Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors have been identified mostly as postsynaptic receptors mediating fast glutamatergic synaptic transmission. However, neurochemical studies based on the modulation of neurotransmitter release have suggested the existence of presynaptic AMPA receptors. We have used a recently described technique that allows a high-purity fractionation of the pre- and postsynaptic proteins of synaptic junctions to evaluate the distribution of the different AMPA receptor subunits in rat hippocampal synapses. Surprisingly, we found very high levels of GluR1- and GluR2/3-like immunoreactivity in the presynaptic fraction, but also in the postsynaptic and extrasynaptic fractions. GluR4-like immunoreactivity was much less abundant but was still detected, predominantly in the postsynaptic fraction. This methodology appears to be far more sensitive than the classical immunogold electron microscopy to determine the localization of synaptic receptors.     

Myelolipoma: A New Adrenal Finding in Carney's Complex?

Pigmented nodular cortical hyperplasia, a rare cause of Cushing’s syndrome, is characterized by resistance to inhibition with dexamethasone and normal sized adrenal glands with multiple, small pigmented nodules. The disorder may be a component of a syndrome inherited as an autosomal dominant pattern that includes intra- and extracardiac myxomas, lentiginous lesions, blue nevi, other functional endocrine tumors, and peripheral nerve tumors (Carney’s complex). We report a patient in whom bilateral myelolipomas were found, in addition to the usual features of this complex. A 29-yr-old man was admitted to the hospital for Cushing’s syndrome of probably more than 15 yr duration. Physical examination showed diffuse facial hyperchromatic macules, 0.2–0.5 cm, predominantly around the lips and on the palmar surfaces of the fingers. Results with dexamethasone suppression nocturnal testing (1 and 8 mg) were compatible with an adrenal adenoma. The computed tomography (CT) of the sella turcica was normal. Adrenal CT showed a tumor in the left gland with a double component: one solid and another suggestive of fat, consistent with an angiomyelolipoma. Following 5 wk treatment with ketoconazole, 800 mg per day po, serum cortisol decreased to 5.9 μg/dL, morning and evening, respectively. Bilateral adrenalectomy was performed. Pathologic examination revealed pigmented nodular cortical hypersplasia and a dominant myelolipoma in the left adrenal. A microscopic myelolipoma was identified in the right adrenal. An echocardiogram showed a mass on the posterior wall of the left ventricle which was a myxoma. Study of the patient's family disclosed two sisters with facial lentigines. Echocardiograms were performed on all available first degree relatives: all were normal. Nocturnal inhibition with dexamethasone revealed that one of the patient’s sisters with lentigines also had hypercortisolism. Myelolipoma has been reported in association to Cushing syndrome in humans and experimentally after pituitary extracts in animals. The relationship between this finding and the Carney’s complex remain elusive.     

Toxoplasma gondii prevents neuron degeneration by interferon-gamma-activated microglia in a mechanism involving inhibition of inducible nitric oxide synthase and transforming growth factor-beta1 production by infected microglia

Interferon (IFN)-gamma, the main cytokine responsible for immunological defense against Toxoplasma gondii, is essential in all infected tissues, including the central nervous system. However, IFN-gamma-activated microglia may cause tissue injury through production of toxic metabolites such as nitric oxide (NO), a potent inducer of central nervous system pathologies related to inflammatory neuronal disturbances. Despite potential NO toxicity, neurodegeneration is not commonly found during chronic T. gondii infection. In this study, we describe decreased NO production by IFN-gamma-activated microglial cells infected by T. gondii. This effect involved strong inhibition of iNOS expression in IFN-gamma-activated, infected microglia but not in uninfected neighboring cells. The inhibition of NO production and iNOS expression were parallel with recovery of neurite outgrowth when neurons were co-cultured with T. gondii-infected, IFN-gamma-activated microglia. In the presence of transforming growth factor (TGF)-beta1-neutralizing antibodies, the beneficial effect of the parasite on neurons was abrogated, and NO production reverted to levels similar to IFN-gamma-activated uninfected co-cultures. In addition, we observed Smad-2 nuclear translocation, a hallmark of TGF-beta1 downstream signaling, in infected microglial cultures, emphasizing an autocrine effect restricted to infected cells. Together, these data may explain a neuropreservation pattern observed during immunocompetent host infection that is dependent on T. gondii-triggered TGF-beta1 secretion by infected microglia.     

