B-50/GAP43 Expression Correlates with Process Outgrowth in the Embryonic Mouse Nervous System

The hypothesis that B-50/GAP43, a membrane-associated phosphoprotein, is involved in process outgrowth has been tested by studying the developmental pattern of expression of B-50/GAP43 mRNA and protein during mouse neuroembryogenesis. B-50/GAP43 mRNA is first detectable at embryonic day 8.5 (E8.5) in the presumptive acoustico-facialis ganglion. Subsequently, both B-50/GAP43 mRNA and protein were co-expressed in a series of neural structures: in the ventral neural tube (from E9.5) and dorsal root ganglia (from E10.5), in the marginal layer of the neuroepithelium surrounding the brain vesicles and in the cranial ganglia (from E9.5), in the autonomic nervous system (from E10.5), in the olfactory neuroepithelium and in the mesenteric nervous system (from E11.5), in a continuum of brain regions (from E12.5) and in the retina (from E13.5). Immunoreactive fibers were always seen arising from these regions when they expressed B-50/GAP43 mRNA. The spatial and temporal pattern of B-50/GAP43 expression demonstrates that this protein is absent from neuroblasts and consistently appears in neurons committed to fiber outgrowth. The expression of the protein in immature neurons is independent of their embryological origin. Our detailed study of B-50/GAP43 expression during mouse neuroembryogenesis supports the view that this protein is involved in a process common to all neurons elaborating fibers.     

Specific involvement of Ca(2+)-calmodulin kinase II in cholinergic modulation of neuronal responsiveness.

1. Muscarinic agonists when applied in the hippocampus at low concentrations suppress intrinsic controls on neuronal excitability through the block of Ca(2+)-activated K conductance(s), gK (Ca), underlying the adaptation of firing and slow afterhyperpolarization (sAHP) in CA1 and CA3 neurons. Carbachol, for example, is effective at 0.1-0.3 microM suggesting activation of a relatively high-affinity receptor. 2. We have examined the mechanism of this action by using a new, highly specific, peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) as well as other kinase inhibitors and show that the muscarinic block of gK (Ca) relies on CaMKII activation in both CA1 and CA3 neurons. Thus phosphorylation of these channels or of an intermediary protein causes the channels to remain closed in the presence of Ca2+ and depolarization. 3. The very similar electrophysiological effects of serotonergic and glutamatergic agonists are mediated either through other kinases or by entirely different processes. 4. Block of intrinsic phosphatase activity by okadaic acid also reduced adaptation and sAHP, and muscarinic agonists had no further effect on these quantities. 5. The removal of presynaptic cholinergic inputs to the hippocampus in animals has a deleterious effect on the performance of tasks requiring spatial memory and is also implicated as a cause of cognitive disorders in humans. By increasing Ca2+ accumulation during electrical activity and promoting CaMKII activity, muscarinic input provides parallel reinforcing pathways for the induction of long-term potentiation, an important cellular memory mechanism. This suggests a possible link between behavioral and cellular approaches to the analysis of learning and memory.     

Developmental expression of G-proteins and adenylyl cyclase in peripheral olfactory systems. Light microscopic and freeze-substitution electron microscopic immunocytochemistry

Summary Light microscopic immunohistochemistry coupled with freeze-substitution electron microscopic immunocytochemistry was used to localize -subunits of G-proteins and type III adenylyl cyclase in developing rat olfactory epithelia. Some cilia immunoreacted with antibodies to G_s and type III adenylyl cyclase as early as prenatal day 15 (E15; El=sperm-positive), but immunolabelling with antibodies to G_olf was not observed until E16. From then on numbers of receptor cells with immunolabelled cilia increased for all three probes. Immunoreactivity for antibodies to the olfactory signal-transduction proteins tended to parallel cilium development, though G_olf lags somewhat behind. Newly formed cilia labelled along their lengths, whereas mature cilia labelled predominantly along their long distal parts. Dendritic knobs and ciliary necklaces showed little or no labelling. While at E22 most multiciliate cells immunolabelled with antibodies to G_s, G_olf, and type III adenylyl cyclase, not all of these cells labelled with antibodies to olfactory marker protein. Olfactory axons immunoreacted more intensely than epithelial surface structures with antibodies to G_s at E15; the reverse occurred by about E18. Immunoreactivity with antibodies to a-subunits of the G-proteins G_o, G_q/G₁₁, and G_i was also found as early as E15. Antibodies to G_o labelled receptor cell dendritic knobs and cilia during development only. Antibodies to G_i labelled Bowman's glands, whereas those to G_q/G₁₁bound to receptor cell cilia and axons (primarily vomeronasal), and supporting cell microvilli. We propose that G_s is the predominant G protein in cilia of immature olfactory receptor cells, while G_oif is predominant in cilia of mature cells. Axonal immunoreactivity for some G-protein antibodies suggests G-protein participation in processing of olfactory axon and/or axon terminal-bound signals.     

