Clonal basis for resurgence of serious Streptococcus pyogenes disease in the 1980s. During the 1980s there was a resurgence of serious Streptococcus pyogenes infections with complications, including rheumatic fever, sepsis, severe soft-tissue invasion, and toxic-shock-like syndrome (TSLS). We have investigated the suggested association between expression of a scarlet fever toxin, SPE A, and systemic toxicity, and the possibility that a new highly virulent clone of S pyogenes has emerged and spread world wide. We studied serotype M1 strains, the serotype most commonly associated with serious complications. 19 isolates from patients with sepsis, with or without TSLS, and 48 from patients with uncomplicated pharyngitis or superficial skin infection were subjected to restriction-enzyme digestion and electrophoresis; 56 isolates (19 serious, 37 uncomplicated disease) were then examined by hybridisation to an speA gene probe. 17 (90%) of the 19 serious-disease isolates had a characteristic ("invasive", I) restriction-fragment profile and were positive for the speA gene. Significantly lower proportions of the isolates from patients with uncomplicated disease had the I profile (21/48 [44%]; p = 0.0035) and speA (20/37 [54%]; p less than 0.001). These findings suggest that the strains from patients with serious disease are a unique clone, which became the predominant cause of severe streptococcal infections in the United States and elsewhere in the late 1980s.


Intestinal epithelial cell protein phosphorylation in enteropathogenic Escherichia coli diarrhoea. The ability of enteropathogenic Escherichia coli (EPEC) to cause diarrhoea in man is associated with the formation of characteristic histopathological lesions in small-intestine enterocytes, with gross cytoskeletal damage and loss of brush-border microvilli. Investigation of enterocyte protein phosphorylation in response to EPEC infection showed that the major phosphorylated protein, identified by immunoprecipitation, is myosin light-chain--an important cytoskeletal protein known to affect actin organisation in non-muscle cells. High enterocyte concentrations of actin and myosin were observed at sites of bacterial infection. Our findings indicate that enterocyte cytoskeletal changes in response to EPEC may be directly triggered by bacterial adherence through signal transduction pathways that stimulate protein kinase activity.


Reversed cerebral asymmetry in women with breast cancer. Altered intrauterine hormonal environment might predispose to both atypical cerebral asymmetry and breast cancer. We therefore investigated computed tomographic scans of 79 right-handed, white patients with breast cancer and 97 controls to assess the pattern of cerebral asymmetry. Women with breast cancer had a reversed pattern of cerebral asymmetry significantly more often than did controls (p less than 0.0001 for both frontal and occipital width. Our findings suggest that an intrauterine or early life factor, probably hormonal, could predispose to breast cancer in adulthood.


Prospective multicentre study of pregnancy outcome after lithium exposure during first trimester. Lithium carbonate is an effective drug for prophylaxis and treatment of major affective disorders. In-utero exposure to lithium during the first trimester of pregnancy might be associated with an increased risk of cardiac malformations, especially the rare Ebstein's anomaly. We prospectively recruited and followed 148 women (mean age 30 years, SD 5 range 15-40) using lithium during the first trimester of pregnancy, who consulted four teratogen information centres in the USA and Canada. Pregnancy outcome was compared with that of controls matched for maternal age. We had complete follow-up of pregnancy outcome in 138 of 148 patients recruited. In the other 10, fetal echocardiograms were available but postnatal follow-up was not done. Mean daily dose of lithium was 927 mg (SD 340). Rates of major congenital malformations did not differ between the lithium (2.8%) and control (2.4%) groups. 1 patient in the lithium group chose to terminate pregnancy after Ebstein's anomaly was detected by a prenatal echocardiogram. There was 1 ventricular septal defect in the controls. Birthweight was significantly higher in the lithium-exposed infants than in the controls despite identical gestational ages (3475 [660] g vs 3383 [566] g, p = 0.02). The true difference in birthweight might have been even larger, since significantly more women using lithium than controls were cigarette smokers (31.8% vs 15.5%, p = 0.002). These results indicate that lithium is not an important human teratogen. Women with major affective disorders who wish to have children may continue lithium therapy, provided that adequate screening tests, including level II ultrasound and fetal echocardiography, are done.


Immune surveillance, organophosphorus exposure, and lymphomagenesis. Prevalence of lymphoproliferative disorders is increased in populations with various chemical exposures, including organophosphorus compounds. Lymphomas are also more common in individuals with a substantially decreased monocyte esterase activity. Organophosphorus compounds inhibit esterases associated with monocytes, natural killer (NK) cells, lymphokine-activated killer (LAK) cells, and cytotoxic T lymphocytes, and these inhibitory effects impair immune surveillance and cytotoxic functions mediated by such cells. Lymphoma development is also associated with Epstein-Barr virus (EBV) and human herpesvirus-6 (HHV-6) infections, which are regulated by cytotoxic immune responses mediated by monocytes, T cells, and NK cells. My hypothesis is that lymphomagenesis is a multistep process, and the absence or inhibition of monocyte esterase and perhaps other immune cell esterases alters esterase-dependent detoxification of a factor critical for the early steps of oncogenesis. Also, such an enzyme deficit might impair the processes that regulate the dissemination and limit the total burden of pathogens such as the lymphoma-associated herpesviruses. An added risk to any viral-mediated lymphoproliferation might be an organophosphorus-induced oncogenic genetic change.


Competitive interactions at [3H]1,3-di(2-tolyl)guanidine (DTG)-defined sigma recognition sites in guinea pig brain. In saturation binding experiments, (+)pentazocine, (+)3-(3-hydroxyphenyl)-N-propylpiperidine (3-PPP), haloperidol and rimcazole did not inhibit the binding of [3H]DTG in a purely competitive fashion. Although Scatchard analysis indicated that [3H]DTG bound to a single site, the inhibition curves of some, but not all, reference compounds exhibited Hill coefficients of less than 0.8. The Scatchard data were consistent with a model of hyperbolic competitive inhibition of binding to the [3H]DTG-defined sigma site, although other possibilities such as negative cooperativity or binding to two sites cannot be definitively excluded. Compounds from numerous pharmacological and structural classes inhibited the binding of [3H]DTG, suggesting that interactions of [3H]DTG with other receptors may have confounded the Scatchard analysis of the binding of [3H]DTG to sigma recognition sites.


Developmental defects of the ear, cranial nerves and hindbrain resulting from targeted disruption of the mouse homeobox gene Hox-1.6. Gene targeting in mouse embryo-derived stem cells has been used to generate mice with a disruption in the homeobox gene Hox-1.6. Mice heterozygous at the Hox-1.6 locus appear normal, whereas Hox-1.6-/Hox-1.6- mice die at or shortly after birth. These homozygotes exhibit profound defects in the formation of the external, middle and inner ears as well as in specific hindbrain nuclei, and in cranial nerves and ganglia. The affected tissues lie within a narrow region along the anteroposterior axis of the mouse but are of diverse embryonic origin. The set of defects associated with the disruption of Hox-1.6 is distinct from and nonoverlapping with that of the closely linked Hox-1.5 gene. But both mutations cause loss, rather than homeotic transformation, of tissues and structures.


Expansion of an unstable DNA region and phenotypic variation in myotonic dystrophy. Myotonic dystrophy is the commonest adult form of muscular dystrophy, with an estimated incidence of 1 per 7,500, although this is likely to be an underestimate because of the difficulty of detecting minimally affected individuals. It is a multisystem autosomal dominant disorder of unknown biochemical basis. No case of new mutation has been proven. We have isolated a human genomic clone that detects novel restriction fragments specific to individuals with myotonic dystrophy. A two-allele EcoRI polymorphism is seen in normal individuals, but in most affected individuals one of the normal alleles is replaced by a larger fragment, which varies in length both between unrelated affected individuals and within families. The unstable nature of this region may explain the characteristic variation in severity and age at onset of the disease. A second polymorphism at this locus is in almost complete linkage disequilibrium with myotonic dystrophy, strongly supporting our earlier results which indicated that most cases are descended from one original mutation.


Detection of an unstable fragment of DNA specific to individuals with myotonic dystrophy. Myotonic dystrophy (DM) is the most common form of adult muscular dystrophy, with a prevalence of 2-14 per 100,000 individuals. The disease is characterized by progressive muscle weakness and sustained muscle contraction, often with a wide range of accompanying symptoms. The age at onset and severity of the disease show extreme variation, both within and between families. Despite its clinical variability, this dominant condition segregates as a single locus at chromosome 19q13.3 in every population studied. It is flanked by the tightly linked genetic markers ERCC1 proximally and D19S51 distally; these define the DM critical region. We report the isolation of an expressed sequence from this region which detects a DNA fragment that is larger in affected individuals than in normal siblings or unaffected controls. The size of this fragment varies between affected siblings, and increases in size through generations in parallel with increasing severity of the disease. We postulate that this unstable DNA sequence is the molecular feature that underlies DM.


Cloning of the essential myotonic dystrophy region and mapping of the putative defect. Myotonic dystrophy is a common dominant disorder (global incidence of 1:8,000) with variable onset and a protean nature of symptoms mainly involving progressive muscle wasting, myotonia and cataracts. To define the molecular defect, we have cloned the essential region of chromosome 19q13.3, including proximal and distal markers in a 700-kilobase contig formed by overlapping cosmids and yeast artificial chromosomes (YACs). The central part of the contig bridges an area of about 350 kilobases between two new flanking crossover borders. This segment has been extensively characterized through the isolation of five YAC clones and the subsequent subcloning in cosmids from which a detailed EcoRI, HindIII, MluI and NotI restriction map has been derived. Two genomic probes and two homologous complementary DNA probes were isolated using the cosmids. These probes are all situated within approximately 10 kilobases of genomic DNA and detect an unstable genomic segment in myotonic dystrophy patients. The length variation in this segment shows similarities to the instability seen at the fragile X locus. The physical map location and the genetic characteristics of the length polymorphism is compatible with a direct role in the pathogenesis of myotonic dystrophy.


Selective inhibition of neurite outgrowth on mature astrocytes by Thy-1 glycoprotein. THY-1, the smallest member of the immunoglobulin superfamily, is a major cell-surface component expressed by several tissues. The protein, carbohydrate and gene structures of this molecule are known, yet its function is not. It is highly expressed in nervous tissue, where it appears on virtually all neurons after the cessation of axonal growth. Here we show that expression of Thy-1 by a neural cell line inhibits neurite outgrowth on mature astrocytes, but not on other cellular substrata which include Schwann cells and embryonic glia. This inhibition of neurite extension on astrocytes can be reversed by low concentrations (nanomolar) of soluble Thy-1. If a similar interaction between neuronal Thy-1 and astrocytes occurs in vivo, it could stabilize neuronal connections and suppress axonal regrowth after injury in the astrocyte-rich areas of adult central nervous system.


