Chronic Alcohol Consumption Increases Cyclo (His-Pro)-Like Immunoreactivity in the Rat Brain

Abstract: Administration of histidyl-proline diketopiperazine (cyclo (HisPro)) to rats attenuates ethanol-induced sleep. To understand the role played by cyclo (His-Pro) in the pathophysiology of prolonged alcohol consumption, we have measured the distribution of this peptide in brains of control and alcohol-treated rats. The data show that prolonged alcohol consumption increases the concentration of cyclo (His-Pro) in hypothalamic as well as extrahypothalamic brain. These changes may reflect a physiologic adaptation of the brain during alcohol consumption.     

Neonatal maternal separation alters adult eyeblink conditioning and glucocorticoid receptor expression in the interpositus nucleus of the cerebellum

Neonatal maternal separation alters learning and memory. Glucocorticoids also modulate adult learning and memory, and neonatal maternal separation alters forebrain glucocorticoid receptor (GR) concentrations. We used eyeblink classical conditioning to assess the effect of neonatal maternal separation on associative learning. We assessed delay eyeblink conditioning, GR expression, and total neuron number in the interpositus nucleus, a critical site of plasticity in eyeblink conditioning, in adult rats that had undergone either standard animal facilities rearing, handling for 15 min, or maternal separation for either 15 or 60 min per day on postnatal days 2-14. At 2-3 months of age, delay eyeblink classical conditioning was assessed. Brains were processed for GR immunohistochemistry, and GR expression in the interpositus nucleus was assessed using a computer-based densitometry system. Neuron counts and nuclear volumes were obtained from an alternate series of thionin-stained sections. Maternal separation significantly impaired eyeblink conditioning in male but not female rats. Handling and maternal separation did not significantly affect interpositus neuron number and volume. However, prolonged maternal separation significantly increased GR expression in the posterior interpositus in males, and increases were correlated with eyeblink conditioning. In female rats, maternal separation and handling did not significantly alter interpositus neuron number, volume, or GR protein expression, and GR expression did not correlate with eyeblink conditioning. Thus, neonatal maternal separation produces adult deficits in eyeblink conditioning and alterations in GR expression in its neural substrate in a sex-dependent manner.     

Repellency of essential oils of some Kenyan plants against Anopheles gambiae

Essential oils of six plants growing in Kenya were screened for repellent activities against Anopheles gambiae sensu stricto. The oils of Conyza newii (Compositeae) and Plectranthus marrubioides (Labiateae) were the most repellent (RD50=8.9 x 10(-5) mg cm(-2), 95% CI) followed by Lippia javanica (Verbenaceae), Lippia ukambensis (Verbenaceae), Tetradenia riparia, (Iboza multiflora) (Labiateae) and Tarchonanthus camphoratus (Compositeae). Eight constituents of the different oils (perillyl alcohol, cis-verbenol, cis-carveol, geraniol, citronellal, perillaldehyde, caryophyllene oxide and a sesquiterpene alcohol) exhibited relatively high repellency. Four synthetic blends of the major components (present in > or = 1.5%) of the essential oils were found to exhibit comparable repellent activity to the parent oils.     

A mass spectrometry assay for detection of endogenous and lentiviral engineered hemoglobin in cultured cells and sickle cell disease mice

Sickle cell disease (SCD) results from a sequence defect in the β-globin chain of adult hemoglobin (HbA) leading to expression of sickle hemoglobin (HbS). It is traditionally diagnosed by cellulose-acetate hemoglobin electrophoresis or high-performance liquid chromatography. While clinically useful, these methods have both sensitivity and specificity limitations. We developed a novel mass spectrometry (MS) method for the rapid, sensitive and highly quantitative detection of endogenous human β-globin and sickle hβ-globin, as well as lentiviral-encoded therapeutic hβAS3-globin in cultured cells and small quantities of mouse peripheral blood. The MS methods were used to phenotype homozygous HbA (AA), heterozygous HbA–HbS (AS) and homozygous HbS (SS) Townes SCD mice and detect lentiviral vector-encoded hβAS3-globin in transduced mouse erythroid cell cultures and transduced human CD34⁺ cells after erythroid differentiation. hβAS3-globin was also detected in peripheral blood 6 weeks post-transplant of transduced Townes SS bone marrow cells into syngeneic Townes SS mice and persisted for over 20 weeks post-transplant. As several genome-editing and gene therapy approaches for severe hemoglobin disorders are currently in clinical trials, this MS method will be useful for patient assessment before treatment and during follow-up.     

