An investigation of the association of benzodiazepines and other hypnotics with the incidence of falls in the elderly.

Clinical and medication data from 2,878 admissions to a Department for Care of the Elderly were examined retrospectively to determine the association between the administration of hypnotics/benzodiazepines and the incidence of falls. Only lorazepam prescribed to females and nitrazepam prescribed to males were associated with a significantly increased incidence of falls. Women fell significantly more frequently than men, and 7.5% of falls resulted in fractures. Stroke was the most common major diagnosis in fall-cases, followed by infection, Parkinsonism and confusion.


Determination of the enantiomers of fenoldopam in human plasma by reversed-phase high-performance liquid chromatography after chiral derivatization.

Fenoldopam, a selective agonist at peripheral dopaminergic (DA-1) receptors, is administered as a racemic mixture and, consequently, an indirect stereospecific high-performance liquid chromatographic assay was developed to study the disposition of the individual enantiomers in human subjects. Fenoldopam enantiomers were extracted from alkalinized plasma into ethyl acetate prior to precolumn derivatization with the chiral reagent 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC). The resulting diastereomers were separated on a reversed-phase butylsilica column and determined using triple-electrode coulometric detection. The limits of determination and detection for the S- and R-enantiomers of fenoldopam were 0.5 and 0.25 ng/ml, respectively. A linear response was observed for (S)- and (R)-fenoldopam concentrations ranging from 0.5 to 50 ng/ml in plasma. The intra-day relative standard deviations (R.S.D.s) for the plasma assay at nominal concentrations of 0.5, 5 and 50 ng/ml were 17.4, 5.2 and 6.9%, respectively, for (S)-fenoldopam and 9.9, 6.2 and 7.4%, respectively, for (R)-fenoldopam. The inter-day R.S.D.s of the method at these concentrations were 9.3, 7.7 and 7.4%, respectively, for (S)-fenoldopam and 9.5, 1.9 and 7.3%, respectively, for (R)-fenoldopam. The mean accuracy of the method at concentrations of 0.5, 5 and 50 ng/ml in plasma was found to be 106.4, 111.8 and 108.9%, respectively, for (S)-fenoldopam and 116.2, 104.2 and 111.2%, respectively, for (R)-fenoldopam. The assay developed was sufficiently sensitive, accurate and precise to support pharmacokinetic studies in human subjects.


Extensive allelic variation in Cryptococcus neoformans.

The orotidine monophosphate pyrophosphorylase (OMPPase) gene locus of the DNA of 13 Cryptococcus neoformans var. neoformans strains, including 10 recent clinical isolates, was studied by using restriction fragment length polymorphisms and nucleotide sequence analysis. The OMPPase locus (URA5) is highly polymorphic, and at least six alleles were identified. The nucleotide sequences of some alleles differed by up to 5%. The majority of the nucleotide polymorphisms in the protein-coding region occurred at the third codon position and were silent. The low frequency of replacement nucleotide substitutions relative to silent nucleotide substitutions implied that there is strong selection against amino acid changes in OMPPase. The allelic variation suggested that there is extensive genomic diversity among C. neoformans clinical isolates from one geographic area. The various alleles are potentially useful markers in the study of the population structure, epidemiology, and pathogenesis of C. neoformans strains.


Use of a dispersed repetitive DNA element to distinguish clinical isolates of Cryptococcus neoformans.

We isolated a recombinant phage from a Cryptococcus neoformans genomic library that contains a member of a dispersed family of repetitive DNA elements. This clone, CNRE-1, hybridized to at least seven chromosomes in C. neoformans on the basis of pulsed-field gel analysis. Hybridization of CNRE-1 to restriction digests of genomic DNA confirmed that there are multiple copies of this element and that restriction fragment length polymorphisms are present in strains from different serotypes of C. neoformans. The utility of this probe as an epidemiologic marker was determined by testing cryptococcal isolates from a single hospital. Five isolates from four patients were closely related to a serotype A reference strain, whereas five other isolates from four additional patients exhibited distinct patterns. In two patients, the isolates obtained during recurrent cryptococcal infections were identical to the original isolates.


Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis.


Rapid and specific detection of the pap, afa, and sfa adhesin-encoding operons in uropathogenic Escherichia coli strains by polymerase chain reaction.

Adhesin-encoding operons (pap, sfa/foc, and afa) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. A quick and sensitive assay to identify these operons was developed by using the polymerase chain reaction (PCR). Three pairs of 25-mer primers were defined from the sequences of the DNA fragments used as probes in hybridization studies to identify each of the three operons, and the six primers were used together in a single reaction of amplification. To validate the PCR approach for detection of adhesin-encoding operons among clinical isolates, we investigated a collection of 97 E. coli isolates with the following characteristics: all isolates originated from the urine of patients with pyelonephritis, and the adhesin responsible for specific binding of the isolates to uroepithelial cells was previously characterized by phenotypic assays, as well as genotypic tests based on hybridization. There was a perfect correlation between the results obtained with the PCR approach and those previously obtained by using DNA probes. These results indicate that the PCR method, which is highly specific and easier to perform than the hybridization method, is a powerful genotypic assay for detection of adhesin-encoding operons. Thus, this assay can be recommended for clinical use to detect virulent urinary E. coli strains, as well as for epidemiological studies.


Ribosomal DNA probe for differentiation of Babesia microti and B. gibsoni isolates.

The objective of this study was to determine whether different isolates of Babesia microti could be distinguished from morphologically similar isolates of B. gibsoni by using a ribosomal DNA (rDNA) probe. A Babesia-specific rDNA probe was obtained by polymerase chain reaction amplification of sequences from B. microti DNA using universal primers directed against highly conserved portions of the eukaryotic 16S-like rRNA gene. The chemiluminescent rDNA probe hybridized to Southern blots of restriction endonuclease-digested DNA preparations of different isolates of B. gibsoni from infected dogs and B. microti from infected humans and white-footed mice. Restriction fragment length polymorphisms served to differentiate these species. Although the hybridization patterns seen with DNAs from six B. microti isolates did not vary, those of the five B. gibsoni isolates did indicate genotypic variation. We concluded that isolates of B. microti and B. gibsoni can be differentiated on the basis of restriction fragment length polymorphism detected with a chemiluminescent rDNA probe.


[Study of the immune profile of pregnant women].

During pregnancy, the fetus is an hemiallograft perfectly tolerated by the mother. With the aim of elucidating the mechanism of this tolerance, we have looked to see whether lymphocyte subsets are modified by pregnancy. Using monoclonal antibodies and flow cytometry, we have studied the changes in the immune profile of normal pregnant women and compared them with non pregnant women. In pregnant women, a significant decrease in the percentage of CD3+ cells is observed in the first trimester (61.1 +/- 14.7% versus 73.8 +/- 6.8%; p less than 0.001). The same data are obtained for CD4+ cells (38.2 +/- 10.7% versus 44.0 +/- 7.0%; p less than 0.05) and CD8+ cells (22.8 +/- 5.6% versus 28.0 +/- 8.9%; p less than 0.05). On the other hand, B lymphocytes (CD19+), monocytes (Leu M3+) and natural killer (NK) cells (Leu7+) remain stable during pregnancy. CD11a+ cells decreased during the 1st and 2nd trimesters. Lastly, activated T lymphocytes (CD3+DR+, CD8+DR+) are not modified. Using absolute numbers, a significant decrease is shown only for CD3+ cells in the 2nd and 3rd trimesters and for CD11a+ cells in the 2nd trimester of pregnancy. The decrease of T lymphocyte subsets, NK and CD11a+ cells during pregnancy partially explains the tolerance for the fetus.


The effect of antihypertensive drugs on in vivo platelet activity in essential hypertension.

To investigate whether antihypertensive drugs have a beneficial effect upon the abnormal in vivo platelet function found in patients with essential hypertension. A cross-sectional study in which plasma beta-thromboglobulin, a marker of in vivo platelet activation, was measured in patients with essential hypertension on various antihypertensive drugs. All were free from any other diseases which might affect platelet function. Plasma beta-thromboglobulin was measured in 24 patients with untreated essential hypertension, 21 normotensive control patients, 16 patients receiving angiotensin converting enzyme (ACE) inhibitors, 16 patients receiving a beta-adrenoceptor blocker, 12 patients receiving calcium antagonists and 12 patients receiving a diuretic alone. Untreated hypertensives had significantly elevated plasma beta-thromboglobulin levels compared with controls. Plasma beta-thromboglobulin levels in patients receiving beta-blockers and diuretics were not significantly different from untreated hypertensives. Treatment with calcium antagonists was associated with lower plasma beta-thromboglobulin levels, but this difference was not statistically significant. In contrast, treatment with ACE inhibitors was associated with significantly lower plasma beta-thromboglobulin levels compared with untreated hypertensives. These results suggest that antihypertensive drugs have different effects upon abnormal in vivo platelet function in patients with essential hypertension. The apparent beneficial effect of ACE inhibitors may mean that they have more impact than other drug groups in the prevention of coronary heart disease.


Incidence of Mycobacterium avium-intracellulare complex bacteremia in human immunodeficiency virus-positive patients.

The product-limit incidence of Mycobacterium avium-intracellulare complex (MAC) bacteremia in 1006 human immunodeficiency virus (HIV)-positive patients followed at one institution over a 3-year period from the day of AIDS diagnosis with monthly lysis-centrifugation blood cultures was 21% +/- 2% SE at 1 year and 43% +/- 3% at 2 years. The product-limit incidence of MAC bacteremia at 1 year after the patients' first CD4 cell count was related to both the CD4 cell count and to whether they had an AIDS diagnosis (both P less than .0001) but not to age, sex, or race. This incidence was 39% +/- 6% for CD4 cell counts of less than 10/mm3, 30% +/- 5% for 10-19/mm3, 20% +/- 4% for 20-39/mm3, 15% +/- 4% for 40-59/mm3, 8% +/- 3% for 60-99/mm3, and 3% +/- 1% for 100-199/mm3. MAC may eventually infect most if not all HIV-positive patients who do not die from another HIV-related event.


Identification of somatostatin-14 and -28 in rat pancreatic juice by a new HPLC method.