High level transient gene expression in human lymphoid cells by SV40 large T antigen boost. High level transient gene expression in lymphoid cells has always been challenging because of the difficulty to efficiently transfect such cells. This has precluded any attempt to clone cDNA encoding proteins by means of their specific biological function in lymphoid cells. We have developed a very efficient transient eukaryotic expression system analogous to the well-known expression system in COS cells. Firefly luciferase and human CD2 genes were used as reporter genes and cloned into the eukaryotic shuttle vector pCDM8 which contains the strong cytomegalovirus promoter and the SV40 origin of replication for autonomous plasmid replication in permissive host cells that express the large SV40 T Antigen. Co-transfection of the reporter plasmids together with an SV40 T Ag expressing plasmid resulted in the several fold amplification of either the Luc activity or the cell surface expression of the CD2 marker in a transient assay. The level of amplification was dependent on the strength of the promoter used to drive the SV40 T Ag expression and was correlated with the extent of autonomous replication of the reporter plasmid in transfected cells. This highly efficient transient gene expression by SV40 T Ag boost was suitable to several human cell lines, making this system of general interest for expression cloning strategies or other gene transfer application that need high level expression.


Identification of a maize nucleic acid-binding protein (NBP) belonging to a family of nuclear-encoded chloroplast proteins. A cDNA encoding a nuclear-encoded chloroplast nucleic acid-binding protein (NBP) has been isolated from maize. Identified as an in vitro DNA-binding activity, NBP belongs to a family of nuclear-encoded chloroplast proteins which share a common domain structure and are thought to be involved in posttranscriptional regulation of chloroplast gene expression. NBP contains an N-terminal chloroplast transit peptide, a highly acidic domain and a pair of ribonucleoprotein consensus sequence domains. NBP is expressed in a light-dependent, organ-specific manner which is consistent with its involvement in chloroplast biogenesis. The relationship of NBP to the other members of this protein family and their possible regulatory functions are discussed.


Cardiostimulatory and antiarrhythmic activity of tubulin-binding agents. Rhythmic, spontaneously pulsating cardiac cells cultured from newborn rats are immediately stimulated to beat faster by addition of a number of tubulin-binding agents but not by their non-tubulin-binding analogues. The tubulin-binding agents tested include vinblastine, vincristine, navelbine, two analogs of vinblastine (S12362 and S12363), nocodazole, colchicine, and podophylotoxin. In addition to binding tubulin, all of the above agents also depolymerize microtubules. In contrast, taxol, a tubulin-binding agent that stabilizes microtubules, does not stimulate cardiac cells. Moreover, the immediate and ensuing cardiac stimulation by vinblastine at 0.05 microgram/ml is completely blocked by pre- and cotreatment with taxol at 1.0 microgram/ml. The time necessary to reverse the cardiostimulatory effect of vinblastine is significantly longer than that required for nocodazole, further implicating depolymerization of microtubules in the cardiac activity of these agents. All of the tubulin-binding agents tested (including taxol) also immediately reverse adriamycin-induced arrhythmias. By using a monoclonal antibody to alpha-tubulin, typical filamentous microtubules are visualized in cardiac muscle and cocultured non-muscle cells by immunofluorescence. When cells are treated for 2 hr with vinblastine at 0.05 microgram/ml, fluorescence is detected in cross-striated patterns in cardiac muscle cells. Overall, these data open the possibility of uncovering an additional relationship between cytoskeletal elements (other than actin and myosin) and the contractility of cardiac muscle. They also suggest an alternative mechanism for affecting cardiac cell function in vitro (namely, by tubulin-binding agents). If these agents are shown to be cardioactive in vivo, they may provide another approach to the treatment and management of cardiac arrhythmias.


p185c-neu and epidermal growth factor receptor associate into a structure composed of activated kinases. The protein product of the neu protooncogene, p185c-neu, is structurally similar to the epidermal growth factor receptor (EGFR). Overexpression of these two receptor tyrosine kinases, but not either separately, leads to transformation and tumorigenicity. Heterodimerization of p185c-neu and EGFR occurs in M1 cells, which express both receptors. We have individually identified the two components of the heterodimer as EGFR and p185c-neu. Analysis of this association with relatively nondenaturing detergents and in the absence of cross-linkers indicates that noncovalent interactions are primarily responsible for heterodimer formation. The rapid reversible heterodimerization was promoted by EGF binding to its receptor. Functionally, the heterodimer is a highly active protein kinase for receptor autophosphorylation and exogenous substrate phosphorylation in vitro. The isolated heterodimer was highly phosphorylated on tyrosine residues in vivo. These results indicate that the physical association between EGFR and p185c-neu is of functional significance and define enzymatic features of complex receptor formation.


T-cell immunity to the joining region of p210BCR-ABL protein. The hallmark of chronic myelogenous leukemia is the translocation of the human c-abl protooncogene (ABL) from chromosome 9 to the specific breakpoint cluster region (bcr) of the BCR gene on chromosome 22. The t(9;22)(q34;q11) translocation results in the formation of a BCR-ABL fusion gene that encodes a 210-kDa chimeric protein with abnormal tyrosine kinase activity. The ABL and BCR genes are expressed by normal cells and thus the encoded proteins are presumably nonimmunogenic. However, the joining-region segment of the p210BCR-ABL chimeric protein is composed of unique sequences of ABL amino acids joined to BCR amino acids that are expressed only by malignant cells. The current study demonstrates that the joining region of BCR-ABL protein is immunogenic to murine T cells. Immunization of mice with synthetic peptides corresponding to the joining region elicited peptide-specific, CD4+, class II major histocompatibility complex-restricted T cells. The BCR-ABL peptide-specific T cells recognized only the combined sequence of BCR-ABL amino acids and not BCR or ABL amino acid sequences alone. Importantly, the BCR-ABL peptide-specific T cells could recognize and proliferate in response to p210BCR-ABL protein. The response of peptide-specific T cells to protein demonstrated that p210BCR-ABL can be processed by antigen-presenting cells so that the joining segment is bound to class II major histocompatibility complex molecules in a configuration similar to that of the immunizing peptide and in a concentration high enough to stimulate the antigen-specific T-cell receptor. Thus, BCR-ABL protein represents a potential tumor-specific antigen related to the transforming event and shared by many individuals with chronic myelogenous leukemia.


Global and local genome mapping in Arabidopsis thaliana by using recombinant inbred lines and random amplified polymorphic DNAs. A population of Arabidopsis thaliana recombinant inbred lines was constructed and used to develop a high-density genetic linkage map containing 252 random amplified polymorphic DNA markers and 60 previously mapped restriction fragment length polymorphisms. Linkage groups were correlated to the classical genetic map by inclusion of nine phenotypic markers in the mapping cross. We also applied a technique for local mapping that allows targeting of markers to a selected genome region by pooling DNA from recombinant inbred lines based on their genotype. We conclude that random amplified polymorphic DNAs, used in conjunction with a recombinant inbred population, can facilitate the genetic and physical characterization of the Arabidopsis genome and that this method is generally applicable to other organisms for which appropriate populations either are available or can be developed.


CD3 zeta dependence of the CD2 pathway of activation in T lymphocytes and natural killer cells. In T lymphocytes, signal transduction through the CD2 adhesion molecule requires surface expression of the CD3-Ti T-cell receptor (TCR) complex. In contrast, in natural killer (NK) cells, CD2 functions in the absence of a TCR. Because the TCR on T lymphocytes and the CD16 low-affinity IgG Fc receptor (Fc gamma receptor type III) complex on NK cells share a common CD3 zeta subunit, we reasoned that CD3 zeta may be critical for CD2 signaling in T lymphocytes and NK cells. Here we show that transfection of a cDNA encoding a transmembrane form of CD16 into TCR- variants of the Jurkat T-cell line results in CD16 expression in association with endogenous CD3 zeta homodimers and restores CD2 signaling function. To test directly the role of CD3 zeta in CD2 triggering, we then transfected human CD2 into each of two murine T-T hybridomas that express TCRs containing either a full-length CD3 zeta subunit or a truncated CD3 zeta subunit incapable of transducing signals. Despite expression of equivalent surface levels of TCR, CD2-mediated signaling is seen only in the T cells containing wild-type CD3 zeta. These findings show that (i) CD16 on NK cells is functionally equivalent to the TCR on T lymphocytes for coupling CD2 to signaling pathways and (ii) CD2 signal transduction depends upon the CD3 zeta subunit of the TCR complex and, by inference, the CD3 zeta subunit of the CD16 complex.


Role of insulin and glucagon in oxytocin effects on glucose metabolism. Oxytocin (OT) infusion in normal dogs increases plasma insulin and glucagon levels and increases rates of glucose production and uptake. The purpose of this study was to determine whether the effects of OT on glucose metabolism were direct or indirect. The studies were carried out in normal, unanesthetized dogs in which OT infusion was superimposed on infusion of either somatostatin, which suppresses insulin and glucagon secretion, or clonidine, which suppresses insulin secretion only. Infusion of 0.2 microgram/kg/min of somatostatin suppressed basal levels of plasma insulin and glucagon and inhibited the OT-induced rise of these hormones by about 60-80% of that seen with OT alone. The rates of glucose production and uptake by tissues, measured with [6-3H] glucose, were significantly lower than those seen with OT alone, and the rise in glucose clearance was completely inhibited. Clonidine (30 micrograms/kg, sc), given along with an insulin infusion to replace basal levels of insulin, completely prevented the OT-induced rise in plasma insulin and markedly reduced the glucose uptake seen with OT alone, but did not reduce the usual increase in plasma glucose and glucagon levels or glucose production. To determine whether the OT-induced rise in plasma insulin was in response to the concomitant increase in plasma glucose, similar plasma glucose levels were established in normal dogs by a continuous infusion of glucose and an OT infusion was superimposed. OT did not raise plasma glucose levels further, but plasma insulin levels were increased, indicating that OT can stimulate insulin secretion independently of the plasma glucose changes. Studies by others have shown that the addition of OT to pancreatic islets or intact pancreas can stimulate insulin and glucagon secretion, indicating a direct effect. Our studies agree with that and suggest that in vivo, OT raises plasma insulin levels, at least in part, through a direct action on the pancreas. These studies also show that OT increases glucose production by increasing glucagon secretion and, in addition, a direct effect of OT on glucose production is likely. The OT-induced increase in glucose uptake is mediated largely by increased insulin secretion.


Taurine uptake into chick B cells.

Taurine uptake into chick B cells. The objective of the present study was to determine whether chick B cells possess a specific transport system for taurine. The Bursa of Fabricius was isolated from newly hatched to 6-week-old chicks and an enriched fraction (86.2%) of B cells was isolated. The chick B cells maintained a high intracellular taurine concentration (0.8-1.12 mM) that decreased with age. The B cells exhibited carrier-mediated and simple diffusion uptake components, but only the carrier-mediated component increased with age. Inhibitor studies indicated taurine uptake was sodium and energy dependent. The data demonstrate that chick B cells possess a specialized taurine transport system and the activity of this system changes during posthatch B cell development.


