Antibodies obtained by xenotransplantation of organ-cultured median eminence specifically recognize hypothalamic tanycytes

Tanycytes are specialized ependymal cells lining the infundibular recess of the third ventricle of the cerebrum. Early and recent investigations involve tanycytes in the mechanism of gonadotropin-releasing hormone (GnRH) release to the portal blood. The present investigation was performed to obtain a specific immunological marker of tanycytes and to identify the compound(s) responsible for this labeling. After 30 days of organ culture, explants of bovine median eminence formed spherical structures mostly constituted by tanycytes. These tanycyte spheres were xenotransplanted to rats, and the antibodies raised by the host animals against the transplanted living tanycytes were used for immunochemical studies of the bovine and rat median eminence. This antiserum immunoreacted with two compounds of 60 kDa and 85 kDa present in extracts of bovine and rat median eminence. The individual immunoblotting analysis of rat medial basal hypothalami showed a decrease in the amount of the 85-kDa compound in castrated rats as compared to control rats processed at oestrus and dioestrus. The antiserum, labeled as anti-P85, when used for immunostaining of sections throughout the rat central nervous system, immunoreacted specifically with the hypothalamic tanycytes. Within tanycytes, P-85 immunoreactivity was exclusively present in the basal processes. It is suggested that the 85-kDa and 60-kDa compounds correspond to two novel proteins selectively expressed by tanycytes. The possibility that they are secretory proteins involved in GnRH release is discussed. Anti-P85 appears to be the first specific marker of hypothalamic tanycytes.     

Clinical and mycological study of scalp white piedra in the State of Paraíba,Brazil

White piedra is a superficial mycoses characterized by nodules on the hair shaft, caused by the basidiomycetous yeasts. In the present study, clinical and mycological findings of scalp white piedra caused by Trichosporon spp. are related. Twenty three cases of scalp white piedra were observed with a high incidence in women (87%) and preschool children from 2 to 6 (74%) years old. These groups presented a relationship of dependence with this infection. Despite the low socio-economic status, poor standards of hygiene, (48% of the patients) as well as the fact that 30.4% of the children shared the same nursery, these factors were not significant for the transmission of the mycosis. These were the first reports of scalp white piedra in Jo?o Pessoa city, Paraíba, Brazil.     

First report of an endoparasitic epicaridean isopod infecting cephalopods

This is the first report of an epicaridean isopod infecting cephalopods. Three cryptoniscus larvae (Isopoda, Epicaridea) were recognized externally and found entirely embedded in the oral bulb at the beginning of the esophagus of two female Patagonian squid (Loligo gahi) sampled in North Peruvian and South Chilean waters, respectively. There was an extremely low prevalence (< 2%) and mean intensity (one) of infection which we believe indicated an accidental infection. However, the tissue location indicated successful penetration and colonization of the squid host.     

Effect of beta-Carotene on the development of the solid Ehrlich tumor in mice

The effect of beta-Carotene on the development of the solid Ehrlich tumor in BALB/c mice was investigated. Male mice received orally, on alternate days, three different doses of beta-Carotene (1, 3.5 or 7 mg/100 g) or corn oil as the control. This protocol started 14 days before tumor inoculation (1.75 x 10(5) cells) into mouse footpad and lasted until 10 days after. The tumor growth was evaluated by daily measurement of the footpad thickness, and the tumor mass was evaluated morphometrically. The proliferation rate of tumor was investigated by counting PCNA (proliferating cell nuclear antigen) positive nuclei in the 10th day of the tumor development. Histopathological examination of the lymphoid tissue: thymus, spleen and popliteal lymph node were also performed. beta-Carotene treatment, at dose 3.5 mg/100 g, increased the tumor growth, proliferative rate and the relative weight of popliteal lymph nodes, showing up an adverse effect only when this intermediate dose was used. No effects were obtained when the smaller (1,0 mg/100 g) or the higher (7.0 mg/100 g) doses were used. These results suggest that depending on the dose, beta-Carotene may determine an undesirable effect upon the tumor growth. This should be taken into account in chemopreventive experiments and human applications.     