Recently we could demonstrate that in rats with pancreatic hypertrophy, somatostatin is secreted in higher concentrations into the pancreatic juice than into the portal vein blood. For measurement of juice somatostatin and to characterize the molecular forms, we established a new reverse-phase HPLC method, which we describe herein. This HPLC method, using a linear gradient system consisting of 0.2% heptafluorbutyric acid in 10 mM sodium acetate and acetonitrile, showed a stable recovery rate of about 85%. Applying the pure juice to this gradient system, we detect somatostatin-14 to be the major form of immunoreactive somatostatin (IRSS) in the pancreatic juice of the rat (5% of total IRSS). The remaining 35% were found to be somatostatin-28. The role of somatostatin in pancreatic juice is not known. It raises the hypothesis that it possibly interacts with the influences intraluminal intestinal growth factors. This study supports the assumption for the existence of an insuloacinar portal system to regulate exocrine pancreatic functions by islet hormones.


Microtiter assay for measurement of factor XIII activity in plasma.

A sensitive, reproducible and rapid quantitative microtiter assay for factor XIII activity is described. The assay is based on incorporation of a soluble amine substrate into casein attached to polystyrol microtiter plates and detection of bound substrate with a monoclonal antibody-enzyme conjugate. Defibrination of plasma samples is not required. The assay shows strong correlation with the immunologic assay for factor XIII catalytic subunit a (r = 0.94), the factor XIII dansylcadaverine assay (r = 0.83), and the factor XIII clot solubility test. Normal values detected in healthy blood donors were in the range of 74% to 117%, with a mean value of 96.9% and a median value of 97.8%; pooled normal plasma was used as a reference. Since the assay is performed in a single microtiter plate and requires few reagents and short incubation periods, it can be easily adapted for clinical use.


Preliminary study on oncogene product immunohistochemistry (c-erbB-2, c-myc, ras p21, EGFR) in breast pathology.

The expression of oncogene products related to cell growth (c-erbB-2, c-myc, ras p21, EGFR) was investigated in benign (15 cases) and malignant breast lesions (20 cases) by means of immunohistochemistry using the avidin-biotin-peroxidase technique with polyclonal and monoclonal antibodies. The aim of this study was to evaluate the relationship between the staining positivity and various morphological and biological features, such as tumour type, grading, hormone receptor status and cell kinetic parameters. In benign breast lesions, as expected, the kinetic parameters were low, both for Ki-67 and LI. All the specimens showed a diploid condition (the DI being equal to 1) and we found a limited degree of immunoreactivity for all the growth factors and oncogene products. In breast cancer we studied the distribution of immunohistochemical positivity for EGFR, c-erbB-2, c-myc, ras p21 and Ki-67, which was related to age, nodal status, ER and PgR receptor status, LI, DI and histopathological grading. A significant positive correlation was found both between ras p21 expression and nodal status and ER-ICA positivity. We observed a strong correlation between LI and Ki-67 and an inverse relation between Ki-67 and ER expression. These findings suggest the importance of studying the relationship between prognostic factors which may provide preoperative prediction in the biological behaviour of breast cancer, not only on biopsy specimens, but also on fine needle aspirates.


Pharmaco-morbidity linkage: a feasibility study comparing morbidity in two pharmacy based exposure cohorts.

The aims were (1) to compare discharge diagnoses and concurrent medication in a pharmacy based cohort of users of H2 receptor antagonists to those in a population of users of other drugs in the same period, who did not use H2 receptor antagonists; (2) to compare these results to those of a similar study performed with the Tayside record linkage scheme. The study was a retrospective cohort study. The morbidity data from the only hospital in one medium sized city (62,000 inhabitants) were linked to the dispensing data of all five community pharmacies on an individual basis (April 1, 1986-December 31, 1989). In the absence of a unique patient identification number, data from pharmacies and hospital were linked by the combination of date of birth, gender, and general practitioner code. For every user of H2 receptor antagonists two controls were obtained from all patients who had not used these drugs, and matched for age (within 5 years), gender, and general practitioner. All discharge diagnoses which followed this first prescription up to December 31, 1989, in a patient in the index cohort, and during the same period in his or her matched controls, were included in the study. In the index cohort (n = 2174) 341 persons were admitted (526 admissions) as against 398 persons (527 admissions) in the control cohort (n = 4348). There was increased morbidity in the index cohort, especially concerning the gastrointestinal system (peptic ulcers and malignancies, abdominal pain, gastrointestinal haemorrhage), but also concerning the musculoskeletal, respiratory, and circulatory systems. The morbidity in the last three groups corresponded with drugs used concomitantly by patients in the index cohort, so it was probably not causally related to the intake of H2 receptor antagonists but was rather an indicator of higher levels of morbidity in the index cohort. The figures were grossly comparable to those of the Tayside record linkage scheme. Probabilistic linking with the patient characteristics of gender, date of birth, and general practitioner code can facilitate the undertaking of postmarketing surveillance studies.


Preliminary evaluation of HER-2/neu oncogene and epidermal growth factor receptor expression in normal and neoplastic human ovaries.

The HER-2/neu oncogene (a member of the Erb-like oncogene family) is distinct from but closely related to the c-erb B gene which encodes the epidermal growth factor receptor (EGFr). HER-2/neu gene amplification was found in a large number of mammary carcinomas and there was a strong correlation between this phenomenon and poor prognosis. In our study HER-2/neu oncogene expression was determined in 16 malignant ovarian tumors, 2 ovarian lymphomas and 5 normal ovaries. The HER-2/neu gene was found both in normal ovaries and malignant tumors, without any apparent difference among the various histological types. In all the specimens examined, HER-2/neu expression did not seem to be related to EGF binding capacity.


Comparison of messenger RNA pools in active and dormant Artemia franciscana embryos: evidence for translational control.

In response to environmental anoxia, embryos of the brine shrimp Artemia franciscana enter a dormant state during which energy metabolism and development are arrested. The intracellular acidification that correlates with this transition into anaerobic dormancy has been linked to the inhibition of protein synthesis in quiescent embryos. In this study, we have addressed the level of control at which a mechanism mediated by intracellular pH might operate to arrest protein synthesis. Two independent lines of evidence suggest that there is an element of translational control when protein synthesis is arrested in dormant embryos. First, as determined by in vitro translation techniques, there were no significant quantitative differences in mRNA pools in dormant as compared to actively developing embryos. In addition, fluorography of the translation products showed that there are no large qualitative changes in mRNA species when embryos become dormant. These data suggest that there was no net degradation of mRNA pools in dormant embryos and that protein synthesis may therefore be controlled more strongly at translation than at transcription. Second, polysome profile studies showed that dormant embryos possess reduced levels of polysomes relative to those found in cells or active embryos. The disaggregation of polysomes is an indication that the initiation step in protein synthesis is disrupted and is further evidence that the mechanism involved in protein synthesis arrest in dormant Artemia involves translational control.


The role of microtubules in contractile ring function.

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.


Laboratory colonization and maintenance of Toxorhynchites moctezuma.

A colony of Toxorhynchites moctezuma was established at the Caribbean Epidemiology Centre in Trinidad in 1984. Toxorhynchites moctezuma was maintained in cages with high humidities. Eggs were deposited most frequently in a cut bicycle tire containing water. A minimum of 42 h was required for hatching, but 94% hatched between 43 and 51 h. Aedes aegypti larvae were supplied as prey. Larval development times varied with the quantity of prey offered, but when fed ad lib, peak developmental time was 18 days. Mean pupal developmental time was 5.5 days. Although only 12% of larvae survived to pupation in 3 years of production, our experience indicates this species would be a likely candidate for mass production and release.


Serum dipeptidyl peptidase (DPP) IV activity in hamster buccal pouch carcinogenesis with 9,10-dimethyl-1,2-benzanthracene.

Dynamic change of serum DPP IV activity in carcinogenesis of hamster buccal pouch epithelium with DMBA was investigated. The serum enzyme level in hamster (21.5 +/- 3.1 IU/l serum) was decreased gradually from the 8th to the 10th wk when papillomas were induced by DMBA application (18.9 +/- 2.7 IU/l serum). The enzyme level was further decreased in the formation of carcinoma in situ or early invasive carcinoma (13.2 +/- 0.5 IU/l serum), and reached to less than half of the normal level at the time when tumors were diagnosed as squamous cell carcinoma histologically (8.1 +/- 1.3 IU/l serum). This enzyme level was increased by tumor excision and decreased again by tumor recurrence toward death. These findings suggested that the decrease of serum DPP IV activity occurred from the early stage of hamster buccal pouch carcinogenesis as a tumor-burden marker.


The adrenergic pharmacology of sympathetically-maintained pain.

The authors seek to highlight some of the recent advances in understanding the pharmacology and pathophysiology of sympathetically-maintained pain, and to develop alternate, and possibly more specific, diagnostic tests for this phenomenon. Mechanical hyperalgesia in sympathetically-maintained pain can be explained by central sensitization so that the activation of A-beta mechanoreceptors now causes pain. The sensitization of central pain-signaling neurons is dynamic and reversible. The authors propose that an ongoing input from peripheral nociceptive afferents is necessary to maintain central sensitization. This nociceptive input may be due to an alpha-adrenoceptor mediated excitatory action of sympathetic efferents on sensory nerves that is independent of neurovascular transmission.


Reduction of left ventricular hypertrophy after longterm antihypertensive treatment with doxazosin.

The aim of this study was to evaluate the effect of antihypertensive treatment with doxazosin on left ventricular anatomy and function. Therefore, after 4 weeks of washout with placebo (phase 1), doxazosin (dosage range from 1 to 16 mg, plus hydrochlorothiazide when necessary) was given to 11 essential hypertensive patients (6 M, 5 F, age range 34-63 years) for 8 weeks (phase 2) in order to achieve diastolic blood pressure values less than 90 mmHg; this dosage was then maintained for a further 20 weeks up to the end of the study (phase 3). Blood pressure was significantly reduced (Anova P less than 0.05), while heart rate did not change. A significant reduction of left ventricular mass index (from 128.5 +/- 26 to 114 +/- 23 g/m2, at the end of phase 1 and 3 respectively, P less than .001)) was observed. Before and during treatment left ventricular systolic function, both at rest and during stress (handgrip and cold pressor tests), evaluated by fractional shortening as related to end-systolic stress, in every case within 95% confidence limits, was calculated in normal subjects. Diastolic function, as evaluated by the ratio between peak early and atrial velocities of transmitral flow examined by pulsed doppler was significantly improved. Plasma catecholamine concentrations, plasma renin activity and plasma aldosterone did not change. A significant reduction of plasma cholesterol concentration was observed. These results confirm that doxazosin is a well tolerated and effective antihypertensive drug, with a favourable effect on blood lipids and they indicate that its longterm administration can induce a significant reduction of left ventricular mass.