Cervical Spondylodiscitis Caused by Blastoschizomyces capitatus

Abstract   A 37-year-old woman, during her second remission of acute myeloid leukemia, presented with severe neck pain and cervico-brachial neuralgia. Investigation revealed a C5–C6 spondylodiscitis. A CT-guided anterior biopsy decompressed the mass, immediately alleviated the symptoms, and isolated a rare yeast: Blastoschizomyces capitatus. To our knowledge, only three cases of spondylodiscitis with this yeast have been described. Six months of voriconazole and liposomal amphotericin B treatment produced a complete resolution on CT and MRI imaging. However, the ongoing severe yeast infection prevented the planned bone marrow allograft.     

Identification of taste receptors and proteomic characterization of the antenna and legs of Tribolium brevicornis,a stored food product pest

Chemoreception plays an important role in mediating a diverse range of behaviours, including predation and food selection. In the present study, we combined anatomical observations, electrophysiology and proteomics to investigate sensilla that mediate chemoreception on the antenna and the legs of Tribolium. Scanning electron microscopy was used to differentiate the coxal and trochanteral segments of the pro‐, meso‐ and metathoracic legs by the presence of sensilla trichoidea and chaetica, while the antennae were covered with five types of sensilla (chaetica, basiconica, trichoidea, squamiformia and coeloconica). Antenna morphology and ultrastructure were similar in both sexes. Electrophysiological recordings allowed us to characterize a row of small sensilla basiconica on the terminal segment of the antenna as taste receptors, responding to sucrose and NaCl. Proteomics investigations of antennae and legs yielded several proteins with specific interest for those involved in chemoreception. Odorant‐binding proteins were antenna‐specific, while chemosensory proteins were detected in both tissues.     

Analogs of Ac-CCK-7 incorporating dipeptide mimics in place of Met28-Gly29.

A series of analogs of Ac-CCK-7 [Ac-Tyr(SO3H)-Met28-Gly29-Trp-Met-Asp- Phe-NH2, (1)] were prepared in which the Met28-Gly29 dipeptide was replaced by omega-aminoalkanoic acids. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors, respectively, and for anorectic activity after intraperitoneal administration to rats. The analog incorporating 4-aminobutanoic acid (5) was only 8 times less potent than 1 in the pancreatic binding assay, was more potent in the striatal binding assay, and was more potent than 1 in reducing food intake in rats. Using a bioactive cyclic analog of Ac-CCK-7 as a template, several rigid spacers were designed and tested as substitutes for the Met28-Gly29 dipeptide. The analogs incorporating 3-aminobenzoic acid (20) and (1S)-trans-2-aminocyclopentanecarboxylic acid (26) proved highly effective in the binding assays and as anorectic agents. We hypothesize that for stimulation of CCK-A receptors, the main function of the N-terminal tripeptide of Ac-CCK-7 is to orient the tyrosine sulfate with respect to Trp30 and that the bioactive arrangement of these elements lies among those which are readily available to both 20 and 26. NOESY and distance-constrained molecular dynamics experiments carried out on 20 and 26 identified conformations in which the relative orientation of the tyrosine hydroxide and the alpha-carbon atom of tryptophan were similar, providing the basis for further drug design efforts.     