Rationale for maintenance pharmacotherapy of opiate dependence. At this time, 27 years after the initial studies on development of methadone for the maintenance treatment of opiate addiction were begun, it has been shown that [table; see text] methadone meets most criteria for a pharmacologic agent for chronic treatment of an addiction. It is effective after oral dosing: it has a long biological half-life in humans, it causes minimal side effects when used in chronic treatment, and it has no true toxic effects or serious side effects. Also methadone has been shown to be very effective when appropriately used in programs which combine pharmacotherapy with the best elements of "drug free" treatment, that is, counseling and psychological support. In addition to pharmacological treatment, there should be access to, if not on-site, medical and behavioral care as needed, as well as linkage to resources for various aspects of rehabilitation. At this time many of the actions, as well as the specific sites of action, and mechanisms of actions of methadone as used in chronic treatment of opiate addiction have been defined by scientific experimentation, both at the preclinical and clinical levels. It is known that methadone prevents abstinence symptoms, prevents drug hunger or craving, blocks euphorogenic effects of other opiates, and prevents relapse to illicit use of opiates. It is known that the site of action of methadone is at specific opioid receptors. Research to date suggests that there is no demonstrable down-regulation or up-regulation of opioid receptors during chronic opioid agonist perfusion, although chronic administration of the opioid antagonist naltrexone does appear to up-regulate opioid receptors. Clinical studies show that chronic use of methadone allows normalization of release and peripheral levels of one of the classes of endogenous opioids, beta-endorphin, and the related peptides derived from POMC released and processed from the anterior pituitary in humans. Also levels of beta-endorphin in cerebrospinal fluid become normal during chronic maintenance treatment, reflecting apparently normal processing and release of beta-endorphin at brain or hypothalamic sites of POMC production. Available data from studies of beta-endorphin indicate that there is a [table; see text] normalization, rather than disruption, of the endogenous opioid system in general during steady state administration of methadone, as contrasted with intermittent dosing and then abrupt withdrawal of short-acting opiates such as heroin. Although there is still much to be learned about the neurobiology of opiate addiction, at this time we do know a great deal about the effects of opiates and opioids.(ABSTRACT TRUNCATED AT 400 WORDS)


[Practical problems in the long-term drug treatment of ischemic heart disease and hypertension]. In choosing drugs for treatment of ischemic heart disease or hypertension one has--next to effects on angina or blood pressure--to consider possible influences on long term course. A simple dosing-scheme with a low number of tablets as well as a thorough information of the patient about the prescribed medication are equally important for the therapeutic success.


Subtentorial subdural empyema: case report. We present a case of an extensive subtentorial subdural empyema of otorhinological origin. Although 3%-6% of all intracranial suppurations are infratentorial, there is no report on extensive multiloculated subtentorial empyema so far.


Compartmental distribution of cytochrome oxidase in the striatum of the rat. Endogenous cytochrome oxidase activity was investigated in the adult rat striatum at the light microscope level to see if it was distributed in accordance with the established striatal patch/matrix compartmentalisation. Striatal sections stained to visualise cytochrome oxidase activity were compared with serial sections stained to visualise tyrosine hydroxylase and calbindinD28k-like immunoreactivity, established markers of the matrix compartment. The distribution of endogenous cytochrome oxidase activity was found to coincide with the immunocytochemical staining pattern seen for tyrosine hydroxylase and calbindinD28k whereby areas of intense tyrosine hydroxylase and calbindinD28k-like immunoreactivity (termed the matrix) corresponded to areas of intense cytochrome oxidase activity. Conversely, areas of less intense tyrosine hydroxylase and calbindinD28k-like immunoreactivity (termed patches) corresponded to areas of low cytochrome oxidase activity. In addition, the distribution of two other oxidative enzymes involved in the regulation of mitochondrial respiration, succinic dehydrogenase and NADH-diaphorase, was examined in the striatum and substantia nigra by using histochemical techniques. Both NADH-diaphorase and succinic dehydrogenase histochemistry showed an uneven pattern of neuropil staining in the striatum. In the substantia nigra a few intensely stained cell bodies were seen in the dorsal-lateral tip of the pars reticulata with both histochemical techniques. By using an anti-cytochrome oxidase antibody an abundance of immunoreactive cell bodies and processes were seen in the substantia nigra, particularly in the dorso-medial rim and dorsal tip of the pars reticulata. The substantia nigra pars lateralis contained many intensely stained cytochrome oxidase-like immunoreactive cell bodies and processes.(ABSTRACT TRUNCATED AT 250 WORDS)


Electrophysiological profile of the new atypical neuroleptic, sertindole, on midbrain dopamine neurones in rats: acute and repeated treatment. Sertindole (Lundbeck code No. Lu 23-174) (1-[2-[4-[5-chloro-1-(4-fluorophenyl)-1H-indol-3-yl]-1-piperidinyl] ethyl]-2-imidazolidinone) is a new potential neuroleptic compound. After 3 weeks of treatment sertindole shows an extreme selectivity to inhibit the number of spontaneously active dopaminergic (DA) neurones in ventral tegmental area (VTA) while leaving the number of active DA neurones in substantia nigra pars compacta (SNC) unaffected. Acute injection of apomorphine or baclofen reverse the inhibition of activity seen after repeated treatment with sertindole. This suggests that sertindole induces a depolarization inactivation of the DA neurones. The depolarization inactivation is reversible since normal activity of DA neurones is found in both SNC and VTA after two weeks withdrawal from repeated treatment with a low dose with sertindole. One or two weeks administration of a high dose of sertindole induced only minor effects on the DA neurones in VTA; i.e., in order to obtain the depolarization inactivation sertindole requires 3 weeks of treatment as has also been reported for other neuroleptics. Three weeks of treatment with clozapine induces a selective inhibition of the active DA neurones in VTA but at much higher doses than seen with sertindole, while haloperidol induces a non-selective decrease of spontaneously active DA neurones in both areas. In acute electrophysiological experiments intravenous (i.v.) administration of sertindole--in contrast to both clozapine and haloperidol--neither reverse d-amphetamine- nor apomorphine-induced inhibition of the firing frequencies of DA neurones in SNC or in VTA. In addition, sertindole does not--even in high doses--increase the firing frequency of DA neurones in SNC or VTA.(ABSTRACT TRUNCATED AT 250 WORDS)


Local application of bicuculline potentiates NMDA-receptor-mediated sensory responses of brain noradrenergic neurons. Direct application of bicuculline methiodide (BIC) to noradrenergic locus coeruleus (LC) neurons potently enhanced their sensory responsiveness. This increased responsiveness was due to the long-lasting expression of a new, N-methyl-D-aspartate (NMDA) receptor-mediated component of the synaptic response. This enhancement only occurred when a high stimulus intensity was used to induce the sensory response. A similar increase in responsiveness was observed with stimulation of the nucleus paragigantocellularis (PGi), one of the major direct afferents to LC. This action of BIC was neither mimicked by picrotoxin, penicillin, or the GABA-B antagonist, 2-hydroxy-baclofen, nor by agents that directly depolarize LC neurons. In addition, the inverse agonist of the benzodiazepine receptor, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), did not mimic this effect of BIC. The BIC-potentiated response component was eliminated by direct application of the neurotransmitter gamma-aminobutyric acid (GABA). These results indicate that BIC, acting at a possibly novel site, unmasks NMDA receptors that can be activated by sensory stimuli. This may reflect a mechanism whereby interactions between two major neurotransmitter systems, excitatory amino acids (EAAs) and GABA, potently modulate signal transmission in the brain.


Reduced tyrosine hydroxylase immunoreactivity in locus coeruleus of suicide victims. Antibodies against tyrosine hydroxylase (TH, the rate-limiting enzyme in norepinephrine synthesis) and dopamine beta-hydroxylase (DBH, the last enzyme in the synthesis) were used for immunohistochemical staining of human brain locus coeruleus sections, obtained postmortem from suicide victims and matched controls. Stain density over individual cells was quantified by a computerized, video-camera-based image analysis system. Mean stain density for TH was significantly lower (by about 30%) in the locus coeruleus of suicide victims. There was no difference between suicides and controls in DBH immunoreactivity or in the number of TH immunoreactive cells. Reduced TH availability, either genetically or environmentally determined, may contribute to the noradrenergic insufficiency postulated to occur in depression and the increased beta-adrenergic receptor concentrations observed in prefrontal cortex of suicide victims.


The current status of the Whipple operation for periampullary carcinoma. In an address delivered before the Boston Surgical Society in 1942, Whipple made the following statement: The radical operation for these tumors of the ampullary region and pancreas, based on the principle of wide, en bloc removal of the tumors, as required in modern cancer surgery, is evidently in an evolutionary stage. Many more cases, with five-year survivals, will be required before valid claims can be made for the operations as done at present. In the nearly 50 years since this address, the Whipple operation has undergone numerous modifications and technical refinements. A number of recent clinical studies clearly demonstrate that this procedure now can be performed with acceptable morbidity and mortality, and that long-term survival is possible even for patients with adenocarcinoma of the head of the pancreas. The decline in operative morbidity and mortality has been achieved largely through advances in surgical technique. Further improvements in survival for patients with periampullary tumors are likely to occur through the development of more effective adjuvant therapy and improved understanding of the biologic behavior of these tumors.