Highly polymorphic XbaI RFLPs of the human 21-hydroxylase genes among Chinese.

A highly polymorphic XbaI restriction site flanking the human 21-hydroxylase genes (21-OHA and 21-OHB) was found with the probe pC21/3c, the cDNA of the 21-hydroxylase gene. From the results of RFLP analysis of 10 subjects with congenital adrenal hyperplasia (CAH) owing to 21-hydroxylase deficiency and their parents, at least four polymorphic fragments (30 kb, 27 kb, 25 kb, and 15 kb) resulting from cleavage at the polymorphic endonuclease sites outside the genes were found, and at least 10 different polymorphic patterns were observed among Chinese. These results indicate that these polymorphic loci are very informative for prenatal diagnosis of 21-hydroxylase deficiency.


Familial screening for genetic haemochromatosis by means of DNA markers.

Genetic haemochromatosis (HFE) is a frequent and potentially fatal disease. Early phlebotomies may prevent complications. The recessive gene for HFE is unknown but closely linked to the HLA-A locus. No direct test for homozygosity for HFE is currently available, apart from HLA typing within the family of a patient with confirmed HFE. During a reverse genetic approach to identify the gene, we found three anonymous genomic probes (P3, P5, and I.82) derived from previously cloned YACs and physically mapped in the HLA class I region. P3 and P5 probes recognise 3 loci (P3A, P3B, P5) and I.82 one locus about 100 kb from HLA-A. Using five biallelic polymorphisms (I.82/BglII, P3B/EcoRV, P3B/PstI, P5/HindIII, P3A/PstI), we tested 198 HLA typed subjects from the families of 22 haemochromatosis patients. The information from the five polymorphisms was sufficient to identify unequivocally extended restriction haplotypes in all families. The restriction haplotypes cosegregate with the HFE allele and enable identification of genotypically identical sibs in all families studied. The linked DNA markers described in this article avoid the disadvantages of HLA serological typing and can be used in genetic counselling of HFE families.


Analysis of murine major histocompatibility complex class II-restricted T-cell responses to the flavivirus Kunjin by using vaccinia virus expression.

The present paper analyzes the influence of major histocompatibility complex (MHC) class II (Ir) genes on MHC class II-restricted T-cell responses to West Nile virus (WNV) and recombinant vaccinia virus-derived Kunjin virus antigens and identifies the immunodominant Kunjin virus antigens. Generally, mice were primed by intravenous infection with WNV or Kunjin virus, and their CD4+ T cells were stimulated in vitro 14 days later with WNV or Kunjin virus antigens to pulse macrophage or B-cell antigen-presenting cells (APC). WNV-specific in vitro T-cell responses from H-2b mice were higher than those from H-2d, H-2k, and H-2q mice. When recombinant vaccinia virus-derived Kunjin virus antigen preparations were tested in vitro, Kunjin virus-immune T cells of H-2b haplotype responded most strongly to structural (prM, C, E) and membrane-associated nonstructural (NS1) proteins encoded by VKV 1031 and showed weaker responses to cytosolic nonstructural protein NS5 (VKV 1022), whereas the responders of H-2k haplotype responded most strongly to the antigens encoded by VKV 1022 and gave lesser responses to VKV 1031. H-2d T cells gave weaker responses than either H-2b or H-2k cells, with responses to VKV 1031 generally being higher than those to VKV 1022. Responses to VKV 1023 or VKV 1024 encoding all of the NS3 to NS5 gene sequence or to VKV 1023 encoding all of NS3 were weak or absent. Within a given inbred strain, B cells and macrophages differed in their abilities to present recombinant vaccinia virus-derived Kunjin virus antigens, both in terms of magnitude of T-cell responses induced and the particular Kunjin virus protein presented. T cells from different non-MHC genetic backgrounds varied in their requirements of macrophage numbers as APC for maximum reactivity, suggesting that the concentration of class II MHC antigens and other molecules affecting APC-T-cell interaction varied in mice with different genetic backgrounds. Regardless of MHC haplotype, responses to VKV 1024, which encompasses VKV 1023 and VKV 1022, were either absent or lower than those to VKV 1022, possibly reflecting differences in the processing requirements of these two proteins. When mice were primed intravenously with recombinant vaccinia virus and when their CD4+ T cells were stimulated in vitro with native Kunjin virus antigens, VKV 1031 primed more efficiently than Kunjin virus and VKV 1022 primed similarly to Kunjin virus.


Alterations of the p53 gene in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignancy which is consistently associated with the Epstein-Barr virus (EBV). The structure of the EBV genome in NPC suggests that NPC is a clonal proliferation of epithelial cells which emerges after EBV infection. The disease develops with high incidence in specific populations in discrete geographic locations, implicating possible genetic or environmental cofactors. Mutations of the p53 gene are among the most frequent genetic changes found in a large variety of human tumors. Mutations in p53 have been shown to abrogate the suppressor function of wild-type p53 and thus contribute to the transformed phenotype. To determine if mutation in p53 participates in the development of the malignant clone in NPC, the structure and sequence of p53 in 42 primary, metastatic, and nude mouse-passaged NPC specimens was analyzed. A high frequency (6 of 9) of mutations was detected in the nude mouse-passaged tumors, while only 2 of 15 metastatic and 0 of the 18 primary tumors harbored mutant p53. The p53 mutations included single-point mutations and more extensive changes such as frame shifts, deletion, duplication, or complete loss of coding sequences. These data indicate that alterations of the p53 gene are unlikely to be involved in the initial genetic events leading to the clonal outgrowth in NPC. However, although it is a rare NPC which can be established in nude mice, this growth advantage appears to be conferred on tumors bearing a mutant p53.


[Studies on the excretion of urinary glycylprolyl dipeptidyl-aminopeptidase (GP-DAP) during normal pregnancy].

Several investigators reported that GP-DAP activity, one of dipeptidyl-aminopeptidases, increased in urine of patients with various renal diseases. In this study, we determined urinary GP-DAP and NAG activities of 165 subjects in normal pregnant women. It was clear that urinary GP-DAP was increased less than NAG with the progress of normal pregnancy. In conclusion, it is suggested that urinary GP-DAP may be a better marker than NAG for the discrimination of renal diseases from normal pregnancy.


[Affected siblings with Alzheimer's disease had missense mutation of codon 717 in amyloid precursor protein gene].

Using reverse genetic techniques, the gene responsible for familial Alzheimer's disease (FAD) is one of the clues to identify the pathogenesis of Alzheimer's disease (AD). Recently a missense mutation in the APP (amyloid precursor protein) gene (generally this mutation was called APP717) was detected in 2 Caucasian AD families and the same mutation was found in 3 Japanese AD families. We experienced brother's cases who were diagnosed as AD. Both of them and one normal person of the next generation had APP717. The first symptom of the elder brother (case 1) was forgetfulness at 52 years old, then dementia was advanced. In his clinical course there were characteristic findings such as the mirror sign, pseudodialog and jargon which has been rarely described in the Japanese literature. Finally he died of pneumonia at 57 years old. He was diagnosed as AD pathologically and physical findings of brain CT, SPECT (single photon emission computed tomography) and EEG supported this diagnosis clinically. The first symptom of the younger brother (case 2) was also forgetfulness at 45 years old, then severe dementia was advanced, at last he died of pneumonia at age 53 old. On the other hand the mother of the brothers died of severe dementia, so it was suspected that brothers died of severe dementia, so it was suspected that she had had AD. The clinical courses and pathological findings were thought to be typical of AD, namely there were no significant differences in comparison with other cases of FAD and sporadic AD.(ABSTRACT TRUNCATED AT 250 WORDS)


Lymphocytotoxic strains of feline leukemia virus induce apoptosis in feline T4-thymic lymphoma cells.

Feline leukemia retrovirus (FeLV) strains with subgroup C env genes kill feline T4 lymphoma 3201 cells by 7 to 12 days after in vitro inoculation, whereas FeLV strains with subgroup A env genes do not. Neither FeLV-A nor FeLV-C kill feline fibroblasts. FeLV-C, but not FeLV-A, is replicated to higher titer by 3201 cells and productive infection precedes death by 3 to 7 days. Transcriptional activity of the FeLV-C long terminal repeat, as assessed by chloramphenicol acetyltransferase activity, is high in feline lymphoid cells but low in feline fibroblasts. Activity of the FeLV-A long terminal repeat is moderate in both cell types. FeLV-C-infected cells form aggregates 1 to 4 days before dying; ultrastructurally, virus particles can be seen approximating the clustered cells. Dying cells demonstrate nuclear condensation, surface blebbing, and fragmentation. DNA fragmentation and laddering compatible with apoptosis occur 1 to 2 days before massive cell death. In FeLV-C-infected 3201 cells, a shift from phospholipid to neutral lipid incorporation of [14C]oleic acid, increases in palmitic acid proportions and decreases in linoleic acid proportions occur 1 to 2 days before peak killing. Exposure of 3201 cells to ultraviolet-inactivated FeLV-KT (200-800 micrograms/10(6) cells) causes cytostasis within 2 days and death within 4 days. Blebbing and nuclear condensation occur but clusters do not form. The induction of programmed cell death in feline thymic lymphoma cells by subgroup C feline retroviruses may be relevant to the pathogenesis of FeLV-induced thymic atrophy, paracortical lymphoid depletion and acquired immunodeficiency in vivo.


Age-associated decrease in proportion and antigen expression of CD8+/CD4+ thymocytes in BALB/c mice.

We studied the distribution of Thy-1.2+, CD8+ and CD4+ thymocytes in 3-, 6.5-, 12- and 18-month-old inbred BALB/c mice by staining cells with specific monoclonal antibodies (mAb) using indirect membrane immunofluorescence. The percentages of Thy-1.2+, CD8+, and CD4+ thymocytes did not vary with age until 12 months, but they exhibited at 18 months a highly significant decline. Staining thymocytes with mAb CD8 and CD4 alone or in a mixture allowed us to show that the age-related decline in proportions of CD8+ and CD4+ thymic cells is associated with the double positive (CD8+/CD4+, immature) subset. Most importantly, we observed a progressive age-related decrease in ratio of bright/dull Thy-1.2+, CD8+ and CD4+ thymocytes. The CD8+ and CD4+ thymocytes that did not adhere to peanut agglutinin (PNA-, which is supposed to represent the mature single positive subset) did not show any age-related change in the proportion of bright and dull cells. Therefore, we concluded that the double positive (CD8+/CD4+, immature, PNA+) thymocytes display reduced expression of CD8 and CD4 antigens with aging. The implications of these findings to the fundamental mechanisms of immunosenescence are discussed.


