytes for their quick functioning. It is also suggested that degenerative changes occur in the dentate gyrus and may be involved in the delayed neural death of CA1 pyramidal cells. These observations indicate that astrocytes play a role in the neural degeneration induced by ischemia and that several types of astrocytes seem to react differently.


Mesostriatal dopaminergic axons transiently express high levels of NILE during development.

The distribution of nerve growth factor-inducible large external (NILE) glycoprotein in the developing mesostriatal dopaminergic system was studied by immunolabeling prenatal, postnatal, and adult rat brains for both NILE and tyrosine hydroxylase (TH). NILE-immunoreactive (NILE-IR) material outlined the TH-immunoreactive (TH-IR) neurons in the ventral mesencephalon at ages E14, E15, and E16 but not at later ages. Fascicles of TH-IR axons in the developing mesostriatal dopaminergic tract were strongly immunoreactive for NILE from E15 through E20. In the postnatal brain, NILE-IR material no longer clearly outlined the fascicles of dopaminergic axons as they ran from the mesencephalon to the striatum. In the striatum of postnatal and adult rats, NILE-IR material was diffusely distributed through the neuropil. Our observations suggest that NILE may play a role in guidance of growing dopaminergic axons in the developing mesostriatal tract and aggregation of the dopaminergic axons into fascicles.


A longitudinal study of age-related loss of noradrenergic nerves and lymphoid cells in the rat spleen.

A longitudinal study of sympathetic noradrenergic (NA) innervation of the spleen was carried out in 3-, 8-, 12-, 17-, 21-, and 27-month-old Fischer 344 (F344) rats using (i) fluorescence histochemistry for localization of norepinephrine (NE); (ii) immunocytochemistry (ICC) for localization of tyrosine hydroxylase (TH)-positive nerve fibers alone, and in combination with specific markers for T and B lymphocytes (OX19 and anti-mu respectively), and macrophages (ED3); and (iii) high-performance liquid chromatography with electrochemical detection for quantitation of NE. Fluorescence histochemistry revealed extensive loss of NA nerve fibers in all compartments of the spleen in 21- and 27-month-old rats. With single-label ICC, a decline in TH+ nerve fibers in all compartments of the spleen was observed by 17 months of age and became more severe with advancing age; these findings suggest that both the rate-limiting enzyme and the transmitter itself (NE) are depleted from sympathetic nerves in aged rat spleen. Double-label ICC demonstrated the loss of TH+ nerve fibers in spleen from 17-, 21-, and 27-month-old rats, and a parallel loss of OX19+ T lymphocytes and ED3+ macrophages in these cellular compartments. Neurochemical measurement of NE demonstrated a decline in NE per wet weight at 27 months of age. The age-related decline in NA innervation of spleen and in the density of specific populations of cells of the immune system (T lymphocytes and antigen-presenting ED3+ macrophages), that follow remarkably similar time courses, supports functional evidence for dynamic interactions between the immune system and NA sympathetic nerves in the spleen, and further suggests a causal relationship between these age-related phenomena, i.e., that age-related immunosenescence promotes sympathetic denervation of the spleen which further compromises immune function. This hypothesis, however, requires further testing.


Evidence for different neurochemical contributions to long-term potentiation and to kindling and kindling-induced potentiation: role of NMDA and urethane-sensitive mechanisms.

Long-term potentiation (LTP) and kindling share a number of features, and it has been suggested that LTP might constitute the cellular mechanism of kindling. This question was approached by assessing the effect of urethane anesthesia (0.75 or 1.5 g/kg) or blockade of NMDA receptors by local infusion of DL-2-amino-5-phosphonovaleric acid (APV; 7.5 micrograms) on LTP, partial kindling, and kindling-induced potentiation (KIP) in the perforant path-dentate gyrus circuit of the intact hooded rat. Urethane anesthesia attenuated but did not block LTP and completely blocked partial kindling and KIP. APV completely blocked LTP but did not block partial kindling or KIP in the unanesthetized rat. These results suggest that different neurochemical mechanisms can support LTP on the one hand, and kindling and KIP on the other. They are consistent with a contribution by NMDA-mediated LTP to kindling and KIP, but they indicate that this contribution is not crucial for kindling and KIP in this circuit.


Distribution of 4a-hydroxytetrahydropterin dehydratase in rat tissues. Comparison with the aromatic amino acid hydroxylases.