Cerebellin and related postsynaptic peptides in the brain of normal and neurodevelopmentally mutant vertebrates

The rat cerebellum was previously shown to contain two polypeptides, a hexadecapeptide termed cerebellin and an apparent metabolite des-Ser1-cerebellin. The cerebellins have a high degree of sequence homology with residues 625-641 of the polyimmunoglobulin (polyIg)-receptor adjacent to its membrane-spanning domain. Since the cerebellins are localized in Purkinje cells and enriched in synaptosomes, this might indicate that cerebellin is a specific proteolytic cleavage fragment of a synaptic protein involved in the transcytosis of an unknown ligand. Using a specific cerebellin radioimmunoassay described here combined with high-performance liquid chromatography, cerebellin immunoreactivity could be demonstrated in the cerebella of all vertebrates examined from man to chicken. Cerebellin immunoreactivity is localized to Purkinje cells in the rat, mouse, and chicken. Furthermore, cerebellin expression is under developmental regulation in both the chicken and mouse. In addition, neurodevelopmental mutations of mice that eliminate granule cells cause a large deficit in cerebellin levels, suggesting some form of transneuronal regulation.     

4-Sulfobenzyl ester – an anionic protecting group for peptide synthesis

The preparation of the 4-sulfobenzyl esters of 18 amino acid derivatives is described. This carboxyl protecting group was introduced according to Hubbuch et al. (1980). The caesium or dicyclohexylammonium salts of N-terminal protected amino acids were reacted with 4-(bromomethyl)benzenesulfonate (1). After N-terminal deblocking, the amino acid-4-sulfobenzyl esters were isolated as zwitterions. The protecting group was removable by catalytic hydrogenation and by saponification. The 4-sulfobenzyl esters could be easily converted to amides and hydrazides. They were stable to 2 M hydrogen bromide in acetic acid as well as to a 10-fold excess of trifluoromethane sulfonic acid in trifluoro-acetic acid. The behaviours of ⁺H₂-Gly-Phe-Leu-OBzl-SO^-₃ and the corresponding methyl, benzyl and 4-nitrobenzyl esters were compared under various conditions.     

Characteristics of patients co-infected with HIV at the time of inpatient tuberculosis treatment initiation in Yaoundé, Cameroon: a tertiary care hospital-based cross-sectional study

Background

Knowledge of the characteristics of patients co-infected with tuberculosis (TB) and human immunodeficiency virus (HIV) when TB treatment is initiated would allow clinicians to improve care and help policy-makers develop relevant and realistic guidelines. The aim of this study was to describe socio-demographic, clinical, and laboratory characteristics of TB/HIV co-infected patients starting inpatient TB treatment in Yaoundé, Cameroon.

Methods

We conducted a retrospective cross-sectional study, collecting data from medical records of HIV-infected patients with TB, aged 15 years old or more, hospitalized in the Infectious Diseases Unit of the Yaoundé Central Hospital, Cameroon from January 1, 2006 to June 30, 2013.

Results

The mean age of 337 patients meeting study inclusion criteria was 39.3 years. More than half were female (53.4%). Most (89.3%) resided in urban areas, 44.2% had a secondary education, and 46.0% were married. The majority was receiving co-trimoxazole prophylaxis (79.5%), and two thirds were taking antiretroviral therapy (67.4%). The mean duration of known HIV infection before TB treatment was 8.4 months. Most (88.1%) had newly diagnosed TB, rather than relapsed disease. Smear-positive pulmonary TB was documented in a third, (35.3%). Laboratory data revealed a median white blood cell count of 5,100 cells/mm³ (IQR 3,300-7,990 cells/mm³), a median hemoglobin level of 8 g/dl (IQR 7–10 g/dl), and a median CD4 cell count of 102 cells/mm³ (IQR 33–178 cells/mm³). Sex differences in our study included older age in the men (p < 0.001), more of whom were married (p < 0.001) and had achieved a higher level of education (p = 0.042). Men had fewer diagnoses of smear-positive pulmonary TB (p = 0.020). They weighed more than the women (p = 0.001) and had higher hemoglobin levels (p = 0.003).

Conclusions

Suboptimal adherence to WHO treatment recommendations in our Cameroonian study reinforces the importance of prescribing co-trimoxazole in HIV infection and ART for all TB/HIV co-infected persons. We urge that Ministries of Health continue implementing and disseminating guidelines for management of TB/HIV co-infected patients, and we call for measures ensuring that healthcare facilities’ stocks of ART and co-trimoxazole are sufficient to meet the need for both.