Susceptibility to low-density lipoprotein oxidation and coronary atherosclerosis in man.

Animal studies indicate a possible role for lipid oxidation in the development of atherosclerosis. We set out to investigate whether there was a relation between the ability of low-density lipoprotein (LDL) to resist oxidation in vitro and the severity of coronary atherosclerosis in man. 35 unselected young (mean [SD] age 39.9 [4.2] years) male survivors of myocardial infarction underwent angiography, and LDL was isolated from their plasma by density gradient ultracentrifugation. In-vitro LDL susceptibility to oxidation was assessed by determination of the lag phase for the formation of conjugated dienes in the presence of copper ions. An inverse relation was found between lag phase and quantitative estimates of global coronary atherosclerosis (r = -0.45; p less than 0.02). Multivariate analysis indicated that the lag phase for oxidative modification of LDL and LDL cholesterol concentration correlated independently with severity of coronary atherosclerosis. The lag phase for oxidation of LDL was also related to the triglyceride content of the LDL fraction (r = -0.55; p less than 0.002). The finding that susceptibility to LDL oxidation is associated with severity of coronary atherosclerosis may indicate that lipid oxidation promotes premature coronary atherosclerosis and that individuals with an LDL enriched in triglycerides are at particular risk.


HIV replication during the first weeks of life.

Diagnosis of HIV infection among children born to HIV-positive mothers can be made in the first 12 months, but few studies have examined HIV status during the first weeks of life. In a prospective longitudinal study of 50 infants born to HIV-1 seropositive women, blood samples were obtained at birth and at 4-9 weeks and 5-9 months of age, and were tested for HIV-1 by the polymerase chain reaction (PCR), viral culture, and p24 antigen measurements. 16 were diagnosed as HIV-infected by the age of 4-9 weeks according to both PCR and culture; by contrast, infection could be detected in only 5 children at birth. No changes in HIV status were observed between 4-9 weeks and 5-9 months in the 44 children who could be retested. Perinatal HIV-1 infection can therefore be diagnosed in the first 2 months of life, either by PCR or viral culture. Our inability to detect HIV-1 infection at birth in almost 70% of babies subsequently found infected suggests an active replication of HIV during the first weeks of life. Our results might favour the hypothesis that transmission of HIV-1 takes place either at the end of pregnancy or at delivery.


Efficiency and accuracy of polymerase-chain-reaction assay for cystic fibrosis allele delta F508 in single cell.

Diagnosis of genetic disorders in the embryo before implantation, though possible by removal of one or two blastomeres at the eight-cell stage, is still experimental because the procedures of gene analysis of DNA from a single cell are not yet reliable enough for clinical application. We have evaluated the efficiency and accuracy of polymerase-chain-reaction (PCR) amplification of a single-copy gene, wild-type or cystic fibrosis delta F508 allele, on single sperm cells from a donor known to be a heterozygous carrier of the delta F508 mutation. DNA from single spermatozoa was decontaminated by restriction-enzyme treatment, then the region around the delta F508 site was amplified by nested PCR. The distribution of the wild-type and mutant alleles (59 [55%] and 48 [45%, respectively]) in the 107 single spermatozoa did not differ from that expected (50% each). 1 sample did not provide an amplified signal. To check that the two alleles would be amplified with equal efficiency when they were both present within a cell, we did PCR for 51 two-sperm samples. Again the distribution did not deviate from that expected (17 [33%] both wild-type; 21 [41%] one wild-type, one delta F508; 13 [26%] both delta F508 vs 25%; 50%; 25% expected). None of the 74 blanks in these experiments was contaminated. We conclude that our delta F508 single-cell assay is efficient and accurate and can be used for analysis of blastomere DNA to diagnose cystic fibrosis before embryo implantation.


Viruses in idiopathic inflammatory myopathies: absence of candidate viral genomes in muscle.

There is indirect evidence that various viruses have aetiological roles in the idiopathic inflammatory myopathies. By means of a sensitive and specific method based on the polymerase chain reaction (PCR), we sought direct evidence for the presence in affected muscle of nucleic acid sequences from Coxsackie virus, mumps virus, encephalomyocarditis virus, adenovirus, human T-lymphotropic virus types I and II, and human immunodeficiency virus. RNA was extracted from muscle biopsy samples obtained from 44 patients with idiopathic inflammatory myopathies a mean of 45 (range 0-216) months after disease onset. All the subjects were older than 16 years at disease onset. The integrity of the mRNA extracted was confirmed by the successful PCR amplification of insulin receptor mRNA in all samples. The PCR method was able to detect between 1 and 20 molecules of added viral nucleic acid for the picornaviruses sought. No detectable virus sequences were found, however, in any of the patients' muscle samples or in samples from 13 controls. We tested for retroviral DNA in 22 samples (17 patients, 5 controls) that met our criterion for adequate DNA extraction (detectable beta-actin DNA by PCR); again no virus sequences were found. Persistence in muscle of these or closely related viruses is unlikely to be a continuing stimulus for disease in the idiopathic inflammatory myopathies.


Response of refractory Hodgkin's disease to monoclonal anti-CD30 immunotoxin.

In Hodgkin's disease, Hodgkin and Reed-Sternberg cells consistently express the antigen CD30. We investigated the possible therapeutic role of an immunotoxin prepared by covalent linking of an anti-CD30 monoclonal antibody (Ber-H2) to saporin (SO6), a type-1 ribosome-inactivating protein. The immunotoxin (0.8 mg/kg in one or two doses) was given to four patients with advanced refractory Hodgkin's disease. In three, there was rapid and substantial reduction in tumour mass (50% to greater than 75%). Clinical responses were transient (6-10 weeks). In-vivo binding of the immunotoxin to tumour cells was shown by immunohistology in two patients. Antibodies to both parts of the immunotoxin developed in all patients.


Population screening for fragile X.

A screening programme to detect fragile X syndrome has been operating in New South Wales, Australia, since 1984. The aim of this programme is to find previously unidentified individuals with the syndrome so that their extended families can be properly informed of the risks before making decisions about childbearing. 14,225 individuals attending adult and child facilities for the intellectually handicapped have been screened, of whom 8172 have been offered testing for the fragile X syndrome with a 79% uptake of the service. 253 probands were found, and in the extended families 818 females at 25-100% risk of being carriers were interviewed and counselled. Continuing contact was maintained and prenatal diagnosis was offered. The effect of the programme was assessed in a subgroup of 90 individuals, most of whom were appreciative of the service and felt that they had been adequately informed. The influence of knowing the diagnosis and its genetic implications were also assessed, the main consequences being a 26% reduction in births and a 61% uptake of prenatal diagnosis. Improved techniques for diagnosis of fragile X have benefited the families identified and counselled, suggesting that systematic screening for fragile X should be an essential component of community genetic services.


Linkage of type 2 diabetes to the glucokinase gene.

Maturity-onset diabetes of the young (MODY) is a subtype of type 2 diabetes that presents from the second decade and has an autosomal dominant mode of inheritance. We have investigated the glucokinase gene, a candidate gene for diabetes, in two MODY pedigrees. In a large 5-generation pedigree (BX) with 15 diabetic members, use of a microsatellite polymorphism revealed linkage of diabetes to the glucokinase locus on chromosome 7p. A peak lod score of 4.60 was obtained at a recombination fraction (theta) of zero. This finding suggests that a defective glucokinase gene contributes to the diabetes phenotype in this pedigree. This is not universal in MODY since linkage to the glucokinase locus was excluded in a second pedigree M (lod score = -7.36 at theta = 0). The affected members in pedigree BX were diagnosed either when young (in pregnancy or on screening) or when they presented symptomatically in middle and old age; most of them were treated by diet alone. Defects in the glucokinase gene may play an important part in the pathogenesis of type 2 diabetes.


Early intervention in psychiatric emergencies: a controlled clinical trial.

In the UK, psychiatric care of patients with acute and chronic disorders has increasingly moved from hospital to the community. We have evaluated in a controlled trial patients with severe mental illness, who were assigned to early intervention by community services or to standard hospital treatment. 100 patients aged 16 to 65 years presenting as psychiatric emergencies to an inner London teaching hospital were randomly allocated to a multidisciplinary community-based team (n = 48) or conventional hospital-based psychiatric services (n = 52) and assessed over a 3-month period. Ratings of psychopathology and social functioning were made before treatment and after 2, 4, and 12 weeks by independent assessors. 85 patients completed all assessments, and all patients had evaluable data beyond 2 weeks. 3 patients died during the study, 2 from natural causes and 1 from an accident. Patients referred to the community service showed greater improvement in symptoms and were more satisfied with services than those in the hospital-based service. Patients treated in the hospital-based service spent eight times as many days as psychiatric inpatients as those treated in the community-based service. Patients both prefer and seem to benefit from community-based psychiatric care, and our early-intervention community service might be a good model for such care.


Pickled vegetables in the aetiology of oesophageal cancer in Hong Kong Chinese.

Oesophageal cancer is common in Chinese populations, but individual-based epidemiological studies have provided little explanation. A case control study with 400 cases and 1598 controls (800 hospital and 798 general practice) was conducted among Hong Kong Chinese. In multivariate analyses, statistically significant effects on risk were detected for several potentially preventable exposures with high attributable risks (ARs). These included preference for consuming drinks or soups at high temperature (AR = 14%), infrequent consumption of green leafy vegetables (AR = 15%) and citrus fruits (AR = 26%), ingestion of pickled vegetables (AR = 29%), tobacco smoking (AR = 44%), and alcohol drinking (AR = 48%). On the assumption of multiplicative risk, the combined AR due to these exposures was 89%. This is the first case control study to show an association between pickled vegetable consumption and oesophageal cancer risk. The finding considerably strengthens the evidence for carcinogenicity of N-nitroso compounds in man and helps to explain the very high risk among Chinese.


Effects of dual-chamber pacing in hypertrophic obstructive cardiomyopathy.