Assessment of QT dispersion in symptomatic patients with congenital long QT syndromes. It has been suggested that QT dispersion recorded on the surface electrocardiogram may be a predictor of arrhythmic events in patients with congenital QT prolongation. To evaluate this, 9 patients (6 female, mean age 17.6 years) with congenital long QT syndromes, all of whom had syncope and documented torsades de pointes, were studied. Patients were studied off treatment and during therapy with beta-blocking agents. Three patients were also studied after left stellate ganglionectomy. An age-matched control group was also studied. Good quality 12-lead electrocardiograms were recorded from all patients. For each lead, QT and RR intervals were measured, and QTc value was calculated. QT and QTc dispersions were calculated for each patient. Patients had a significantly longer mean QT interval compared with that of the control group (450 +/- 100 vs 359 +/- 63 ms; p = 0.015) at similar mean RR intervals (736 +/- 231 vs 783 +/- 289 ms), with a longer mean QTc value (0.53 +/- 0.08 vs 0.41 +/- 0.02 s1/2; p = 0.004). Patients also had longer QT and QTc dispersions compared with those of the control group (110 +/- 45 vs 43 +/- 12 ms [p = 0.004], and 0.108 +/- 0.03 vs 0.05 +/- 0.02 s1/2 [p = 0.002], respectively). QT and QTc dispersions on and off beta-blocking agents were not significantly different. Comparing patients with frequent and those with infrequent symptoms, there was no difference in QT or QTc dispersion either off treatment or during therapy with beta-blocking agents.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular characterization of hemoglobin C in Sicily. Analysis of polymorphisms of the beta-globin gene cluster was performed on 12 families and on one unrelated individual of Sicilian origin who carried hemoglobin C (Hb C). Two different haplotypes were found in association with beta c Sicilian alleles, corresponding to haplotypes I and II previously described in American blacks. In our population, the more frequent one (haplotype I) was linked to the lack of a polymorphic HpaI site 3' to the beta gene (13.0-kb fragment), similarly to haplotype I in blacks, while the less frequent one was linked to a 7.0-kb HpaI fragment attributable to a site that had never been previously described in linkage with beta c alleles. In Italy, these two haplotypes have been found in rare cases in association with beta A alleles. These findings provide new insights into the origin of Hb C present in Sicily, suggesting that (1) the beta c mutation detected in Sicily derived from African black chromosomes and does not represent a new mutation; and (2) Hb C may have originated either by multiple mutational events on separate chromosomes or by mutation in the HpaI site 3' to the beta gene in a pre-existing beta c chromosome.


Serum prolactin levels in homosexual and bisexual men with HIV infection. Prolactin is a neurohormone that may be secreted in response to stress and also has regulatory effects on the immune system. Some, but not all, studies suggest that prolactin levels are higher than normal in persons with HIV infection. The authors measured prolactin levels in HIV-positive and HIV-negative homosexual and bisexual men to assess possible differences in levels and then examined relationships between prolactin level and measures of medical status, anxiety, depression, stress, and neuropsychological test performance. Blood for prolactin level determination was obtained from 121 HIV-seropositive and 79 HIV-seronegative homosexual and bisexual men enrolled in a longitudinal study. The men also underwent a daylong assessment that included medical, immunological, psychiatric, psychosocial, psychosexual, and neuropsychological evaluations. There was no statistically significant difference in serum prolactin level among the seronegative men, the seropositive men with no or minimal physical symptoms, and the seropositive men with significant physical symptoms of HIV infection. Furthermore, within the HIV-seropositive group, the correlations between serum prolactin level and measures of depression, anxiety, stress, and neuropsychological test performance were all nonsignificant. Serum prolactin level does not seem to respond to HIV infection or to be related to stress or psychiatric symptoms in HIV-infected men. As none of the subjects had AIDS, the possibility cannot be ruled out that prolactin level increases in very late stages of HIV infection.


Racial/ethnic identity and amount and type of psychiatric treatment. The purpose of this study was to examine the relationship of racial/ethnic identity to the amount and type of psychiatric treatment received by white, black, Latino, and Asian patients in the Los Angeles County mental health system. The patients studied (N = 19,400) consisted of all adult inpatients and outpatients seen in all county mental health facilities between January 1983 and August 1988. Multiple regression analysis was used to test the relationship between race/ethnicity and four measures of treatment received: number of treatment sessions, treatment modality, treatment setting, and therapist's discipline. The covariates included in the analyses were age, sex, socioeconomic status, primary language, diagnosis, and measures of treatment when these were logical predictors and were not acting as dependent variables. Race/ethnicity did not have a consistent significant relationship to the treatment variables studied. However, diagnosis had a consistent and highly significant relationship to all four measures of treatment. A psychotic diagnosis was related to receiving more treatment sessions, greater use of medication, greater use of inpatient treatment, and less treatment by a professional therapist. Socioeconomic status and primary language also had consistent and significant relationships to three of the treatment variables. In considering modifications to the service delivery system, clinicians must evaluate whether the type of treatment provided to psychotic patients is the treatment of choice in terms of effectiveness and efficiency or whether it involves bias in service delivery. Similarly, the issue of bias in treatment of lower socioeconomic patients must be addressed.


Vitamin E in the treatment of tardive dyskinesia: a double-blind placebo-controlled study. The authors compared vitamin E with placebo in a double-blind randomized crossover study of 27 patients with tardive dyskinesia. Each treatment period lasted for 6 weeks. Vitamin E showed no differences from placebo in the treatment of tardive dyskinesia.


[The effect of sufentanil on regional and global cerebral circulation and cerebral oxygen consumption in the dog]. The intracranial hemodynamic and metabolic effects of 20 micrograms/kg sufentanil were studied in ten mongrel dogs. Anesthesia was maintained with 0.7 vol.% end-tidal isoflurane and 50% nitrous oxide in oxygen. Catheters were inserted into both femoral arteries and veins, the superior sagittal sinus, the left atrium, and the lateral cerebral ventricle for blood pressure measurement, arterial and sagittal sinus blood sampling, radioactive microsphere injections, and intracranial pressure (ICP) monitoring. Cardiac output (CO) was measured using an electromagnetic flow probe on the pulmonary artery. Following baseline measurements, sufentanil was injected and data were recorded at 5, 15, and 30 min. RESULTS. In group 1 (n = 5) blood pressure was not controlled, while in group 2 (n = 5) blood pressure was maintained at baseline level with a phenylephrine infusion. Arterial blood pressure decreased by 32% in response to sufentanil in group 1 and remained constant in group 2 according to the protocol. CO decreased by 40%-50% in both groups. Regional and global cerebral blood flow (CBF) decreased by 25%-40% with no difference between groups. The cerebral hemodynamic changes were associated with a decrease of 35%-40% in cerebral oxygen consumption. ICP did not change over time. DISCUSSION. These data are in contrast to studies in dogs, where sufentanil produced non-dose-dependent increases in CBF and ICP. Our results are more consistent with studies in humans and rats where administration of sufentanil was associated with either no change or decreases in cerebral hemodynamics, metabolism, and ICP. We conclude that in dogs with normal intracranial physiology sufentanil decreases regional and global CBF in response to a decrease in cerebral metabolic demand without significantly affecting ICP.


Fatal rodenticide poisoning with brodifacoum. The increased prevalence of rodents resistant to warfarin led to the development of the hydroxycoumarin anticoagulant brodifacoum. A 25-year-old man attempted suicide by consuming four boxes of d-CON Mouse-Prufe II; each box contains 42 g of bait that is 0.005% brodifacoum. He presented to a hospital nine days later with syncope, hematochezia, gross hematuria, epistaxis, anemia, and a severe coagulopathy. Radiographic studies were consistent with pleural, pericardial, and mediastinal hemorrhages. Vitamin K and fresh frozen plasma were given, and he was later discharged on oral phytonadione (vitamin K1). The patient's coagulopathy recurred, necessitating multiple plasma transfusions and prolonged treatment with oral phytonadione. Fifteen weeks after hospital discharge, he presented again with a history of additional brodifacoum ingestion. Neurologic status was initially normal, but in the emergency department he suddenly became comatose soon after emesis was induced with syrup of ipecac. Computed tomography of the brain revealed a subarachnoid hemorrhage that led to brain death less than 24 hours later. This case demonstrates the severe and prolonged coagulopathy that can result from ingestion of brodifacoum, a compound that has a toxic potency about 200-fold that of warfarin and a half-life as much as 60 times longer.


Specificity and mode of action of the muscle-type protein-arginine deiminase. The primary and secondary specificities and mode of action of the muscle-type protein-arginine deiminase (PAD) were investigated using various derivatives of Arg and its homologues, as well as Arg-containing peptides by quantitative analyses of the reaction products on reverse-phase HPLC. The enzyme converted benzoyl-D-Arg-p-nitroanilide into its citrulline derivative at 18% of the rate of the L-isomer, while the D-Arg residues in peptides were not deiminated to a significant extent. This suggests that PAD does not have strict stereospecificity and it is dependent on the structure of the residues or groups on both sides of the target Arg residue. In contrast, the benzoyl-/-ethyl ester derivatives of homoarginine, alpha-amino-beta-guanidino-propionic acid, canavanine, and NG-methyl-Arg, exhibited poor PAD susceptibility, suggesting that the length and nature of the arm as exactly three CH2 groups, and the integrity of the guanidyl group are quite strict specificity determinants. The enzyme action on Arg residues in peptides depends greatly on their position in the sequence, and on the nature of the neighboring residues. For example, deimination of Arg residues situated at positions 1-3 from the NH2-terminus, except for those preceded by a carbobenzoxy- or benzoyl-group, were in most cases very slow, whereas those at the COOH-terminus were deiminated relatively faster. A single Arg residue sandwiched between two Pro residues was not deiminated at all, while a pair of Arg residues between two Pro were deiminated moderately. Consequently, PAD exhibited a variety of modes of action on more than one Arg residues in the peptides tested. The results suggest the applicability of PAD, albeit quite limited, for selective modification of certain Arg residues in peptides and proteins by appropriately controlling reaction time and several other parameters. The PAD's mode of action was compared with those of three Arg-bond cleaving proteases.


Pharmacokinetics and antibacterial activity of daily gentamicin. Twenty full term neonates with suspected bacterial infection were randomly assigned to a once daily or a twice daily dosage regimen with gentamicin (4 mg/kg/day). Concomitantly all patients were treated with ampicillin (200 mg/kg/day). The gentamicin concentration time curves were analysed by an open two compartment model under steady state conditions on day 4 of treatment. The mean theoretical maximum serum concentration in the group taking gentamicin once daily (10.9 micrograms/ml) was significantly higher than in the group taking it twice daily (7.4 micrograms/ml). Potentially toxic serum concentrations were never reached. Mean trough concentrations were comparable in both groups (once daily 0.8 micrograms/ml; twice daily 1.0 micrograms/ml). Urinary alanine aminopeptidase excretion increased during and even two days after end of treatment in both groups without any significant differences. The results of the dynamic in vitro model revealed that both dosage schedules showed comparable bactericidal effects on pathogens inhibited by low concentrations of gentamicin like Escherichia coli and Staphylococcus aureus. However the once daily regimen was significantly superior in isolates with high minimal inhibitory concentrations.


Demonstration of passenger leukocytes in a case of Epstein-Barr virus posttransplant lymphoproliferative disorder using restriction fragment length polymorphism analysis. Lymphocytic populations of the posttransplant lymphoproliferative disorder in a completely matched renal allograft and a recipient's regional lymph node were examined, using restriction fragment length polymorphism analysis with probe YNH24 to the variable number tandem repeat D2S44. The donor's DNA was found in lymphocytes extracted from both the grafted kidney and the recipient's lymph node 26 days after engraftment. In both these organs, the results of in situ hybridization with a terminally biotin-labeled oligonucleotide probe to the Not I tandem repeat region of the Epstein-Barr virus (EBV) genome, as well as those of Southern blot hybridization to the EBV nuclear antigen region of the EBV genome, were positive. These findings confirmed the presence of the donor's lymphocytes ("passenger leukocytes") in the host nodal tissue in human renal transplantation and implicated EBV as playing a role in the development of posttransplant lymphoproliferative disorder. It is speculated that the EBV proliferative stimulus contributed to the recipient and the donor lymphocyte expansions. Alternatively, the proliferation of both lymphocyte populations could result from a mutual stimulation by minor histocompatibility or other antigens.