Evaluation of PCR-restriction profile analysis and IS2404 restriction fragment length polymorphism and amplified fragment length polymorphism fingerprinting for identification and typing of Mycobacterium ulcerans and M. marinum

Mycobacterium ulcerans and M. marinum are emerging necrotizing mycobacterial pathogens that reside in common reservoirs of infection and exhibit striking pathophysiological similarities. Furthermore, the interspecific taxonomic relationship between the two species is not clear as a result of the very high phylogenetic relatedness (i.e., >99.8% 16S rRNA sequence similarity), in contrast to only 25 to 47% DNA relatedness. To help understand the genotypic affiliation between these two closely related species, we performed a comparative analysis including PCR restriction profile analysis (PRPA), IS2404 restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) on a set of M. ulcerans (n = 29) and M. marinum (n = 28) strains recovered from different geographic origins. PRPA was based on a triple restriction of the 3' end region of 16S rRNA, which differentiated M. ulcerans into three types; however, the technique could not distinguish M. marinum from M. ulcerans isolates originating from South America and Southeast Asia. RFLP based on IS2404 produced six M. ulcerans types related to six geographic regions and did not produce any band with M. marinum, confirming the previous findings of Chemlal et al. (K. Chemlal, K. DeRidder, P. A. Fonteyne, W. M. Meyers, J. Swings, and F. Portaels, Am. J. Trop. Med. Hyg. 64:270-273, 2001). AFLP analysis resulted in profiles which grouped M. ulcerans and M. marinum into two separate clusters. The numerical analysis also revealed subgroups among the M. marinum and M. ulcerans isolates. In conclusion, PRPA appears to provide a rapid method for differentiating the African M. ulcerans type from other geographical types but is unsuitable for interspecific differentiation of M. marinum and M. ulcerans. In comparison, whole- genome techniques such as IS 2404-RFLP and AFLP appear to be far more useful in discriminating between M. marinum and M. ulcerans, and may thus be promising molecular tools for the differential diagnosis of infections caused by these two species.     

Linkage analysis in Usher syndrome type I (USH1) families from Spain.

Usher syndrome (USH) is an autosomal recessive hereditary disorder characterised by congenital sensorineural hearing loss and gradual visual impairment secondary to retinitis pigmentosa (RP). The disorder is clinically and genetically heterogeneous. With regard to Usher type I (USH1), several subtypes have been described, the most frequent being USH1B located on chromosome 11q13.5. Of 18 USH1 families studied by linkage analysis, 12 (67%) showed significant lod score values for locus D11S527 (Zmax=14.032, theta=0.000) situated on chromosome 11q. Our findings suggest considerable genetic heterogeneity in the Spanish USH1 population. It is important to note that one of our families linked to the USH1B locus shows interesting intrafamilial clinical variability. As regards the remaining six USH1 families, the linkage analysis did not provide conclusive data, although two of them show slight linkage to markers located on chromosome 3q (Zmax=1.880, theta=0.000 for D3S1279), the same location that had previously been assigned to some USH3 families.     

Identification of a nucleocapsid protein as a specific serological marker of human herpesvirus 6 infection.

Enveloped whole virions and nucleocapsids of human herpesvirus 6 (HHV-6) strain Z29 were purified from supernatant fluids of infected human cord blood lymphocytes by filtration through polyvinylpyrrolidone-treated filters, banding on a Nycondenz step gradient, and centrifugation through two successive continuous sucrose gradients. More than 20 proteins ranging in molecular weight from less than 30,000 to more than 200,000 were identified in preparations of purified whole virions labeled with [35S]methionine and [35S]cysteine. Immunogenic virion proteins of HHV-6 were identified in immunoblot assays with human immune sera, immune sera generated from mice immunized with purified whole virions or purified nucleocapsids, and a monoclonal antibody generated from a mouse immunized with purified nucleocapsids. The sera and the monoclonal antibody reacted strongly with a 101-kilodalton protein in the immunoblots, suggesting that the protein is a component of the nucleocapsid. Human sera lacking HHV-6-specific antibodies and seropositive for one or more of the other human herpesviruses failed to react with this protein, indicating that it is a specific serologic marker for HHV-6 infection.     

A project to reduce the burden of diabetes in the African-American Community: Project DIRECT.