Although attempts have been made to treat hypertrophic obstructive cardiomyopathy with right ventricular pacing, the usual treatment for those refractory to medical therapy is open heart surgery. To assess in detail the value of non-surgical therapy the effects of acute and long-term dual-chamber pacing were investigated in 13 patients with hypertrophic obstructive cardiomyopathy refractory to medical treatment. In the first part of the study, atrioventricular (AV) sequential pacing was found to reduce peak subaortic pressure gradient in 12 of the 13 patients, from 82 (SD 42) to 47 (34) mm Hg (p less than 0.002), without concomitantly reducing aortic blood pressure or cardiac output. This effect was related to AV interval. In the second part of the study, a dual-chamber pacemaker was implanted in 8 patients and programmed to the optimum AV interval for the individual (50-90 ms). Patients were followed up for up to 62 months. Pacing resulted in a significant and long-lasting reduction in severity of angina pectoris (from NYHA class 3 to 1) and dyspnoea (from NYHA class 3 to 2). Echocardiography showed no significant change in septal thickness or left ventricular contractility but there was a trend to a spontaneous decrease in obstruction. In patients with hypertrophic obstructive cardiomyopathy, synchronised and ventricular pacing at optimum AV interval for the individual reduces the intraventricular pressure gradient and improves functional tolerance. Since the effect is longlasting, such pacing should be deemed an alternative therapy to surgery in selected cases.


Abnormalities of surfactant in children with recurrent cyanotic episodes.

The mechanism of recurrent cyanotic episodes in infants and children is not known, but a deficiency of surfactant is a possible cause. We have measured the amount of surfactant collected by bronchoalveolar lavage from two children with recurrent cyanotic episodes and from two controls with anatomical airway obstructions. We also assessed the physical properties of the surfactant by changing the surface area (A) of a monolayer and measuring its surface tension (gamma). The cases had lower amounts of surfactant extracted, which could explain some of the abnormalities of the gamma/A loops. However, the finding that the cases had reversed loops (ie, the surface tension is higher during monolayer compression than during expansion) shows that there is also a qualitative abnormality. These features suggest a possible diagnostic test if not a mechanism for this disorder.


Shockwave lithotripsy of salivary duct stones.

Surgical extirpation of the affected gland has been necessary for cases of sialolithiasis in which the stone cannot be removed by dilatation or dissection of the salivary duct. The ability of the piezoelectric lithotripter to deliver shockwaves to a small focus makes extracorporeal shockwave lithotripsy of salivary gland stones potentially safe. Its safety and efficacy have been assessed in 51 patients with symptomatic solitary salivary stones that could not be removed by conservative measures. The stones had a median diameter of 8 (range 4-18) mm and were located in the submandibular gland in 69% of patients and in the parotid gland in 31%. A total of 72 shockwave treatment sessions (maximum 3 per patient) were given under continuous sonographic monitoring. In 45 patients (88%) complete fragmentation (fragments less than or equal to 3 mm) of the concrements was achieved. No patient needed anaesthesia, sedatives, or analgesics. The only untoward effects were localised petechial haemorrhages after 10 (13%) out of 72 treatments and transient swelling of the gland immediately after delivery of shockwave in 2/72 (3%) sessions. 20 weeks after the first session 90% (46/51) of patients were free of discomfort, and 53% (27/51) were stone free. Stone-clearance rate was higher among patients with stones in the parotid gland (81%) than among those with stones of the submandibular gland (40%). Auxiliary measures such as dilatation or dissection of the salivary duct were required only in patients with stones in the submandibular gland (20%). No long-term damage to the treated salivary gland or to adjacent tissue structures was noted during the median follow-up of 9 (1-24) months. Extracorporeal piezoelectric shockwave therapy seems likely to be safe, comfortable, and effective minimally-invasive, non-surgical treatment for salivary stones.


Value of the alert and action lines on the partogram.

A partogram based on a World Health Organisation model has been used for many years in the peripheral maternity clinics of Pikine, Senegal, to monitor labour. We have assessed the value of the partogram and efficacy of the alert and action lines. 1022 pregnant women were monitored by partogram during 4 months. The alert line was crossed in 100 (9.8%) of these cases and the frequency of neonatal resuscitation was higher for this group (relative risk 4.0, 95% confidence interval 2.3-7.1; p less than 0.0001), as was the number of "fresh" stillbirths (5.3, 1.8-15.6; p less than 0.01) (recent death may have occurred during labour). Among the women who crossed the alert but not the action line, neonatal resuscitation was also four times more likely than for the normal labour group (4.0, 2.1-7.6; p less than 0.001), and the fresh stillbirth rate was higher but not significantly so. For women who crossed both lines, the fresh stillbirth rate was ten times higher than for women in the normal labour group (9.9, 2.8-34.7; p less than 0.001). Crossing the alert line had a sensitivity of 27%, a specificity of 93%, and a positive predictive value of 17% for neonatal resuscitation. If the action line was chosen as the decision level, the positive predictive value remained the same but the sensitivity was only 8%. Health workers intervened (eg, artificial rupture of the membranes, administration of oxytocics) in half the dystocic cases. Of the women who did not receive any such treatment 44% crossed the action line compared with only 26% of those who did receive treatment (p = 0.06). The results show the usefulness and efficacy of the partogram and underscore the value of medical intervention as soon as the alert line is crossed.


[Drug therapy of chronic inflammatory bowel disease--reliable standards and new developments].

The standard therapy of ulcerative colitis and Crohn's disease is based on the treatment with corticosteroids, sulfasalazine and 5-aminosalicylic acid (mesalazine). Depending on the localization and the extent of bowel inflammation these drugs are given topically (proctosigmoiditis, left-side colitis) or systemically (total and subtotal colitis). Azathioprin and metronidazole are considered to be reserve drugs (the latter one having proven to be effective only in Crohn's disease). During the last years, the efficacy of several new agents has been investigated. At present, their routine administration cannot be advised. The clinical usefulness of new topical corticosteroids showing both high antiinflammatory effect and no or at least minor systemic side effects is promising.


Construction and analysis of Bordetella pertussis mutants defective in the production of fimbriae.

Although the role of fimbriae in bacterial disease has been well established, little is known about the function of Bordetella pertussis fimbriae. To study this function, well-defined fimbrial mutants were constructed. B. pertussis harbours three fimbrial genes, fim2, fim3 and fimX, and strains were constructed in which one or more fimbrial genes were inactivated by means of gene replacement. Analysis of these strains by means of immunoblotting suggested the presence of a fourth fimbrial gene, tentatively designated fimY. A fimbrial mutant was analysed in a mouse respiratory infection model, together with a strain harbouring a deletion in the gene for the filamentous haemagglutinin. Both mutants were affected in their ability to persist in the trachea. Persistence in the nasopharynx was only affected by the mutation in the filamentous haemagglutinin gene. Neither the filamentous haemagglutinin nor the fimbrial mutants were affected in their ability to persist in the lung. Our results suggest that the filamentous haemagglutinin plays a more crucial role than fimbriae in the colonization of the upper respiratory tract of the mouse.


Comparative efficacy of autolysin and pneumolysin as immunogens protecting mice against infection by Streptococcus pneumoniae.

Previous studies on Streptococcus pneumoniae have established that the pneumococcal proteins autolysin (N-acetylmuramyl-L-alanine amidase) and pneumolysin both contribute significantly to the virulence of the organism. In the present work, autolysin and a defined toxoid derivative of pneumolysin were tested, individually and in combination, for efficacy in a mouse model as antigens protecting against challenge with virulent, wild-type S. pneumoniae. While each antigen alone provided significant protection, the degree of protection was not increased when the antigens were administered together. In an additional experiment, mice were challenged with a genetically-modified mutant strain of pneumococcus unable to express active pneumolysin. Pre-immunization of such mice with autolysin failed to provide any significant protection against the challenge. The results of this study suggest that the most important contribution made by autolysin to the virulence of S. pneumoniae may be its role in mediating the release of pneumolysin from the pneumococcal cytoplasm during infection.


Effect of insertional inactivation of the genes encoding pneumolysin and autolysin on the virulence of Streptococcus pneumoniae type 3.

Derivatives of Streptococcus pneumoniae type 3 deficient in production of either pneumolysin or autolysin were constructed. This was achieved by transformation of type 3 pneumococci with DNA from derivatives of a rough strain (Rx1), in which the respective genes had been interrupted by insertion-duplication mutagenesis using internal fragments of the cloned genes in the vector pVA891. Southern blot analysis confirmed that the pneumolysin or autolysin genes in the respective transformants had been interrupted by insertion of the plasmid-derived sequences. Both the pneumolysin-negative and the autolysin-negative strains had significantly reduced (P less than 0.0001) virulence in mice, as judged by survival time after intraperitoneal challenge. The median survival time of mice challenged with type 3 pneumococci in which either pneumolysin or autolysin production had been reconstituted by back-transformation of the mutants with an intact copy of the respective cloned gene (with concomitant elimination of plasmid-derived sequences), was indistinguishable from that of mice challenged with the wild-type strain. These results establish the importance of both pneumolysin and autolysin to the virulence of type 3 pneumococci.


Geographic mitochondrial DNA variation in the rock hyrax, Procavia capensis.

Restriction-fragment-length polymorphisms in mitochondrial DNA (mtDNA) were used to evaluate geographic population genetic structure in the rock hyrax, Procavia capensis, a species which occurs widely, though restricted to rocky habitat, throughout South Africa. Ten restriction endonucleases were employed to assay mtDNAs from 55 specimens representing 10 localities. Haplotypes showed strong geographic patterning, and estimates of nucleotide sequence divergence indicate two major clades thought to be dispersing along separate routes. The divergence time of approximately 2 Myr between clades is relatively high for intraspecific variation. We speculate that the marked genetic break distinguishing the northwestern populations from those constituting the south/central clade may be indicative of two species in what has conventionally been regarded as P. capensis.


The role of pili in colonization of the rabbit intestine by Vibrio parahaemolyticus Na2.

Vibrio parahaemolyticus Na2 and its pili were studied in relation to intestinal colonization. The isolated pili were adhesive to the intestinal epithelium. The adhesion of the organisms was blocked by masking the epithelial receptor with the purified pili, or by treating the organisms with anti-pilus antibody (Fab fraction). These results suggest that the pili of V. parahaemolyticus Na2 play an important role in the adhesion of the organisms to the rabbit intestine.


Proposal for clinical trials using anti-inflammatory drugs in the therapy of angina pectoris, myocardial infarction and coronary restenosis after angioplasty and bypass grafting.