Global ischaemia induces a biphasic response of the mitochondrial respiratory chain. Anoxic pre-perfusion protects against ischaemic damage. Studies of Langendorff-perfused rat hearts have revealed a biphasic response of the mitochondrial respiratory chain to global ischaemia. The initial effect is a 30-40% increase in the rate of glutamate/malate oxidation after 10 min of ischaemia, owing to an increase in the capacity for NADH oxidation. This effect is followed by a progressive decrease in these oxidative activities as the ischaemia is prolonged, apparently owing to damage to Complex I at a site subsequent to the NADH dehydrogenase component. This damage is exacerbated by reperfusion, which causes a further decrease in Complex I activity and also decreases the activities of the other complexes, most notably of Complex III. Perfusion for up to 1 h with anoxic buffer produced only the increase in NADH oxidase activity, and neither anoxia alone, nor anoxia and reperfusion, caused loss of Complex I activity. Perfusing for 3-10 min with anoxic buffer before 1 h of global ischaemia had a significant protective effect against the ischaemia-induced damage to Complex I.


Interaction between secretory leucocyte proteinase inhibitor and bronchial mucins or glycopeptides. Physiopathological implications for the protection of mucins against proteolysis by human leucocyte elastase. The interaction of secretory leucocyte proteinase inhibitor with bronchial mucins and glycopeptides was studied by means of c.d. spectroscopy. The interaction with mucins was characterized by an increase in organized structure of alpha-helical type, as evidenced by the appearance in the difference spectra of two positive bands at 208 and 218 nm. This phenomenon was correlated with the amount of inhibitor present in the mixtures, suggesting that the change was inherent to the inhibitor. Surprisingly, when the inhibitor was mixed with acid glycopeptides, difference c.d. spectra showed a decrease in organized structure, characterized by a negative minimum at 196 nm. Glycopeptides treated with neuraminidase gave similar profiles of difference spectra in three different mixtures, indicating that the interaction was smaller. The interaction between the inhibitor and mucins was also studied for its ability to modify in vitro the proteolytic activity of human leucocyte elastase. Mucins alone were degraded by that proteinase into glycopeptides of Mr 400,000-500,000, whereas mucins mixed with inhibitor before adding elastase were proteolysed to a lesser extent. These data demonstrate that the secretory leucocyte proteinase inhibitor interacts with mucins and consequently is capable of protecting the mucins against proteolysis by elastase.


Novel genetic markers of rheumatoid arthritis in Chilean patients, by DR serotyping and restriction fragment length polymorphism analysis. The analysis of genetic markers of rheumatoid arthritis (RA) in a population in which the DR4 serotype is not strongly associated with the disease. Chilean RA patients (56 seropositive and 22 seronegative) and 141 controls were studied by serotyping. Southern blot analysis of Bam HI restriction fragment length polymorphism (RFLP) was done in genomic DNA from 46 patients with seropositive RA, 17 patients with seronegative RA, and 45 controls, using a complementary DNA probe specific for DRB1 genes. The prevalence of the HLA-DR9 haplotype was strikingly higher in seropositive RA patients (21%) than in controls (3%) (Pcorr less than 0.0008, by Fisher's exact test; relative risk [RR] = 9.34). The prevalence of DR4 and DR1 haplotypes, although slightly increased, did not achieve a significant preponderance. The simultaneous presence of two Bam HI fragments (3.6 kb and 4.5 kb) was found with higher prevalence in seropositive patients (83%; RR = 9; Pcorr less than 0.00002) than in controls (36%), and seemed higher in seronegative RA patients as well (71%; RR = 4). Furthermore, its prevalence remained increased in comparisons of DR4 positive controls (36%) with DR4 positive seropositive patients (100%; RR = 67; Pcorr less than 0.0002) and DR4 positive seronegative patients (100%; RR = 36; Pcorr less than 0.006), even after excluding the DR9 positive individuals. A tendency toward higher association with DR1 seropositive RA patients (67%; RR = 12), a group with no DR4 or DR9 positive individuals, than in DR1 positive controls (14%), was also observed. The HLA-DR9 haplotype was definitively consolidated as a very strong genetic marker exclusively for seropositive RA in Chilean patients, as suggested by our previous observations. RFLP analysis showed that the simultaneous presence of 3.6-kb and 4.5-kb Bam HI fragments constituted a better RA marker than did any of the heretofore studied haplotypes. These fragments together would be linked to RA independently of the DR1, DR4, and DR9 haplotypes. The overall evidence indicates that Chilean seropositive RA patients display a genetic background that is different from that underlying RA susceptibility in other populations and suggests the existence of common, as well as distinct, genetic elements predisposing to seronegative and seropositive RA.


Inactivation of placental factor XIIIa by acrylamide. Acrylamide rapidly and irreversibly inactivates thrombin activated Factor XIIIa, without affecting neither the intact zymogen nor its proteolytic activation. The inactivation is strictly dependent on the presence of calcium ions and is accompanied by a decrease in the number of free and total thiol residues, suggesting that cysteine residue(s), whose reactivity is modulated by calcium, is (are) probably responsible for the acrylamide-directed inactivation.


Autoradiographic localization of dopamine uptake sites in the rat brain with 3H-GBR 12935. The regional distribution of dopamine (DA) uptake sites in the rat brain has been studied by quantitative autoradiography using [3H]GBR 12935 as a ligand. The binding of [3H]GBR 12935 to striatal sections was saturable and of high affinity (Kd = 1.6 nM); it occurred at a single population of sites and possessed the pharmacological features of the DA uptake sites. The highest densities of [3H]GBR 12935 binding sites were found in the caudate-putamen, nucleus accumbens, olfactory tubercle, ventral tegmental area and substantia nigra (especially in the pars compacta). Moderate levels of [3H]GBR 12935 binding were observed in globus pallidus, thalamus, hypothalamus, hippocampus, amygdala (basolateral nucleus) and prefrontal and singular cortices. This regional distribution of [3H]GBR 12935 binding closely correlated with the reported distribution of dopaminergic nerve terminals. The topographical distribution of [3H]GBR 12935 has also been studied in detail in striatal subregions and this distribution was compared, using quantitative TH immunoreactivity, to the density of striatal dopaminergic nerve terminals. There is good overlapping between these two regional distributions, the highest density of both markers was found in the lateral part of the striatum and a similar rostro-caudal gradient has been observed. A dopaminergic denervation caused a complete loss of [3H]GBR 12935 in basal ganglia ipsilateral to the lesion.


Risks versus benefits of inhaled beta 2-agonists in the management of asthma. The therapeutic goal for the treatment of asthma should be to suppress bronchial mucosal inflammation with preventive drugs such as inhaled corticosteroids, and to relieve symptoms of wheezing and breathlessness with bronchodilator drugs. The lower recommended doses of inhaled beta 2-agonists produce rapid effective bronchodilatation without systemic adverse effects; higher doses may produce substantial improvements in airway response which may help patients with more severe airflow obstruction. Higher doses of inhaled beta 2-agonists also cause dose-related systemic adverse beta 2 effects including tremor, tachycardia, hypokalaemia and associated electrocardiographic sequelae. In this respect, although fenoterol appears to cause greater extrapulmonary beta 2-mediated adverse effects at higher doses, there is no evidence to suggest that it is any less beta 2-selective. There is also some evidence to suggest that use of regular inhaled beta 2-agonists may cause increased bronchial hyperreactivity and possibly deterioration in disease control. Patients who require such regular use should therefore be given additional anti-inflammatory therapy with inhaled corticosteroids. The recent availability of novel, longer-acting inhaled beta 2-agonists such as salmeterol and formoterol will also make necessary a careful reappraisal of their long term use in patients with asthma.


The contribution of the different binding sites of the N-methyl-D-aspartate (NMDA) receptor to the expression of behavior. The effects of competitive (CGP 37849 and CGP 39551) and non-competitive (dizocilpine) N-methyl-D-aspartate (NMDA) antagonists were tested in three animal models (catalepsy, sniffing, locomotion) and, in addition, the modulation of these effects by an agonist of the strychnine-insensitive glycine binding site was investigated. Both competitive and non-competitive NMDA antagonists reduced neuroleptic-induced catalepsy. Weak sniffing was induced by the competitive antagonist but strong sniffing by the non-competitive NMDA antagonist. Due to muscle relaxation the competitive antagonist reduced locomotion, in contrast to stimulation of locomotor activity induced by the non-competitive NMDA antagonist. The glycine agonist (D-cycloserine) potentiated the effects of the non-competitive but antagonized those of the competitive NMDA antagonist.


An inside look at a small cell. Yeast Cell Biology sponsored by Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, August 13-18, 1991. It should be apparent from this review that new genes important for cell function are rapidly being discovered in yeast. In the not too distant future, the sequence of the yeast genome will provide us with a list of all of the proteins needed to make a cell. The challenge will then be to continue to develop biochemical and genetic approaches to uncover the function of these proteins. Clearly, yeast is going to play a major role in the future of cell biology and the power of this small eukaryote is just beginning to be tapped.


Development shows some backbone. HFSP Workshop on Genetic Control of Vertebrate Development cosponsored by the Human Frontier Science Program, European Science Foundation, and European Molecular Biology Organization, Les Diablerets, Switzerland, May 26-30, 1991. This meeting aptly illustrated the power of a combined analysis of development in a range of vertebrate systems. Each system has its own inherent strengths: the mouse has gene transfer technology and targeted mutagenesis, the frog and chick have experimental embryology, and the zebrafish has genetics. It is the synergistic effect of considering all of these systems in combination that is without measure. In the past, the study of vertebrate development has been relegated to a largely descriptive phase. Initially, this was through analysis of morphological changes taking place during development. More recently, this has taken the form of cataloging the expression patterns of genes transcribed in development. It is clear that we are now entering an era when a functional analysis of development can get underway.