A 4a-carbinolamine intermediate is generated stoichiometrically during the tetrahydrobiopterin-dependent phenylalanine hydroxylation reaction catalyzed by phenylalanine hydroxylase. The dehydration of the carbinolamine is catalyzed by the enzyme, 4a-hydroxytetrahydropterin dehydratase. We have now examined the distribution of the dehydratase activity in various rat tissues by activity measurements and by immunoblot analysis to explore the possibility that the dehydratase may also play a role in tyrosine and tryptophan hydroxylation. The only two tissues that express relatively high dehydratase activity are liver and kidney, which are also the only two tissues that express phenylalanine hydroxylase activity. The dehydratase activity was generally very low in those tissues which contain high levels of tyrosine and tryptophan hydroxylase activity, except for the pineal gland. These results suggest that the dehydratase may not play an important role in the regulation of the synthesis of those neurotransmitters which are derived from the hydroxylated aromatic amino acids.


Variability of the insulin gene in American blacks with NIDDM. Analysis by single-strand conformational polymorphisms.

Previous studies of the insulin gene--utilized restriction-fragment--length polymorphisms as markers for potential mutations at this locus. This indirect type of analysis could not define the number of variants that might exist within the structural portions and regulatory regions of the gene in non-insulin-dependent diabetes mellitus (NIDDM) patients. New technology has allowed us to examine insulin genes at the single nucleotide level from 100 American black NIDDM patients. Genomic DNA from patients was amplified by the polymerase chain reaction with primers flanking four regions of the gene: 1) the proximal promoter from positions -182 to 42 (including most of exon 1); 2) exon 1 from 14 to 259, which included the rest of exon 1 and all of the 1st intron; 3) exon 2 from 216 to 452; and 4) exon 3 from 1188 to 1433. One of the primers in each reaction was 32P-end labeled and the resulting products denatured into single strands and electrophoresed on nondenaturing sequencing gels such that mobility was a function of composition and size (single-strand conformational polymorphism or SSCP). Under these conditions, single-base changes in fragments up to 245 nucleotides were detected. Analysis of the proximal promoter region revealed several SSCP patterns in individuals. Direct genomic sequencing of DNA representative of these patterns showed the presence of a common C to G change at position -56 and a C deletion at position -90 in three patients.(ABSTRACT TRUNCATED AT 250 WORDS)


Colocalization of GLUT2 glucose transporter, sodium/glucose cotransporter, and gamma-glutamyl transpeptidase in rat kidney with double-peroxidase immunocytochemistry.

Glucose is reabsorbed from the glomerular filtrate in the proximal segment of the renal tubule in two stages. The first stage is uphill transport across the brush border membrane by Na(+)-glucose cotransport and the second stage is downhill transport across the basolateral membrane by facilitated diffusion. Genes for both a renal Na(+)-glucose cotransporter (SGLT1) and a renal facilitated glucose transporter (GLUT2) have been cloned and sequenced. To examine whether SGLT1 and GLUT2 colocalize to the same tubular epithelial cells in rat kidney, double-immunoperoxidase studies with dual chromogens and paraformaldehyde perfusion-fixed frozen sections of rat kidney were performed. Antipeptide antisera were prepared against rat GLUT2 (amino acids 510-522) and rabbit SGLT1 (amino acids 402-420). Proximal tubules were identified immunocytochemically with an antiserum raised against a synthetic peptide corresponding to the 21 amino acids at the COOH-terminal of the heavy chain of rat gamma-glutamyl transpeptidase, which is a proximal tubule-specific enzyme. The anti-GLUT2 antiserum strongly stained the basolateral membrane of 46% of cortical tubules, whereas the SGLT1 antiserum stained the brush border of 56% of the cortical tubules. The gamma-glutamyl transpeptidase antiserum also stained the brush border of 51% of the cortical tubules. GLUT2 and SGLT1 colocalized to 40% of cortical epithelium, but 16% of cortical epithelial cells were immunopositive for brush border SGLT1 and immunonegative for basolateral GLUT2. These gamma-glutamyl transpeptidase staining results suggest that at least 50% of the tubules in the cortex are proximal tubules and that SGLT1 and GLUT2 colocalize to most proximal tubules. The fact that SGLT1 antiserum immunoreacted with tubules unreactive to the GLUT2 antiserum suggests that either the SGLT1 epitope is conserved on a related brush border protein or that there is another GLUT transporter responsible for the exit of sugar from these proximal tubule cells.


Immunochemical localization of aminopeptidase M in the alimentary tract of the guinea pig and rat.