The importance of inflammatory phenomena in atherosclerosis is now appreciated. Here, a clinical trial to be conducted using anti-inflammatory drugs (sulfasalazine, griseofulvin and colchicine) in angina pectoris, myocardial infarction and coronary restenosis after angioplasty and bypass grafting is proposed. Patients who have both atherosclerosis and a disease responsive to anti-inflammatory drugs (ulcerative colitis or Crohn's disease, dermatomycosis, necrotizing vasculitis, Behcet's disease, gout or other colchicine-sensitive diseases), are desirable targets of the present proposal.


Alzheimer's disease: a 'cobalaminergic' hypothesis.

An association between Alzheimer's Disease (AD) and low CSF and serum vitamin B12 (B12) has recently been described (1, 2, 3). This is apparently independent of nutritional intake (4). It has been suggested that such patients may exhibit an atypical form of cobalamin deficiency (3, 4). It is therefore proposed that these deficiencies may be aetiologically important, at least in sub-groups of AD, and a mechanism is described whereby B12 deficiency may result in the characteristic neurotransmitter changes of the disease. The hypothesis generates predictions regarding biochemical evaluation of such patients and suggests associations between the neurochemical disturbances and structural abnormalities of AD.


Molecular neuroanatomic mechanisms of Parkinson's disease: a proposed therapeutic approach.

Current concepts suggest that parkinsonian bradykinesia is a consequence of an imbalance between striatopallidal and striatonigral output pathways. Dopamine appears to oppositely effect these neurons due to the selective localization of the D1 receptor on striatonigral neurons and the D2 receptor on striatopallidal neurons. Studies measuring changes in gene regulation suggest how selective D1 and D2 dopamine agonist treatments may be used to restore the normal balance between the striatal output pathways.


Dopaminergic agonists in the treatment of Parkinson's disease.

Dopaminergic agonists are drugs that directly stimulate the dopamine receptors of the striatum and have proved useful in treating Parkinson's disease. In the United States, they are primarily used in conjunction with levodopa. Most have long half-lives and have been particularly useful in controlling and attempting to prevent motor fluctuations.


An integrated approach to patient management in Parkinson's disease.

New concepts about the pathogenesis and pathophysiology of Parkinson's disease have emerged. For these concepts to be useful, they must be understood, and for them to be applied, the psychology of the patient and the patient's family must be understood. The initial consultation is crucial in establishing a successful relationship between a patient, family, and physician. This consultation is analyzed and ways of avoiding errors and misconceptions delineated. Emphasis is placed on imaginitive questioning using the format of the ADL portion of the UPDRS in establishing the diagnosis and following treatment. The rational for starting treatment with selegiline at this time is discussed in the context of the role that increased MAO-B activity plays in the progression of Parkinson's disease. After making the diagnosis and starting treatment with selegiline, deciding when to start levodopa is the next crucial decision. Often as important as deciding when to start levodopa is overcoming the resistance of the patient to accept this treatment. The next crucial decision occurs after the patient develops response fluctuations on levodopa. A format for assessing the fluctuations is presented, and the merits of different treatments, including selegiline, dopamine agonists (bromocriptine and pergolide), and sustained-release or controlled-release levodopa preparations (Sinemet CR), discussed. The management of patients with depression, sleep problems, and advanced disease including postural instability and mental changes are reviewed.


Protein kinase-C activation increases the quantity and poly(A) tail length of corticotropin-releasing hormone messenger RNA in NPLC cells.

We have studied the effect of protein kinase-C activation on the regulation of CRH gene expression in the human hepatoma cell line NPLC/PRF/5 (NPLC), the only cell line known to express the endogenous CRH gene. Incubation of NPLC cells with 100 nM 12-O-tetradecanoyl phorbol 13-acetate (TPA), a phorbol ester that activates protein kinase-C, resulted in a rapid (1-h) and prolonged (72-h) increase in CRH mRNA levels, with the maximum increase of 16-fold observed at 24 h. In addition, TPA treatment increased the size of CRH mRNA by approximately 100 nucleotides. This size increase, which was blocked by protein synthesis inhibitors, occurred within 1 h of TPA addition and lasted at least 8 h, with a return toward the baseline size by 24 h. Structural analysis of CRH mRNA revealed two poly(A) addition sites and, as found in human placenta, multiple transcription start sites. The increase in CRH mRNA size was not due to changes in the sites of either transcription initiation or poly(A) addition, but, rather, to a 3-fold increase in the length of the poly(A) tail itself. The ability of TPA to increase CRH mRNA levels in NPLC cells suggests that the protein kinase-C second messenger pathway may be involved in the physiological regulation of CRH gene expression. Increases in CRH mRNA poly(A) tail length potentially may influence CRH mRNA stability or translatability and, thus, may represent a general mechanism by which the protein kinase-C pathway can influence gene expression.


Phylogenetic specificity of prolactin gene expression with conservation of Pit-1 function.

In mammals, the pituitary POU homeodomain protein, Pit-1, binds to proximal and distal 5'-flanking sequences of the PRL gene that dictate tissue-specific expression. These DNA sequences are highly conserved among mammals but are dramatically different from PRL 5' sequences in the teleost species, Oncorhynchus tschawytscha (chinook salmon). To analyze the molecular basis for pituitary-specific gene expression in a distantly related vertebrate, we transfected CAT reporter gene constructs containing 2.4 kilobases (kb) 5'-flanking sequence from the salmon PRL (sPRL) gene into various cell types. Expression of the sPRL gene was restricted to pituitary cells, but in rat pituitary GH4 cells levels of expression were at least 90-fold lower than those obtained with a -3 kb rat PRL (rPRL) construct. Conversely, in primary teleost pituitary cells, -2.4 kb sPRL/CAT was expressed at levels about 10-fold higher than -3 kb rPRL/CAT. To determine whether species-specific transactivation by Pit-1 was sufficient to explain these species differences in PRL gene expression, we isolated a cDNA clone encoding the salmon Pit-1 POU domain and constructed a rat Pit-1 expression vector that contained salmon Pit-1 POU domain sequences substituted in frame. The chimeric Pit-1 encoded 14 amino acids unique to salmon. Coexpression of rat Pit-1 with salmon or rat PRL/CAT in transfected HeLa cells resulted in specific and strikingly comparable levels of promoter activation. Moreover, the specificity and efficacy of the chimeric salmon/rat Pit-1 was similar to wild type rat Pit-1 in activating salmon and rat PRL/CAT.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of tyrosine aminotransferase gene expression by retinoic acid.

Regulation of tyrosine aminotransferase (TAT) gene expression was examined in RALA255-10G, a simian virus-40 tsA mutant-immortalized adult rat hepatocyte line. At the nonpermissive temperature (40 C), these hepatocytes exhibited a differentiated phenotype and actively expressed the TAT gene, but only in the presence of dexamethasone (DEX). The glucocorticoid-mediated TAT expression was inhibited by cycloheximide, a protein synthesis inhibitor, and by RU486, a glucocorticoid antagonist, suggesting that glucocorticoid induction requires protein synthesis and may be mediated through hormone receptors. (Bu)2cAMP (Bt2cAMP) or retinoic acid, individually or in combination, failed to increase TAT mRNA levels. However, Bt2cAMP greatly potentiated the induction by DEX, whereas retinoic acid inhibited the induction by DEX or DEX/Bt2cAMP. Nuclear run-on assays demonstrated that the induction of TAT expression by DEX or DEX/Bt2cAMP in RALA255-10G cells is regulated primarily at the transcriptional level. In contrast, retinoic acid antagonized the DEX- or DEX/Bt2cAMP-mediated induction without affecting the rate of TAT gene transcription. Instead, retinoic acid destabilized TAT mRNA. The half-life values of TAT mRNA in DEX/Bt2cAMP- and DEX/Bt2cAMP/retinoic acid-treated cells were approximately 235-270 min and 90-100 min, respectively. Our results indicate that inhibition of TAT expression by retinoic acid was regulated primarily at the posttranscriptional level.


A dominant repressor of cyclic adenosine 3',5'-monophosphate (cAMP)-regulated enhancer-binding protein activity inhibits the cAMP-mediated induction of the somatostatin promoter in vivo.

The transactivation of genes through the cAMP-regulated enhancer (CRE) is proposed to occur by the binding and phosphorylation of the transcription factor CREB (CRE-binding protein). Originally believed to be a single protein, more than 10 different CREB proteins have been cloned. The contributions of each of these factors to gene regulation have yet to be determined unambiguously. We have isolated a CREB cDNA that contains a mutation of a single amino acid in the DNA-binding domain. In gel shift assays, this mutant, designated KCREB, is unable to bind to the somatostatin (SS) CRE. In addition, KCREB acts as a dominant repressor of the wild-type factor, blocking the ability of wild-type CREB to bind to the CRE when present as a KCREB:CREB heterodimer. The KCREB mutant also acts as a dominant repressor in vivo, completely blocking the ability of wild-type CREB to mediate induction by protein kinase-A of a SS CRE reporter gene in F9 teratocarcinoma cells. We have used this mutant to analyze the participation of CREB in the induction of the SS promoter in CA-77 cells, a medullary thyroid carcinoma cell line that produces high levels of SS. Although KCREB can block a portion of the cAMP induction of the SS promoter in CA-77 cells, approximately 45% of the induction remains insensitive to the mutant. These data support the paradigm that CREB is involved in the cAMP induction of SS in vivo. Furthermore, the inability of KCREB to completely block cAMP-mediated SS expression in CA-77 cells suggests that additional factors may contribute to the cAMP regulation of CRE function.


Levodopa-induced dyskinesias in Parkinson's disease: clinical and pharmacological classification.

Levodopa-induced dyskinesias (LID) in Parkinson's disease (PD) may be classified into three main categories: "On" dyskinesias, diphasic dyskinesias (DD), and "off" periods. The study of 168 parkinsonian patients showed that about half (n = 84) showed one pattern of LID only. A combination of two was present in 68, and 16 had the three presentation patterns. A fairly good correlation between type of dyskinesia and presentation pattern was established. Chorea, myoclonus, and dystonic movements occurred during the "on" period. Dystonic postures, particularly affecting the feet, were mainly present in the "off" period, but a few patients had a diphasic presentation. Repetitive stereotyped movements of the lower limbs always corresponded to DD. Acute pharmacological tests using dopamine agonists (subcutaneous apomorphine 3-8 mg; intravenous lisuride 0.1-0.15 mg) and dopamine antagonists (intravenous sulpiride 200-400 mg and intravenous chlorpromazine 25 mg) were performed in 40 patients. Dopamine agonists enhanced "on" dyskinesias and markedly reduced or abolished "off" period dystonia and DD. Dopamine antagonists reduced all types of LID but usually aggravated parkinsonism. These clinical and pharmacological results indicate that LID in PD are a heterogeneous phenomenon difficult to explain on the basis of a single pathophysiological mechanism.