Sensational science. Sensory Transduction: 45th Annual Symposium of the Society of General Physiologists, Marine Biological Laboratory, Woods Hole, MA, USA, September 5-8, 1991. In the course of several days of formal and informal talks, in the idyllic setting of Woods Hole, the impression grew among many of the participants that useful common themes have emerged for comparison among sensory transduction systems. Many of these were made explicit in a talk on biophysical principles of sensory transduction by Steven Block (Cambridge, MA, USA). In one hour, Block summarized the rest of the symposium and much more, in a dazzling tour through the senses. One of his points was that all sensory transducers must fulfill common goals: detection of the signal, which involves the functions of collecting, selecting or tuning, and capture of the stimulus; amplification, to raise the signal energy (without adding noise) for transmission to other parts of the organism; adaptation or feedback, to extract behaviorally useful parts of the signal; termination, to re-prime the system for the next signal; and encoding, which puts the information in a useful form for downstream processing or effector elements. Another useful comparison was between quantum-detecting systems, such as photoreception and olfaction, where the energy of the stimulus quantum (photon or odor ligand) is large and a uniform response is desired, and noise-limited systems, such as auditory transduction or magnetoreception, where thermal noise is larger than the smallest stimuli and time-averaging helps pull the signal out of the noise. A third observation from Block was that sensory transduction systems--while often performing at physical limits--have not necessarily been perfected by the process of evolution.(ABSTRACT TRUNCATED AT 250 WORDS)


Downstream of the homeotic genes.

Downstream of the homeotic genes. The homeotic genes of Drosophila melanogaster determine which structures form in each of the body segments. Disrupting the function of the homeotic genes causes body parts found in one domain of the animal to be replaced by body parts normally found elsewhere. Each of the homeotic genes encodes a protein, or a closely related family of proteins, which is capable of binding DNA and controlling the transcriptional activities of downstream genes. The homeotic genes are in the middle of a complex regulatory network, and many of the genes that control homeotic expression have been well characterized. However, very little is known about what comes after the homeotic genes, the downstream genes whose activities are regulated by the homeotic genes. Here, we review the known relationships between the homeotic proteins and the few identified target genes. The details of these interactions may be characteristic and may thus guide the search for additional targets.


5-Chlorolevulinate modification of porphobilinogen synthase identifies a potential role for the catalytic zinc. Porphobilinogen synthase (PBGS) is a Zn(II) metalloenzyme which catalyzes the asymmetric condensation of two molecules of 5-aminolevulinate (ALA). The nitrogen of the first substrate ends up in the pyrrole ring of product (P-side ALA); by contrast, the nitrogen of the second substrate molecule remains an amino group (A-side ALA). A reactive mimic of the substrate molecules, 5-chlorolevulinate (5-CLA), has been prepared and used as an active site directed irreversible inhibitor of PBGS. Native octameric PBGS binds eight substrate molecules and eight Zn(II) ions, with two types of sites for each ligand. As originally demonstrated by Seehra and Jordan [(1981) Eur. J. Biochem. 113, 435-446], 5-CLA inactivates the enzyme at the site where one of the two substrate molecules binds, and modification at four sites per octamer (one per active site) affords near-total inactivation. Here we report that 5-CLA-modified PBGS (5-CLA-PBGS) can bind up to four substrate molecules and four Zn(II) ions. Contrary to the conclusion of Seehra and Jordan, we find that the preferential site of 5-CLA inactivation is the A-side ALA binding site. On the basis of the dissociation constants, the metal ion binding sites lost upon 5-CLA modification are assigned to the four catalytic Zn(II) sites. 5-CLA-PBGS is shown to be modified at cysteine-223 on half of the subunits. We conclude that cysteine-223 is near the amino group of A-side ALA and propose that this cysteine is a ligand to the catalytic Zn(II). The vacant substrate binding site on 5-CLA-PBGS is that of P-side ALA. We have used 13C and 15N NMR to view [4-13C]ALA and [15N]ALA bound to 5-CLA-PBGS. The NMR results are nearly identical to those obtained previously for the enzyme-bound P-side Schiff base intermediate [Jaffe et al. (1990) Biochemistry 29, 8345-8350]. It appears that, in the absence of the catalytic Zn(II), 5-CLA-PBGS does not catalyze the condensation of the amino group of the P-side Schiff base intermediate with the C4 carbonyl derived from 5-CLA. On this basis we propose that Zn(II) plays an essential role in formation of the first bond between the two substrate molecules.


Hemophilia B caused by five different nondeletion mutations in the protease domain of factor IX. Factor IX is a multidomain protein and is the proenzyme of a serine protease, factor IXa, essential for hemostasis. In this report, we describe the molecular basis of hemophilia B (deficiency of factor IX activity) in five patients who have neither deletions nor rearrangements of the factor IX gene. By enzymatic amplification and sequencing of all exons and promoter regions, the following causative mutation in the protease domain of factor IX was identified in each patient: IXSchmallenberg: nucleotide 31,215G----T, Ser365Ile; IXVarel: nucleotide 31,214A----G, Ser365Gly; IXMechtal: nucleotide 31,211G----C, Asp364His; IXDreihacken: nucleotide 30,864G----A, Arg248Gln; and IXMonschau: nucleotide 30,855A----T, Glu245Val. In IXVarel, nucleotide 31,213T was also replaced by C, which results in a silent mutation (GAT----GAC) at Asp-364. Thus, this patient has a double base-pair substitution of TA to CG at nucleotides 31,213 and 31,214 but only a single amino acid change of Ser-365 to Gly. This patient also developed an antibody to factor IX during replacement therapy, which suggests that deletion of the factor IX gene is not necessary for development of the antibody in hemophilia B patients. The levels of plasma factor IX antigen in the patients ranged from 40% to 100% except for IXDreihacken (Arg248Gln), in which case it was approximately 4% of normal. The Ser365Gly and Ser365Ile mutants are nonfunctional because of lack of the active site serine residue. Mutant Asp364His is inactive because it cannot form the hydrogen bond between the carboxylate group of Asp-364 and the alpha-amino group of Val-181 generated after activation. As observed in other homologous serine proteases, this hydrogen bond is essential for maintaining the correct active site conformation in normal factor IXa (IXaN). Purified Arg248Gln had approximately 41% and Glu245Val had approximately 17% of the activity of normal factor IX (IXN) in a partial thromboplastin time (aPTT) assay. In immunodot blot experiments, the isolated Glu245Val mutant did and the Arg248Gln mutant did not bind to an anti-IXN monoclonal antibody that has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. We have recently shown that a high-affinity calcium binding site exists in the protease domain of IXN; among the proposed Ca(2+)-binding ligands is the carboxyl group of Glu-245. Further, a part of the epitope for the above antibody was shown to be contained in the 231 to 265 residue segment of factor IX.(ABSTRACT TRUNCATED AT 400 WORDS)


Inhibition of normal B-cell function by human immunodeficiency virus envelope glycoprotein, gp120. Despite the occurrence of hypergammaglobulinemia in human immunodeficiency virus (HIV) infection, specific antibody production and in vitro B-cell differentiation responses are frequently impaired. In this study, we have examined the effects of HIV envelope glycoprotein gp120 on T-helper cell function for B cells. In the culture system used, B-cell functional responses were dependent on T-B-cell contact, since separation of T and B cells in double chambers by Transwell membranes rendered the B cells unresponsive in assays of antigen-induced B-cell proliferation and differentiation. Cytokines secreted by T cells were also essential, since anti-CD3 monoclonal antibody (mAb)-activated, paraformaldehyde-fixed T-cell clones failed to induce B-cell proliferation and differentiation. Pretreatment of the CD4+ antigen-specific T cells with gp120 was found to impair their ability to help autologous B cells, as determined by B-cell proliferation, polyclonal IgG secretion, and antigen-specific IgG secretion. The gp120-induced inhibition was specific in that it was blocked by soluble CD4. Furthermore, only fractionated small B cells (which are T-cell-dependent in their function) manifested impaired responses when cultured with gp120-treated T cells. Antigen-induced interleukin (IL)-2 and IL-4, but not IL-6, secretion were markedly reduced in gp120-treated T-cell clones. Addition of exogenous cytokines failed to compensate for defective helper function of gp120-treated T cells. The findings in this study indicate that gp120 impairs helper functions of CD4+ T cells by interfering with T-B-cell contact-dependent interaction; the inhibitory effects of soluble envelope proteins of HIV may contribute to the immunopathogenesis of the HIV-associated disease manifestations.


Testicular function in patients with hypospadias associated with enlarged prostatic utricle. Of 485 patients with hypospadias who underwent urethrography, enlarged prostatic utricles were found in 173 (35.7%). Utricles with a higher grade of enlargement were seen mainly in patients with a severe degree of hypospadias. Serum testosterone levels before and after 3 days' treatment with human chorionic gonadotrophin were determined by radioimmunoassay in 97 prepubertal boys with hypospadias and enlarged prostatic utricles. Lower levels of serum testosterone were found after hormonal stimulation in 22 patients with high grade utricular enlargement when compared with 5 controls and 75 patients with low grade enlargement; the difference was statistically significant. This finding supports the hypothesis that utricular enlargement may be due to androgenic insufficiency during the critical period of organogenesis.


Effect of alpha-adrenergic blockade with prazosin on large coronary diameter during exercise. Exercise-induced dilation of coronary resistance vessels is limited by alpha-adrenergic mechanisms. However, the effect of alpha-adrenergic mechanisms on large coronary arteries during exercise is not known. In the present study, sonomicrometry was used to measure circumflex coronary arterial diameter during treadmill exercise before and after alpha 1-adrenergic blockade with prazosin in eight instrumented dogs. Before infusion of prazosin, exercise caused a fall in coronary vascular resistance (2.1 +/- 0.4 to 1.6 +/- 0.2 units, p less than 0.05) and dilation of the circumflex coronary artery (4.66 +/- 0.37 to 4.79 +/- 0.34 mm, p less than 0.05). Intracoronary infusion of prazosin during exercise caused a further decrease in coronary vascular resistance (1.6 +/- 0.2 to 1.4 +/- 0.2 units, p less than 0.05) and a further increase in circumflex coronary arterial diameter (4.79 +/- 0.34 to 4.83 +/- 0.34 mm, p less than 0.05). Intracoronary infusion of vehicle without prazosin during exercise did not cause a further decrease in coronary vascular resistance or increase in coronary diameter. Prazosin caused no significant increase in heart rate, aortic pressure, or coronary blood flow. Therefore, both small coronary resistance vessels and large epicardial coronary arteries dilated during exercise and dilated further after alpha-adrenergic blockade. This finding indicates that alpha 1-adrenergic activity during exercise limits dilation of both large and small coronary arteries.