Aminopeptidase M (APM) was localized in the kidney and alimentary tract of guinea pigs and rats by indirect immunohistochemistry. APM was detected in the brush border of the epithelium of the proximal convoluted tubule of the kidney and of the small intestine, and it was localized to cells scattered throughout lymphoid tissue in the small intestine and colon. The gastric mucosa was unstained. APM was localized to numerous fibers supplying the myenteric plexus of the stomach, small intestine, and colon. The submucosal plexus was sparsely supplied by immunoreactive fibers. Occasional cell bodies were stained in the myenteric plexus. Staining was abolished by preabsorption of the primary antibody with APM. APM was characterized in membranes prepared from the muscle and mucosa of the guinea pig and rat stomach, small intestine, and colon by Western blotting. The major immunoreactive protein identified in membranes prepared from all tissues had an apparent molecular weight of 140, corresponding to the monomer of APM. In the brush border APM has a digestive function, whereas in neural tissue it may degrade and inactivate neuropeptides.


c-Ki-ras mutations in dysplastic fields and cancers in ulcerative colitis.

A sensitive restriction fragment length polymorphism assay and DNA sequencing were used to detect c-Ki-ras mutations in 56 specimens of colonic epithelium from 18 patients with chronic ulcerative colitis. Mutations were not detected in biopsy specimens that were negative or indefinite for dysplasia. In 4 of 8 patients with high-grade dysplasia, a c-Ki-ras codon 12 or 13 mutation was detected. In three colectomy specimens, a wide area of dysplastic cells (greater than 10 cm2) contained a specific ras mutation. In two of these specimens, an invasive cancer contained a c-Ki-ras mutation identical to that found in adjacent dysplastic epithelium. These studies indicate that mutations of c-Ki-ras may be an excellent molecular genetic marker to map dysplastic fields and invasive cancer in ulcerative colitis.


Genetic and blood coagulation characterization of "Swedish" families with von Willebrand's disease types I and III: new aspects of heredity.

Twenty-five patients with von Willebrand's disease (vWD) type III were analysed with regard to blood coagulation variables and possible deletions. Nine of the probands and their families were further investigated with DNA linkage analyses. Different patterns of heredity can be suggested in our families with vWD type III, on the basis of blood coagulation analyses. The findings suggest homozygosity in five families and the possibility of compound heterozygosity or a new mutation in the proband in three families. The linkage analyses confirm the results of the coagulation analyses. The segregation of the von Willebrand factor (vWF) gene can be followed in the families, and carrier diagnosis can be made in several of the probands' relatives. The possibility of large deletions in the vWF gene of the probands and their parents was investigated with probes representing the whole vWF cDNA. No deletions were found.


Isolation and mapping of polymorphic cosmid clones used for sublocalization of the multiple endocrine neoplasia type 1 (MEN1) locus.

Multiple endocrine neoplasia type 1 (MEN1) is characterized by neoplasia of the parathyroids, the pancreas, and the pituitary. Tumorigenesis involves unmasking of a recessive mutation at the MEN1 locus, which has been mapped to the centromeric part of chromosomal region 11q. In order to localize the MEN1 gene further and to make its isolation possible, a number of new markers were isolated. Two radiation-reduced somatic cell hybrids were identified that only contained markers close to and flanking the MEN1 region. DNA from these hybrids was used for the construction of a cosmid library, and clones containing human inserts were isolated. In addition, cosmid clones were isolated for locus expansion of 7 other markers that were mapped to the 11q12-13.2 region. The 33 newly isolated clones together with 25 previously published markers from this region were analyzed in a panel of radiation-reduced somatic cell hybrids. From the hybridization pattern, the region was divided into 11 parts. New restriction fragment length polymorphisms were identified in 7 of the newly isolated cosmid clones and in one plasmid. These were then used to sublocalize meiotic cross-overs more precisely in two MEN1 families, thus refining the mapping of the disease gene.


Wiskott-Aldrich syndrome carrier detection with the hypervariable marker M27 beta.

Whole-blood cells of obligate carriers of the X-linked Wiskott-Aldrich syndrome (WAS) exhibit nonrandom inactivation of the X-chromosomes. However, because of the limited polymorphism of the probes available, the X-methylation pattern can only be determined in a restricted proportion of females. We thus analysed a large set of normal females and members of WAS families, using the recently described marker M27 beta, which detects the hyperpolymorphic locus DXS255. The probe was used to detect differences in methylation between the active and inactive X-chromosome, and the findings were compared with the pattern obtained using the well-documented probes from the 5' end of the PGK and HPRT genes. All the normal females were found to use either X-chromosome randomly, and there was complete correlation between the three probes in the populations studied. Segregation analysis performed with M27 beta and other related markers in the WAS families was fully in accordance with the X-inactivation data. The use of M27 beta, for both X-inactivation and segregation analysis of WAS kindreds, provides a basis for genetic counselling in the majority of families, including those with no surviving males.