Clozapine prevents recurrence of psychosis in Parkinson's disease.

Psychosis secondary to dopaminergic therapy can limit the ability to manage motor symptoms of advanced Parkinson's disease (PD). We report the results of an open label 3-month trial that evaluated the antipsychotic effects of clozapine in eight PD patients with drug-induced psychosis. Response was quantified using a simplified brief psychiatric rating scale and two PD scales. Clozapine significantly improved psychiatric scores at low doses. The use of every other day regimens (not previously utilized) led to good control of symptoms and minimized side effects. Clozapine also had a positive sleep effect in four patients and improved dyskinesia in one. Finally, this treatment prevented recurrence of psychosis while levodopa doses were significantly increased and while other antiparkinsonian medications were added. Motor disability related to PD improved as a result of these treatment adjustments. We conclude that clozapine is effective in treating drug-induced psychosis in PD and allows for safe optimization of antiparkinsonian therapy.


Ethosuximide and tremor in Parkinson's disease: a pilot study.

Following the demonstration of an anti-tremor effect of ethosuximide in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkey model, we have tested the effect of this drug in 10 patients with typical parkinsonian tremor. Six patients suffered from Parkinson's disease with prominent, relatively drug-resistant rest tremor. The other four patients had a drug-induced parkinsonian tremor due to neuroleptic agents taken for a psychiatric condition. Five of the six parkinsonian patients and three of the four psychiatric patients reported within a few days a marked and intolerable exacerbation of their tremor. One patient in each group was improved, and this improvement was verified in repeated examinations. Thus, for unknown reasons, the effect of ethosuximide was not as predicted by the monkey model. Among possible explanations is the fact that we had chosen patients with drug-resistant tremor.


Effect of ethosuximide on rest tremor in the MPTP monkey model.

Based on the hypothesis that low-threshold calcium conductance in the thalamus might be involved in the pathophysiology of parkinsonian tremor, ethosuximide was given chronically to a monkey previously treated with MPTP and displaying exceptionally a typical rest tremor. After 5 days of daily treatment, the tremor was reduced by 60%. Diltiazem and verapamil which act on different calcium channels had no such effect. Ethosuximide also potentiated the anti-tremor effect of the dopamine D2 agonist LY-171555.


Postvaccinal parkinsonism.

A 5-year-old boy, with a history of fever beginning 15 days after a vaccination for measles, developed a rigid-akinetic syndrome 3 days after the fever began. A spinal tap obtained 1 week after the onset of fever showed pleocytosis with a monocellular pattern. A CT scan of the head and EEG did not disclose any abnormality. An MRI performed 3 months after the event, however, showed clear-cut evidence of bilateral substantia nigra lesions, suggesting secondary gliosis. The response to levodopa was good, but adverse reactions appeared early. The child is now 7 years old. Bromocriptine, deprenyl, and levodopa have produced a remarkable improvement of the parkinsonian features.


Neuromuscular function and polymorphism of the acetylcholine receptor gamma gene.

Examination of inbred and wild mice has shown that the gene coding for the acetylcholine receptor gamma subunit is polymorphic and the polymorphism correlates with differences in neuromuscular function tested by two different methods. Polymorphism of the AChR gamma gene was demonstrated after digestion of genomic DNA with Pstl and Xbal. These two restriction enzymes demonstrated RFLP patterns specific for C57Bl/6J (PSXS) and C3H/HeJ (PRXR) mice, respectively. Examination of F1 (C3H/HeJ x C57Bl/6J) and F2 hybrid populations, and other murine inbred strains, showed the inheritance and strain specificity of the RFLPs. Testing wild mice demonstrated that the PSXS form of the AChR gamma gene is the most common in the wild. The AChR gamma and AChR delta subunits are closely linked and carried on the same chromosome as several contractile proteins. Because these genes are essential for correct neuromuscular development and activity, we investigated the possibility that the observed AChR gamma polymorphisms mark a haplotype which correlates with variations in neuromuscular function. Analysis of exercise times showed a correlation between AChR gamma polymorphism and neuromuscular function. To our knowledge, this is the first description of AChR polymorphism cosegregating with variations in neuromuscular function.


[Influence of succinate on the effectiveness of glutamatergic synaptic transmission in surviving slices of rat hippocampus].

Sodium succinate has been studied for its influence on the efficiency of glutamate transmission in the Schaffer collateral synapses and duration of its potentiation state caused by high-frequency stimulation in surviving hippocampal slices. It is shown that administration of sodium succinate (50 mg/kg i. p.) to rats 30 min prior to decapitation stabilizes glutamate transmission in field CA1 of the rat hippocampal slices and increases duration of its potentiation state.


Effect of diabetes on in vivo and in vitro hypothalamic somatostatin release.

In order to better understand the mechanisms underlying the reduction in growth hormone (GH) secretion in diabetic rats, we studied hypothalamic somatostatin secretion both in vivo (into hypophysial portal blood) and in vitro (from hypothalamic fragments) 5, 9 and 30 days after induction of diabetes. Experimental diabetes was induced by an intraperitoneal injection of streptozotocin (STZ) at a dose of 65 mg/kg. Basal plasma GH was significantly reduced in diabetic rats at all stages. Somatostatin levels in hypophysial portal blood was unaffected in 5-day STZ-diabetic rats and significantly increased 9 days after STZ administration. Chronic insulin replacement therapy in diabetic animals partly normalized somatostatin levels as well as plasma GH and glucose levels. A good correlation was observed between in vivo and in vitro experiments. Indeed, somatostatin release from hypothalamic fragments did not change 5 days after STZ-induced diabetes and significantly increased 9 and 30 days after STZ administration. The in vitro increase in hypothalamic somatostatin secretion was observed in 10 as well as in 33 mM glucose concentration in the incubation medium. In the same experiment, the in vitro hypothalamic corticotropin-releasing factor secretion was lowered 5 and 9 days after diabetes induction. We conclude that hypothalamic somatostatin release increases in diabetic rats. These changes may contribute to the reduction in GH secretion in these animals. However, since these changes occur after the onset of plasma GH decrease, a factor(s) other(s) than somatostatin may play a causal role in the reduction in GH secretion.


Hypothalamic regulation of impaired growth hormone secretion in diabetic rats. 1. Studies in streptozotocin-induced diabetic rats.

Growth hormone (GH) secretion is blunted in diabetic rats. In the present experiment we observed that pituitary GH concentrations and the plasma GH response to an exogenous dose of growth hormone-releasing hormone (GHRH) is decreased in streptozotocin-induced diabetic rats (p less than 0.02) with respect to normal rats. In an attempt to determine if increased somatostatin (SRIF) secretion is responsible for the decreased GH secretion, we studied the effect of modulating SRIF tone on the GH response to GHRH in normal and streptozotocin-induced diabetic rats. Rats were pretreated with either normal sheep serum and saline (NSS+SAL), somatostatin antibodies (SRIF-Ab), or pyridostigmine (PD), an acetylcholinesterase inhibitor hypothesized to reduce hypothalamic SRIF secretion. Pretreatment of normal rats with SRIF-Ab or PD resulted in an increased GH response to exogenous GHRH in comparison to NSS+SAL-pretreated normal rats at 5 min postinjection. In contrast, pretreatment of diabetic rats with SRIF-Ab or PD did not alter the GH response to exogenous GHRH when compared to NSS+SAL-pretreated diabetic animals. These results suggest that the blunted GH response to exogenous GHRH observed in streptozotocin-induced diabetic rats may not be due to an increase of endogenous SRIF tone.


Hypothalamic regulation of impaired growth hormone secretion in diabetic rats. 2. Studies in spontaneously diabetic BB Worcester rats.

In the present study we investigated the effects of modulating endogenous somatostatin (SRIF) on the GH response to growth hormone-releasing hormone (GHRH) in spontaneously diabetic BB/Wor rats and nondiabetic littermates. Plasma growth hormone (GH) concentrations following injection of GHRH (500 ng/kg, i.v.) were measured in the rats after pretreatment with either normal sheep serum+saline (NSS+SAL), somatostatin antibody (SRIF-Ab), or pyridostigmine bromide (PD), an acetylcholine esterase inhibitor hypothesized to decrease hypothalamic SRIF tone. The GH response to GHRH in spontaneous diabetic rats pretreated with NSS+SAL was significantly lower (p less than 0.05) than the response observed in the nondiabetic group. SRIF-Ab pretreatment reversed the blunted GH response observed in the diabetic rats. However, PD pretreatment was not effective. These results indicate that the blunted GH response observed in BB/Wor diabetic rats is reversed by neutralization of endogenous SRIF with SRIF-Ab and leads to the conclusion that SRIF plays an active role in modulating GH secretion in spontaneously diabetic rats. The failure of PD to modulate the GH response suggests this acetylcholine agonist is ineffective in this animal paradigm.


Partial characterization of a neurotransmitter pathway regulating the in vivo release of prolactin.

Nicotinic cholinergic, opiate and serotonergic agonists as well as dopaminergic antagonists induce the release of pituitary prolactin. The purposes of the present studies were to determine if nicotine, morphine and the serotonin1A (5-HT1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) utilize a common synaptic pathway to release prolactin and, if so, to establish the serial order of the receptors involved. We also sought to determine whether the pathway under investigation leads to the secretion of prolactin via a mechanism involving dopamine, the prolactin inhibitory factor. Male rats with indwelling jugular catheters were pretreated with saline, mecamylamine, naltrexone, methysergide or bromocriptine. In the saline-treated animals, administration of nicotine, morphine, 8-OH-DPAT and haloperidol resulted in significant increases in plasma prolactin levels. Mecamylamine pretreatment prevented the prolactin response to nicotine only. Naltrexone blocked the stimulation of prolactin release by morphine and by nicotine. Methysergide inhibited the effects of 8-OH-DPAT, morphine and nicotine but not haloperidol. Bromocriptine blocked the prolactin secretion induced by haloperidol as well as by each of the above agonists. Also, in dual-immunocytochemically stained sections, tyrosine hydroxylase-immunoreactive cells and serotonin-immunoreactive processes were detected in close anatomical proximity in the dorsomedial arcuate nucleus. These data indicate that nicotine, morphine and 8-OH-DPAT act to release prolactin via a common synaptic pathway expressing nicotinic cholinergic, opiate, and 5-HT1A receptors at synapses arranged serially in that functional order. Furthermore, the data indicate that the in vivo secretion of prolactin via this pathway may ultimately occur through the inhibition of dopamine release.