Catalytic and immunologic similarities between monkey and human liver cytochrome P-450db1 (human cytochrome P-450 2D6). In vivo pharmacogenetic studies have suggested that the monkey may be an animal model for the human polymorphism of cytochrome P-450 2D6 (also called cytochrome P-450db1). In the present study, the catalytic, immunologic, and electrophoretic properties of cytochrome P-450db1 in liver microsomes from African green monkeys (Cercopithecus aethiops) were examined and compared with P-450db1 in human liver microsomes. Using sparteine as the substrate, the activity of microsomal P-450db1 from the two sources was indistinguishable in terms of the pattern of sparteine metabolites produced, the apparent Ki values of 8 competitive inhibitors (r = 0.94, p less than 0.001), and the extent of immunoinhibition by anti-rat P-450db1 antibody. Kinetic analyses demonstrated that the apparent KM values of the high affinity component of sparteine oxidation in monkey liver microsomes fell within the range observed in human livers; the Vmax of this component was as much as six times greater than the highest value reported for human liver. Western immunoblots showed a protein band in monkey liver microsomes that co-migrated with P-450db1 in human liver. The high degree of similarity observed here between P-450db1 of monkey and human liver microsomes suggests that the monkey will be a good animal model for P-450db1 enzyme studies, and possibly for studies of the role of this enzyme in drug abuse and dependence.


Pharmacokinetics and tissue distribution of 2-fluoro-beta-alanine in rats. Potential relevance to toxicity pattern of 5-fluorouracil. Clinical pharmacokinetic studies in our laboratory demonstrated that 2-fluoro-beta-alanine (FBAL), the major catabolite of fluorouracil (FUra), has a prolonged elimination with an approximately 150-fold longer half-life than that of the unchanged drug in humans [Heggie et al.: Cancer Res. 47, 2203-2206 (1987)]. Recent studies have suggested that FUra catabolites, such as FBAL, may have a role in neurotoxicity and cardiotoxicity that may occur during FUra chemotherapy [Okada et al.: Acta Neuropathol. 81, 66-73 (1990)]. This study was undertaken to determine the kinetics and tissue distribution of FBAL in rats following iv bolus administration of radiolabeled FBAL. Plasma disappearance curves for FBAL could be described by the sum of three exponentials, with half-lives of 0.26, 12.1, and 8426 min. Radioactivity, consisting mainly of FBAL-bile acid conjugates, was excreted in bile within 30 sec of iv bolus administration of FBAL and continued throughout the experimental period at concentrations 10-100-fold higher than that of the corresponding plasma level. Urinary excretion, consisting mainly of free FBAL, represented the major pathway of elimination of FBAL, with 40% of the administered dose excreted within 24 hr and approximately 70% over 192 hr. Fecal excretion was a minor pathway of elimination of FBAL, with approximately 10% of the administered dose excreted over 192 hr. During the initial 30 min, the highest levels of tissue radioactivity were found in the kidneys, liver, spleen, lungs, and heart. Radioactivity was retained over longer time periods in the enterohepatic circulation, central nervous system, heart, and skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)


Disposition of the novel serotonin agonist, LY228729, in monkeys and rats. The disposition of a novel 5HT-1a agonist, LY228729, was studied in rats after oral administration and in monkeys after both i.v. and oral administration of a radiolabeled drug. Plasma concentrations of LY228729 declined with a half-life of 2.3 and 1.5 hr in monkeys after oral dosing and i.v. administration, respectively, and 1.9 hr in rats dosed orally. Peak plasma concentrations of the N-despropyl metabolite were greater than the parent drug following oral administration in both rats and monkeys and declined with a half-life of 3.2-3.5 hr. Plasma levels of total radioactivity rapidly exceeded that of the parent drug in both species. Radioactivity was eliminated more slowly, with terminal half-lives of 39.4 hr in the monkey and 48.6 hr in the rat. The parent drug and its despropyl metabolite accounted for only a small percentage of the total radioactivity in the plasma. Following i.v. and oral administration, radioactivity was eliminated predominantly in the urine of monkeys, but was distributed evenly between the urine and feces of rats. Parent drug and the N-despropyl metabolite were the major products in rat urine. In the monkey, the major metabolite was an uncharacterized polar compound.


Comparative metabolism and covalent binding of procainamide by human leukocytes. Activated neutrophils and monocytes were found to metabolize procainamide to a reactive hydroxylamine. In contrast, there was little or no metabolism by lymphocytes or platelets. Therefore, it appears that only leukocytes that contain myeloperoxidase can metabolize procainamide to a significant degree. There was no difference in the degree to which neutrophils from males or females metabolized procainamide; however, monocytes from males formed significantly more hydroxylamine than did monocytes from females. By use of radiolabeled procainamide, covalent binding of procainamide to leukocytes was detected, and the degree of binding correlated with the cells' ability to oxidize procainamide. These findings suggest that myeloperoxidase is the major enzyme involved in the formation of reactive metabolites by leukocytes, a pathway that we propose may be responsible for procainamide-induced lupus and agranulocytosis.


Substituted pyridines: nonsteroidal inhibitors of human placental aromatase cytochrome P-450. The binding to human placental aromatase cytochrome P-450 and resulting extent of inhibition was examined for pyridine, pyridines substituted at the 2-, 3-, or 4-positions with phenyl or benzoyl groups, and the nonpyridinic structural analogs biphenyl and benzophenone. Spectral binding studies with partially purified aromatase indicated that pyridine, 3- and 4-phenylpyridines, and 3- and 4-benzoylpyridines interact at the active site via ligation of the pyridinic nitrogen to heme iron, as judged by the type II difference spectra. The apparent dissociation constants for these compounds are 2.1, 0.11, 0.09, 2.6, and 0.27 mM, respectively. Biphenyl, benzophenone, and 2-benzoylpyridine show exclusively a hydrophobic interaction in the presence of the substrate, androst-4-ene-3,17-dione, to give reverse type I difference spectra, with apparent spectral dissociation constants of 0.14, 0.42, and 1.8 mM, respectively. 2-Phenylpyridine shows a unique spectrum, previously not observed with cytochrome P-450, that has components of both type I and type II spectra. As measured by tritium ion release from [1 beta-3H]androst-4-ene-3,17-dione, competitive inhibition of human microsomal aromatase activity was observed with pyridine, 2-, 3-, and 4-phenylpyridines, biphenyl, and benzophenone, with Ki values of 320, 62, 1.48, 0.36, 750, and 130 microM, respectively. In contrast, 2-, 3-, and 4-benzoylpyridines exhibited complex kinetic behavior from which inhibition constants could not be obtained.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparisons of hepatic and renal cytochromes P-450-dependent monooxygenases from fuzzy and Sprague-Dawley rats. The differences in monooxygenase activities between the fuzzy rat, a variant of the hairless rat, and the Sprague-Dawley rat were examined. Benzo(a)pyrene hydroxylase and benzphetamine N-demethylase activities and cytochrome P-450 contents were significantly lower in the livers of fuzzy rats than in the livers of the Sprague-Dawley rats. The hydroxylase activity in the kidneys of the two strains were not significantly different from each other. In comparison to the Sprague-Dawley rats, the induction of these enzymic activities by phenobarbital and 3-methylcholanthrene were of a lesser magnitude in the fuzzy rats, indicating that regulation of the hepatic monooxygenases differed in the two strains. Immunoblot data showed that cross-reactivity between antirat P-450IIB1 and microsomal protein from phenobarbital-treated rats appeared to be lower in the fuzzy rat when compared with the Sprague-Dawley rat. These differences in the constitutive enzymes and the differences in the induction of hepatic monooxygenases between the two strains of rats may be important determinants in studies involving metabolism of drugs and other chemicals following cutaneous exposure.


Pharmacokinetic evaluation of two human epidermal growth factors (hEGF51 and hEGF53) in rats. The pharmacokinetic profiles of two iodinated human epidermal growth factors (125I-hEGF51 and 125I-hEGF53) in rats receiving a single intravenous dose have been investigated using three independent bioanalytical techniques. Based on a tetrachloroacetic acid precipitation methodology, hEGF51 and hEGF53 were found to have distribution half-lives of 0.80 +/- 0.2 and 0.80 +/- 0.18 min, and elimination half-lives of 79.8 +/- 24.1 and 77.9 +/- 21.1 min, respectively. Evaluated by immunoprecipitation, distribution half-lives were 0.59 +/- 0.09 and 0.63 +/- 0.15 min, and elimination half-lives were 117.8 +/- 22.9 and 118.7 +/- 38.8 min, respectively. Both precipitation techniques produced similar, parallel plasma concentration-time curves, and there were no significant differences in other calculated kinetic parameters, including clearance and volume of distribution evaluated by either technique. Plasma disposition profiles of both peptides were also confirmed by visualization with SDS-PAGE and autoradiography, and were found to be similar to those generated by tetrachloroacetic acid and immunoprecipitation methods. Thus, three independent methods strongly suggest that both peptides have the same disposition profile, which exhibits a very rapid disappearance rate in the distribution phase followed by a much slower elimination process. These results also indicate that the pharmacokinetic behavior of human epidermal growth factor is not altered by deletion of two amino acids from the carboxyl terminus. In addition, the incubation study suggests that about 23% of the exogenous peptides were associated with red blood cells.


Microbial models of mammalian metabolism. Biotransformations of N-methylcarbazole using the fungus Cunninghamella echinulata. The fungal metabolism of N-methylcarbazole (NMC) was investigated during the development of microbial models of mammalian metabolism. NMC was metabolized by the fungus Cunninghamella echinulata (ATCC 9244) to generate four metabolites: carbazole, N-hydroxymethylcarbazole (NHMC), 3-hydroxycarbazole, and 3-hydroxy-NHMC. Carbazole and NHMC are two major metabolites previously identified in mammalian systems, while 3-hydroxycarbazole and 3-hydroxy-NHMC have not been previously reported as metabolites of NMC. Structural identification of the four metabolites was based upon spectral (UV, MS, and NMR) and chromatographic (TLC and HPLC) comparisons with synthetic standards. These studies demonstrate that C. echinulata catalyzes the two principal biotransformations observed in mammalian systems with NMC: aliphatic hydroxylation to yield the (relatively) stable carbinolamine NHMC (with slow decomposition to the N-dealkylated product, carbazole) and aromatic hydroxylation in the 3-position.


Pharmacokinetic evaluation of (-)-6-aminocarbovir as a prodrug for (-)-carbovir in rats. The recently synthesized carbocyclic 2',3'-didehydro-2',3'-dideoxy-6-deoxy-6-amino-guanosine [(-)6AC] was evaluated as a prodrug for carbovir, carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine [(-)CBV] in seven male Sprague-Dawley rats. A randomized three-way cross-over design was used. Rats were assigned to receive the following treatments: a 20 mg/kg (-)6AC infusion, 40 mg/kg (-)6AC orally, and a 20 mg/kg (-)CBV infusion. Blood samples were collected over 480 min, and urine was collected for up to 48 hr. A 2- to 3-day washout period was observed between treatments. Following i.v. infusion, (-)6AC concentrations in the blood declined rapidly in a monoexponential pattern with an elimination half-life of 11.3 +/- 3.3 min (mean +/- SD, n = 7). The time-averaged total body clearance was 115.7 +/- 32.6 ml/min/kg. The fraction of the dose excreted unchanged in urine was 0.28 +/- 0.06. The fraction of the (-)6AC dose metabolized to (-)CBV was 0.48 +/- 0.14. Following oral administration of (-)6AC, the bioavailability of (-)CBV was 46.2 +/- 9.9% (n = 6) in comparison with the bioavailability of approximately 20% previously obtained after an oral dose of (-)CBV. The Cmax of (-)CBV after a 40 mg/kg oral dose of (-)6AC was 1.65 +/- 0.7 micrograms/ml as compared with the previously reported Cmax of 1.00 microgram/ml obtained after a 60 mg/kg oral dose of (-)CBV. (-)6AC has considerable potential for the improvement of the extent of absorption of (-)CBV from oral dosing.