Project DIRECT (Diabetes Interventions Reaching and Educating Communities Together) is the first comprehensive community diabetes demonstration project in the United States in an African-American community. This article describes its intervention components and evaluation design. The development and implementation of Project DIRECT has included the community since the project''s beginning. Interventions are targeted in three areas: health promotion (improving diet and physical activity levels), outreach (improving diabetes awareness, detection of undiagnosed diabetes, and ensuring that persons with diabetes who are not receiving continuing diabetes care are integrated into the health-care system), and diabetes care (improving self-care, increasing access, and improving the quality of diabetes preventive care received within the health-care system). Evaluation will be internal (conducted by Project DIRECT staff to assess process outcomes in persons directly exposed to each specific intervention) and external (review of outcomes to assess the impact of the multi-intervention program at the level of the entire community). Because diabetes exacts a disproportionate toll among African Americans, the findings from this project should aid in developing strategies to lessen the burden of this disorder, particularly among minority populations.     

Surfactant protein A enhances Mycobacterium avium ingestion but not killing by rat macrophages

Mycobacterium avium complex (MAC) is a significant cause of opportunistic infection in patients with acquired immunodeficiency syndrome. Although the major route of entry of MAC is via the gastrointestinal tract, MAC can infect humans through the respiratory tract and eventually encounter alveolar macrophages within the lung. Once in the lung, MAC can potentially interact with surfactant protein A (SP-A), an important component of the pulmonary innate-immune response. Previous work on other pulmonary pathogens including Mycobacterium bovis Bacillus Calmette-Guerin (BCG) suggests that SP-A participates in promoting efficient clearance of these organisms by alveolar macrophages. In the present study, we investigated the role of SP-A in clearance of MAC by cultured rat macrophages. SP-A bound to MAC organisms and enhanced the ingestion of the mycobacteria by macrophages. Infection of macrophages with SP-A-MAC complexes induced the production of nitric oxide (NO) and tumor necrosis factor-alpha. However, intracellular survival of MAC was not altered by preopsonization with SP-A. In addition, inhibitors of inducible NO synthase did not alter MAC clearance. These results suggest that SP-A can bind to and enhance the uptake of MAC by alveolar macrophages, similar to previous findings with BCG and Mycobacterium tuberculosis.However, unlike BCG and other pulmonary pathogens that are cleared effectively in the presence of SP-A via a NO-dependent pathway, macrophage-mediated clearance of MAC is not enhanced by SP-A.     

A sodium-activated potassium current in intact ventricular myocytes isolated from the guinea-pig heart

A current generated by the Na-activated K channel has been identified in whole cell currents recorded from isolated guinea-pig ventricular myocytes. A partial activation of this current can be achieved near to the physiological range of intracellular sodium concentration [( Na+]i) when it contributes significantly to the global outward current. The decline of the Na-activated K current, the lengthening of the action potential duration and the recovery of [Na+]i occur with a similar time course during recovery from Na loading.     

The Na+-activated K+ channel contributes to K+ efflux in Na+-loaded guinea-pig but not rat ventricular myocytes

Activation of the Na+-activated K+ channels (KNa channels) has been suggested to contribute to the ischaemia-induced accumulation of extracellular K+ (K+e) in the mammalian myocardium. Recent evidence shows that these channels are not present in rat ventricular myocytes [9]. We have therefore investigated the effect of raised intracellular Na+ activity (aiNa) on intracellular K+ activity (aiK) in guinea-pig myocytes, which possess the channels, and on rat ventricular myocytes which do not. The Na+-activated K+ current was activated by an increase in aiNa induced by removing extracellular Ca2+ and Mg2+ and inhibiting the Na-pump. The aiNa increased and the aiK decreased in both guinea-pig and rat myocytes superfused with Ca2+- and Mg2+-free Tyrode. The new steady-state increase in aiNa and decline in aiK were similar in both species. Inhibition of the Na-pump resulted in an additional increase in aiNa and decrease in aiK in both species. However, both the increase in aiNa and decrease in aiK were greater in guinea-pig myocytes and the decline in aiK in guinea-pig myocytes followed the development of a large Na+-activated K+ current. When Li+ replaced Na+ in the superfusate the Na+-activated K+ current did not develop and the fall in aiK was reduced. In Na+-loaded rat myocytes, which do not have a Na+-activated K+ current, the decline in aiK was reduced and blocked by 2 mM Mg2+ suggesting that a Mg2+-sensitive non-specific cation channel may be involved in the K+ efflux from rat myocytes [12]. These data suggest that KNa channels are a major route for K+ efflux from Na+-loaded guinea-pig myocytes.