The human fumarylacetoacetase gene: characterisation of restriction fragment length polymorphisms and identification of haplotypes in tyrosinemia type 1 and pseudodeficiency.

Deficiency of human fumarylacetoacetase (FAH) activity results in hereditary tyrosinemia type I. Using the restriction enzymes BglII, KpnI and StuI and a 1.3-kb cDNA probe for the FAH gene, we have found 6 restriction fragment length polymorphisms (RFLPs). These RFLPs were utilised in 3 tyrosinemia families in which one or both parents are carriers of both a tyrosinemia and a pseudodeficiency gene for FAH. Full information was obtained in two of these families. The polymorphisms identified 6 haplotypes. The haplotype distribution was significantly different in 32 unrelated tyrosinemia patients compared with a reference population of 100 individuals. The combined polymorphism information content was 0.77.


Prenatal diagnosis of familial hypercholesterolemia caused by the "Lebanese" mutation at the low density lipoprotein receptor locus.

Here, we report the prenatal diagnosis of familial hypercholesterolemia in a Christian-Arab family that carries the "Lebanese" mutation, a single base substitution that creates a HinfI restriction site, at the low density lipoprotein (LDL) receptor locus. Polymerase chain reaction amplification and restriction analysis were performed on genomic DNA extracted from a chorionic villus sample. In conjunction with karyotype analysis, the fetus was identified as a heterozygous female. Analysis of LDL receptor restriction fragment length polymorphisms confirmed the presence of a male parent marker and revealed that the fetus inherited the mutant gene from its mother. This technique offers a simple and rapid diagnostic tool that can be carried out at an early stage of gestation. It is recommended for families and population groups with molecularly defined LDL receptor mutations.


A possible association between basic amino acids of position 13 of DRB1 chains and autoimmune hepatitis.

Fifty-one patients with autoimmune hepatitis have been studied for HLA association by conventional serology and also by modified polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genotyping. HLA-DR4 was significantly associated with autoimmune hepatitis (46 of 51 patients, 90.2%). DNA typing of the DRB1 gene for 43 DR4-positive patients by using the PCR-RFLP technique revealed that of 43 patients, 33 had DRB1*0405 (Dw15), five had DRB1*0406 (DwKT2), four had DRB1*0403 (Dw13a), two had DRB1*0401 (Dw4), two of 43 had DRB1*0407 (Dw13b) and one had DRB1*0408 (Dw14b). Thus, there was no significant difference in Dw frequencies between DR4-positive patients and DR4-positive healthy subjects. These findings suggest that the DR4-specific sequence (Val 11 and His 13 at amino acid positions 11 and 13, respectively), but not particular Dw-associated DR4 sequence, in the first domain of the DRB1 chain contributes to susceptibility to autoimmune hepatitis among Japanese. Interestingly, all five of the DR4-negative patients had the DR2 specificity (DRB1*1502 or 1601). Taken together, these results imply that the basic amino acids at position 13, which is present only on the DR2 and DR4 B1 molecules (Arg on DR2 and His on DR4), are most important for determining the predisposition to autoimmune hepatitis.


Signals delivered via the Qa-2 molecule can synergize with limiting anti-CD3-induced signals to cause T lymphocyte activation.

Qa-2 is a glycolipid anchored, MHC encoded class I molecule expressed at high levels on all murine peripheral T lymphocytes. Anti-Qa-2 antibodies have previously been found to stimulate T cells to proliferate in the presence of crosslinking antibody and PMA. We have examined the effect of anti-Qa-2 antibodies on T cells stimulated with a suboptimal concentration of immobilized anti-CD3. When anti-Qa-2 antibodies were co-immobilized with limiting anti-CD3, in the absence of PMA, a clear augmentation of T cell proliferation was seen. Interestingly, the co-stimulatory anti-Qa-2 antibodies could be directed against epitopes mapped to either the alpha 3 or the alpha 1/alpha 2 Qa-2 domains. As was the case with activation induced by soluble/crosslinked anti-Qa-2 antibodies plus PMA, CD8+ T cells were less able to be costimulated with anti-Qa-2 antibodies than CD4+ cells. Surprisingly, Ca2+ mobilization was only seen when two anti-Qa-2 antibodies reactive to separate structural domains were co-crosslinked on the surface of Indo-1 loaded T cells with a suboptimal concentration of anti-CD3. Collectively these results raise questions regarding the mechanism of Qa-2 mediated signaling and its potential role in T cell activation.