Neurotransmitter-induced hypothalamic-pituitary-adrenal axis responsiveness is defective in inflammatory disease-susceptible Lewis rats: in vivo and in vitro studies suggesting globally defective hypothalamic secretion of corticotropin-releasing hormone.

The susceptibility of female Lewis (LEW/N) rats to the development of streptococcal cell wall (SCW)-induced arthritis and other autoimmune phenomena is associated with the inability of their hypothalamic-pituitary-adrenal (HPA) axis to adequately respond to inflammatory stimuli. In contrast, resistance to the development of SCW-induced arthritis and other inflammatory autoimmune manifestations in histocompatible female Fischer rats (F344/N) is related to their intact HPA axis response to inflammatory mediators. To evaluate the mechanism and the specificity of the HPA axis defect in LEW/N rats, we examined the ability of three major excitatory neurotransmitter systems to activate the HPA axis in both Lewis and Fisher rats. The responsiveness of plasma ACTH and corticosterone to the cholinergic muscarinic receptor agonist arecoline, the alpha 1-adrenergic receptor agonist methoxamine and the serotonin (5-HT) type 2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane were significantly blunted and/or abolished in LEW/N compared to F344/N rats. To localize the HPA axis defect to the hypothalamic CRH neuron, we evaluated the ability of explanted hypothalami from the two strains to secrete immunoreactive CRH in vitro, in response to acetylcholine (ACh), norepinephrine (NE), 5-HT and the 5-HT agonist quipazine. LEW/N hypothalami released less immunoreactive CRH (iCRH) in response to ACh, NE, 5-HT and quipazine than F344/N hypothalami. The dose-response curves of these compounds in the former were shifted to the right and/or abolished, suggesting decreased sensitivity of LEW/N hypothalami to these neurotransmitters.(ABSTRACT TRUNCATED AT 250 WORDS)


Opposite effects of neurotensin on dopamine inhibition in different regions of the rat brain: an iontophoretic study.

Anatomical, biochemical and behavioral data suggest functional interactions between dopamine and neurotensin in regions of the brain receiving a co-existent and/or distinct innervation by these two transmitters. We therefore measured and compared the effects of iontophoretically applied dopamine and neurotensin in the prefrontal and anterior cingulate cortex (co-existent innervation) vs the nucleus accumbens and neostriatum (distinct innervation) of urethane-anesthetized rats. In every region, the firing rate of most spontaneously active neurons was depressed by dopamine. Neurotensin had no effect on the same cells, except for a few nucleus accumbens units which were inhibited by the peptide. When dopamine and neurotensin were concomitantly applied, the magnitude of maximal inhibitions induced by dopamine was modified in the majority of neurons tested. A significant decrease in dopamine inhibition was observed in 100% of anterior cingulate, 74% of prefrontal cortex and 48% of accumbens units. On the contrary, in neostriatum, dopamine inhibition was significantly increased in 60% of the units tested. In every region, the remaining neurons showed less than 30% changes in dopamine responsiveness, and were therefore considered unaffected by neurotensin. In the anterior cingulate cortex, inhibitions, respectively, induced by the dopamine D1 agonist, SKF 38393, and the D2 agonist, LY 171555, were also decreased by simultaneous application of neurotensin. Together with currently available data on the cellular localization of neurotensin receptors in rat brain, these results suggest that the modulation of dopamine inhibition by neurotensin may have opposite effects depending on whether the neurotensin receptors are located postsynaptically on target neurons (antagonistic effects) or presynaptically on dopamine terminals (potentiating effects).


Chemoreceptor A-fibres in the human carotid body contain tyrosine hydroxylase and neurofilament immunoreactivity.

Previous retrograde tracing studies on rat and guinea-pig showed a projection of sensory tyrosine hydroxylase-immunoreactive neurons to the region of the carotid bifurcation via the carotid sinus nerve. In the present study, focussing on the sensory innervation of the human carotid body, antisera to tyrosine hydroxylase and other catecholamine synthesizing enzymes were applied for an immunohistochemical investigation of carotid bodies obtained at autopsy. In addition, an array of antisera directed to non-enzyme antigens known to be present in viscero-afferent neurons were incorporated in the study. The glomic lobules consisting of glomus cells and sustentacular cells contained a variable number of enzyme-immunoreactive glomus cells. Arteries were supplied by nerve fibres displaying the full phenotype of sympathetic noradrenergic axons, i.e. immunoreactivity to tyrosine hydroxylase, aromatic-L-amino-acid-decarboxylase and dopamine-beta-hydroxylase. The glomic lobules, however, were densely innervated by tyrosine hydroxylase-immunoreactive axons lacking immunoreactivity to aromatic-L-amino-acid-decarboxylase and dopamine-beta-hydroxylase. These fibres reacted with neurofilament 160kD-antibody but were devoid of immunoreactivity to all neuropeptides tested (calcitonin gene-related peptide, somatostatin, substance P). Ultrastructurally, tyrosine hydroxylase/neurofilament 160kD-immunoreactive axons gave rise to large axonal swellings filled with mitochondria and vesicles, and established extensive contacts to glomus cells. Nerve bundles surrounded by a perineural sheath contained both myelinated (2.0-2.8 microns in diameter) and unmyelinated (0.14-3.0 microns) tyrosine hydroxylase-immunoreactive axons. Most of the unmyelinated immunoreactive axons were running singularly within a Schwann cell-sheath. Judged from the pattern of immunoreactivities as well as their preterminal and terminal ultrastructure, tyrosine hydroxylase-immunoreactive axons innervating glomus cells are of sensory origin. Although final proof by retrograde tracing cannot be presented in man, this conclusion is supported by experimental evidence in laboratory animals. The myelinated immunoreactive axons correspond to chemoreceptor A-fibres whereas the classification of the large unmyelinated immunoreactive axons has yet to be established. The lack of immunoreactivity to the dopamine-synthesizing enzyme, aromatic-L-amino-acid-decarboxylase, in this fibre type does not support the view of dopamine being the primary transmitter of chemoreceptor afferents.


When does Parkinson's disease begin?

A long preclinical or asymptomatic period may occur in Parkinson's disease (PD). Many long-latency parkinsonian syndromes exist. The presence of early-life risk factors is consistent with a long prodromal period. Marked degeneration of the substantia nigra and loss of striatal dopamine are necessary before clinical symptoms develop. Lewy bodies, the histological hallmark of PD, occur in 10% of normal individuals over age 50. Clinical symptoms develop slowly and are often intermittent in early PD. Nonmotor signs, eg, depression or sensory changes, often precede motor signs by many years. Reduction of striatal dopamine can be detected with PET in "at-risk" asymptomatic individuals. Individual sensitivity to drug-induced parkinsonism also suggests a preclinical state. Biologic markers may eventually be able to detect preclinical PD.


Dopamine agonists used as monotherapy in de novo PD patients: comparisons with selegiline.

Studies indicate that selegiline, a monoamine oxidase type B inhibitor, slows progression of Parkinson's disease (PD) and delays the need for levodopa. While dopamine agonists also delay the need for levodopa because of their symptomatic, antiparkinsonian effect, only recently has it been proposed that agonists may also have a protective effect. When dopamine agonists are used as monotherapy in newly diagnosed PD patients, fewer patients improve than on levodopa, but fewer patients develop response fluctuations. This might indicate that dopamine agonists have a protective, as well as a symptomatic, effect, as the lack of response fluctuations may indicate that dopamine agonists protect nigral neurons. Response fluctuations may result from destruction of nigral neurons.


The role of the regulatory enzymes of catecholamine synthesis in Parkinson's disease.

The major neuropathology of Parkinson's disease (PD) is the degeneration of nigrostriatal dopamine (DA), resulting in a deficiency of DA, and of the enzyme tyrosine hydroxylase (TH), which catalyzes the synthesis of L-dopa. The symptomatic treatment of PD consists of replenishing DA by administering L-dopa, which is enzymatically converted to DA in the striatum. The increase of TH activity by modification of the enzyme leads to an increased synthesis of striatal L-dopa, and thereby replenishes the missing DA more efficiently. The activity of TH is increased by protein kinase-dependent phosphorylation of the enzyme or by inhibition of dephosphorylation with specific phosphatase inhibitors. Thus, modification of TH results in an activated form of the enzyme, which might provide a basis for developing new strategies in the treatment of PD. The extraneuronal enzyme, catechol-O-methyl transferase (COMT), inactivates catecholamines by O-methylation, and its inhibition leads to increased levels of striatal DA. The availability of selective and nontoxic COMT inhibitors makes it possible to assess their therapeutic role in treatment of PD. The intraneuronal enzymes, monoamine oxidase (MAO)-A and MAO-B, inactivate catecholamines and other biogenic amines, such as serotonin, by deamination. Inhibition of these enzyme activities leads to increased levels of striatal DA. The irreversible MAO-B inhibitor selegiline was shown to exert antiparkinsonian activity, especially in the early stages of parkinsonism. Selegiline also prevents the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism in MPTP-treated mice and monkeys. Its role in the prevention of the disease is under investigation in several clinical centers.


Kainic acid depresses the ex vivo release of dynorphin B and glutamate from rat hippocampal mossy fiber synaptosomes.

This experiment examined the effects of intracerebroventricularly (i.c.v.) administered kainic acid (KA) on the subsequent ex vivo release of L-glutamate (Glu) and dynorphin B-like immunoreactivity (Dyn B-LI) from isolated rat hippocampal mossy fiber (MF) synaptosomes at 4.5 h, 20 h or 48 h after administration of 0.5 microgram/microliter KA. The Dyn B-LI content in the synaptosomal fraction initially decreased at 4.5 h and then rebounded and remained elevated above control levels at 20 h and 48 h. The K(+)-evoked release of Dyn B-LI from the synaptosomes was markedly depressed at 4.5 h after KA and remained significantly below control levels at 20 h and 48 h. In contrast, KA caused no change in the K(+)-evoked release of Glu at 4.5 h as compared to control levels, but did result in a significant decrease in Glu release at 20 h and 48 h. These data indicate a persistent effect of i.c.v. KA on neurotransmission at MF-CA3 synapses in rat hippocampus, resulting in a suppression of the release of Glu as well as the opioid peptide, Dyn B.