Susceptibility of the legume flower thrips to Metarhizium anisopliae on different varieties of cowpea

The susceptibility of Megalurothrips sjostedtito Metarhizium anisopliae when reared on susceptible, tolerant, and moderately resistant varieties of cowpea at different constant temperatures was evaluated in the laboratory. Insects were exposed either to direct spray of the conidia or to fungus-treated floral tissues. Mortality was significantly higher on the moderately resistant variety at all temperatures compared to the susceptible and tolerant varieties. Correspondingly, lethal time and lethal concentration values were significantly shorter and lower, respectively, on the moderately resistant variety compared to the other varieties, thus indicating that the two control methods are compatible as part of an integrated pest management strategy. Thrips raised on the tolerant variety incurred an exceptionally low level of mortality when the inoculum was sprayed directly on the insects or when the insects were exposed tofungus-treated floral tissues. Observations on the effects of airborne volatiles and crude extracts of this variety revealed an inhibitory effect on fungal germination, colony forming units and growth. This suggests the existence of anti-fungal substances in the tolerant variety. This revised version was published online in July 2006 with corrections to the Cover Date.     

Light sensitive zinc content of protein fractions from bovine rod outer segments

Bovine retinas, isolated rod outer segments and emulphogene extracts of rod outer segments have been shown to contain appreciable amounts of Zn²⁺, Ca²⁺ and Mg²⁺ when isolated in the absence of added metal ions. Chromatography of emulphogene extracted rod outer segments in Sephadex G-25 showed virtually all the Ca²⁺, Zn²⁺ and protein to elute with the void volume. Levels of Zn²⁺ but not Ca²⁺ were light sensitive. The Zn²⁺ contents of protein fractions were about 60% higher when samples were bleached. Under optimal conditions protein fractions contained 1.4 – 1.8 g atoms Zn²⁺/mole rhodopsin for dark adapted samples and 2.1 to 3.2 g atoms Zn²⁺/mole of rhodopsin for bleached samples.     

Genotypic Diversity Is Associated with Clinical Outcome and Phenotype in Cryptococcal Meningitis across Southern Africa

Cryptococcal meningitis is a major cause of mortality throughout the developing world, yet little is known about the genetic markers underlying Cryptococcal virulence and patient outcome. We studied a cohort of 230 Cryptococcus neoformans (Cn) isolates from HIV-positive South African clinical trial patients with detailed clinical follow-up using multi-locus sequence typing and in vitro phenotypic virulence assays, correlating these data with clinical and fungal markers of disease in the patient. South African Cn displayed high levels of genetic diversity and locus variability compared to globally distributed types, and we identified 50 sequence types grouped within the main molecular types VNI, VNII and VNB, with 72% of isolates typed into one of seven ''high frequency'' sequence types. Spatial analysis of patients’ cryptococcal genotype was not shown to be clustered geographically, which might argue against recent local acquisition and in favour of reactivation of latent infection. Through comparison of MLST genotyping data with clinical parameters, we found a relationship between genetic lineage and clinical outcome, with patients infected with the VNB lineage having significantly worse survival (n=8, HR 3.35, CI 1.51-7.20, p=0.003), and this was maintained even after adjustment for known prognostic indicators and treatment regimen. Comparison of fungal genotype with in vitro phenotype (phagocytosis, laccase activity and CSF survival) performed on a subset of 89 isolates revealed evidence of lineage-associated virulence phenotype, with the VNII lineage displaying increased laccase activity (p=0.001) and ex vivo CSF survival (p=0.0001). These findings show that Cryptococcus neoformans is a phenotypically heterogeneous pathogen, and that lineage plays an important role in cryptococcal virulence during human infection. Furthermore, a detailed understanding of the genetic diversity in Southern Africa will support further investigation into how genetic diversity is structured across African environments, allowing assessment of the risks different ecotypes pose to infection.     

Overproduction of human M₃ muscarinic acetylcholine receptor: an approach toward structural studies

Human M(3) muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral nervous system, is involved in several neurodegenerative and autoimmune diseases. Recently, M3R overexpression has been suggested to play a role in certain forms of cancer, showing promise as a new potential pharmacological target. However, the lack of structural information hampered to develop a new potent selective and potent antagonist. We describe here different strategies for overexpressing functional M3R on the perspective of future biophysical studies. To achieve this goal, four tagged M3R genes were engineered and codon optimized. Different heterologous expression systems, including mammalian cells and viral transfection, were employed to overexpress M3R. Although codon optimization resulted in only twofold to threefold increase of M3R expression, we found that epitope tagging of the synthetic M3R, especially with hemagglutinin and Flag epitope tags, could improve M3R expression levels. On the other hand, viral transfection led to a yield of 27 pmol/mg protein that is the highest level reported so far for this receptor subtype in mammalian cells. Taking together several of the strategies used can help increasing M3R expression, not only to start purification efforts but also for secondary structural analysis trial and functional analyses.