Deacetylation of cinobufagin by rat liver. The enzyme system mediating the deacetylation of cinobufagin (CB) at the 16-position to give deacetylCB was characterized in the rat. Tissue distribution studies showed that the highest activity of CB deacetylation was mainly localized in the liver microsomal fraction. Some activity was also detected in the serum and intestine. Kinetic studies of the enzymatic reaction carried out by microsomes demonstrated that the formation of deacetyl-CB increased linearly with time up to 60 min and with protein content up to 10 mg. Apparent Km and Vmax values calculated from Lineweaver-Burk plots were 2.7 x 10(-4) M and 4.17 nmol/mg of protein/min, respectively. Low concentrations of several metal salts (AgNO3, MgCl2, CoSO4, and CuCl2) did not affect CB deacetylase activity. Microsomal CB deacetylation was inhibited by the organophosphorus compounds cyanox and fenitrothion at 1.0 x 10(-6) M. Eserine sulfate, disulfiram, rifampicin, and phenacetin at 1.0 x 10(-4) M also decreased CB deacetylase activity. Aspirin, sodium fluoride, and EDTA at 1.0 x 10(-3) M did not inhibit the deacetylation. In vivo treatment of rats with phenobarbital resulted in a 2-fold increase in microsomal CB deacetylase activity. All of these results suggest that the enzyme responsible for CB deacetylation is somewhat different from the other characterized esterases.


Characterization of the cytochrome P-450 gene family responsible for the N-dealkylation of the ergot alkaloid CQA 206-291 in humans. The ergot alkaloid CQA 206-291 (CQA) was converted by human liver microsomes (n = 16) almost exclusively to the N-deethylated metabolite (I), as identified by the on-line coupling of liquid chromatography and mass spectroscopy. Metabolite I formation exhibited monophasic and linear enzyme kinetics (2.9-300 microM), and a 5.6-fold interindividual variability (7.2-40.2 nmol/mg/hr). Chemical inhibition experiments revealed that imidazole antimycotic agents (ketoconazole, miconazole, and clotrimazole) were potent inhibitors of this N-deethylation. Polymorphically metabolized substrates (sparteine and phenytoin), well-established cytochrome P-450 probe substrates (antipyrine and tolbutamide), and steroid hormones (estradiol and testosterone) were noninhibitory, indicating that their metabolism is catalyzed by forms of cytochrome P-450 that do not catalyze this route of CQA biotransformation. The ergot alkaloids--dihydroergotamine, bromocriptine, and SDZ 208-911--were competitive inhibitors of metabolite I formation, suggesting that these compounds are metabolized by similar enzymes. Cyclosporine A was a potent competitive inhibitor of CQA metabolism, providing initial evidence that formation of metabolite I was catalyzed by proteins of the CYP3 gene family. This was substantiated by the finding that CQA metabolism was completely inhibited by a polyclonal antibody directed against a pregnenolone 16 alpha-carbonitrile-inducible cytochrome P-450 of rat liver. The rate of CQA metabolism correlated significantly to the level of CYP3A4 expression, the rate of cyclosporine A metabolism to each of the primary metabolites (M-1, M-17, and M-21), and the rate of midazolam 4-hydroxylation. COS 1 cells transfected with human CYP3A4 and CYP3A5 provided direct evidence that these enzymes catalyze the metabolism of CQA.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of human liver cytochromes P-450 involved in theophylline metabolism. Theophylline is metabolized in the liver by one or more cytochrome P-450 enzymes. To assess the amounts and types of these human cytochromes P-450, we incubated theophylline with microsomes prepared from 22 different human livers in the presence of NADPH, and measured simultaneous rates of 1- and 3-N-demethylations to 3-methylxanthine (3-MX) and 1-methylxanthine (1-MX), respectively; and 8-hydroxylation to 1,3-dimethyluric acid (1,3-DMU). Under optimal conditions, 3-MX, 1-MX, and 1,3-DMU formation proceeded with mean Km values of 2.05, 1.93, and 5.34 mM and Vmax values of 2.28, 2.48, and 23.4 pmol/mg/min, respectively. Formation of 3-MX and 1-MX correlated best with amounts of the immunoreactive protein HLd (P-450IA2) (p less than 0.05), whereas formation of 1,3-DMU correlated with the microsomal content of HLp (P-450IIIA3) and HLj (P-450IIE1). In immunoinhibition experiments, incubations conducted with a polyclonal anti-rat P-450c/d antibody, the formation of all the three theophylline metabolites (p less than 0.05) was significantly inhibited. However, addition of isoform-specific anti-rat-P-450d antibodies to the microsomal mixture significantly inhibited 1-N-demethylation, selectively, with little (if any) inhibition of 3-N-demethylation or 8-hydroxylation. Nonspecific cytochrome P-450 inhibition was ruled out by showing that erythromycin N-demethylation, an activity catalyzed by HLp, was unaffected by either anti-P-450c/d (P-450IA1/IA2) or anti-P-450d. Anti-rat-P-450p antibodies failed to block formation of theophylline metabolism, but did inhibit erythromycin N-demethylase.(ABSTRACT TRUNCATED AT 250 WORDS)


Pharmacokinetics of 2-ethyl-1,3-hexanediol. II. Nonsystemic disposition following single percutaneous or peroral doses in Fischer 344 rats. The pharmacokinetics of [1,3-14C]-2-ethyl-1,3-hexanediol (EHD) were investigated following single percutaneous doses of 150 mg/kg, applied to male and female Fischer 344 rats, or single peroral doses of 1.5 or 150 mg EHD/kg given by gavage to male Fischer 344 rats. EHD-derived radioactivity was slowly absorbed through skin and relatively rapidly excreted through the urine in a first-order manner over 48 hr postdosing. Skin penetration of 14C was sufficiently slow that the terminal rate constant for the plasma concentration data had to be derived from the absorption phase of this curve, based on the terminal rate constant for a comparable intravenous dose plasma curve [Frantz et al.: Drug Metab. Dispos. 19, 881 (1991)]. Plasma data from perorally doses rats exhibited dose-linearity over a 1.5-150 mg/kg range, with plasma 14C concentration vs. time plots for oral doses of EHD resembling the iv time-course data. This resulted from a very rapid absorption phase (5.5 min t1/2), with plasma 14C levels for both dose levels decreasing in a biexponential manner. The major route of excretion after peroral doses was in urine, making this mode of excretion consistent for both routes of administration evaluated in this study and including the doses given in previous iv work. Kinetic analysis confirmed that this route of excretion was first-order. HPLC analysis of urine from both routes demonstrated that EHD was metabolized and excreted as at least two major, water-soluble urinary metabolites; these metabolites were not identified in this investigation. No unmetabolized EHD was detected in urine, indicating that EHD may be completely metabolized in the rat. Overall, EHD was absorbed, distributed, metabolized, and eliminated from the Fischer rat in a first-order manner following either cutaneous or peroral doses. The results of this study indicate that the kinetic patterns observed experimentally will be dose-proportional for doses administered in the range of 1.5-150 mg/kg.


Cytochrome P-450 isozymes involved in the oxidative metabolism of delta 9-tetrahydrocannabinol by liver microsomes of adult female rats. Oxidative metabolism of delta 9-tetrahydrocannabinol (THC) by liver microsomes was studied in female rats. Delta 9-THC was mainly biotransformed to 11-hydroxy-delta 9-THC (11-OH-delta 9-THC) and 9 alpha,10 alpha-epoxy-hexahydrocannabinol (EHHC) by liver microsomal fraction of adult female rat. Two isozymes of cytochrome P-450 (P-450) [F-1 (IIC6) and F-2 (IIC12)] were purified from liver microsomes of female rats and oxidation activities toward delta 9-THC were assessed in the reconstituted system containing NADH-P-450 reductase and cytochrome b5. P-450 F-1 showed considerable activity toward 11-OH-delta 9-THC formation (10.62 nmol/min/nmol of P-450), whereas P-450 F-2 did not show any activity toward delta 9-THC oxidation under the conditions used. Preincubation of microsomes with antiserum against P-450 F-1 obtained from rabbits caused a marked decrease in 11-OH-delta 9-THC formation, whereas antiserum against P-450 F-2 did not exhibit any inhibitory effect on the oxidation of delta 9-THC by liver microsomes of adult female rats. Further, antiserum against P-450 F-1 or F-2 did not affect the microsomal formation of 9 alpha,10 alpha-EHHC from delta 9-THC. These results indicate that P-450 F-1 and its immunochemically related P-450 isozyme(s) play important roles in the formation of an active metabolite, 11-OH-delta 9-THC, from delta 9-THC by liver microsomes of adult female rats.


Disposition of 1-naphthol in the channel catfish (Ictalurus punctatus). The pharmacokinetics, tissue distribution, and metabolism of 1-naphthol were examined in the channel catfish (Ictalurus punctatus). Catfish were administered [1-14C]1-naphthol intravascularly at 1, 5, or 25 mg/kg or orally at 1 mg/kg. Plasma data for 1-naphthol were fitted by a three-compartment pharmacokinetic model. There were dose-related changes in the area under the plasma concentration vs. time curve, apparent volume of distribution, and total body clearance after intravascular dosing. After oral dosing, peak plasma concentrations of 1-naphthol occurred at 1 hr; parent compound made up less than 15% of the total radioactivity, and the bioavailability was 32%. Plasma protein binding was 92% and was independent of concentration. 1-Naphthol and metabolites were rapidly eliminated from the tissues after oral dosing; less than 1% of the administered dose remained at 24 hr. Renal excretion was the primary route of elimination of total 14C. Approximately 60% of the oral dose was excreted in the urine within 48 hr. Parent 1-naphthol made up 1% of the urinary 14C. Major metabolites in the urine were sulfate and glucuronide conjugates, which composed 65 and 28% of the total 14C, respectively. Biliary excretion accounted for 7% of the oral dose. The glucuronide conjugate and an unidentified polar metabolite made up the majority of the biliary 14C. The high capacity of channel catfish for conjugative metabolism of 1-naphthol was demonstrated. The dose dependency of pharmacokinetic values could not be explained by saturable metabolism or plasma protein binding.