Reciprocal protein kinase A regulatory interactions between cystic fibrosis transmembrane conductance regulator and Na+/H+ exchanger isoform 3 in a renal polarized epithelial cell model

Although Cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to regulate the activity of NHE3, the potential reciprocal interaction of NHE3 to modulate the protein kinase A (PKA)-dependent regulation of CFTR in epithelial cells is still unknown. In the present work, we describe experiments to define the interactions between CFTR and NHE3 with the regulatory, scaffolding protein, NHERF that organize their PKA-dependent regulation in a renal epithelial cell line that expresses endogenous CFTR. The expression of rat NHE3 significantly decreased PKA-dependent activation of CFTR without altering CFTR expression, and this decrease was prevented by mutation of either of the two rat NHE3 PKA target serines to alanine (S552A or S605A). Inhibition of CFTR expression by antisense treatment resulted in an acute decrease in PKA-dependent regulation of NHE3 activity. CFTR, NHE3, and ezrin were recognized by NHERF-2 but not NHERF-1 in glutathione S-transferase pull-down experiments. Ezrin may function as a protein kinase A anchoring protein (AKAP) in this signaling complex, because blocking the binding of PKA to an AKAP by incubation with the S-Ht31 peptide inhibited the PKA-dependent regulation of CFTR in the absence of NHE3. In the A6-NHE3 cells S-Ht31 blocked the PKA regulation of NHE3 whereas it now failed to affect the regulation of CFTR. We conclude that CFTR and NHE3 reciprocally interact via a shared regulatory complex comprised of NHERF-2, ezrin, and PKA.     

The bare area of the upper conjunctiva: a closer look at the anatomy of transconjunctival upper blepharoplasty

Transconjunctival upper blepharoplasty is a novel technique for which the overall experience has been limited. The authors have used this technique in 42 patients for eyelid rejuvenation, with predictable results and low morbidity. The authors previously described the "bare" area in the medial upper conjunctiva, which was initially noted during their clinical series. This area serves as an anatomic window to access the medial upper eyelid fat pad with a high degree of safety. To clarify the anatomy of this approach, an anatomic study of the upper medial eyelid was performed on the orbits of 10 cadavers (20 orbits). The bare area was further elucidated during these dissections and its microscopic components described in detail. Its spatial relationship to the medial orbital wall and trochlea was also examined during this study. The authors hope that these findings will aid surgeons planning to use this technique.     

Purification and characterization of recombinant protein kinase CK2 from Zea mays expressed in Escherichia coli

Recombinant protein kinase subunits rmCK2alpha-1 and rmCK2beta-1 from Zea mays were expressed separately in Escherichia coli and assembled to a fully active tetrameric holoenzyme complex in vitro. The obtained maize holoenzyme was purified to homogeneity, biochemically characterized, and compared to CK2 from human. Kinetic measurements of the recombinant maize holoenzyme (rmCK2) revealed k(cat) values for ATP and GTP of 4 and 2s(-1), respectively; whereas the recombinant maize catalytic subunit showed almost equal values for ATP and GTP, i.e., ca. 0.8s(-1). A comparison of the k(cat)/K(m) ratio between the maize holoenzyme and the catalytic subunit from CK2 maize shows that the incorporation of the catalytic subunit into the holoenzyme leads to a 14-fold activation in the case of ATP and 8-fold activation in the case of GTP. The maize holoenzyme is about 10 times more sensitive towards CK2 inhibitor heparin, on the other hand, it is stimulated only 0% by polylysine as compared to the human counterpart. The maize holoenzyme activity is more sensitive towards NaCl concentrations higher than those of rhCK2 and treatment with urea showed that rmCK2 holoenzyme was denatured more readily than the human holoenzyme.     

Prime-boost immunization schedules based on influenza virus and vaccinia virus vectors potentiate cellular immune responses against human immunodeficiency virus Env protein systemically and in the genitorectal draining lymph nodes

Vaccines that elicit systemic and mucosal immune responses should be the choice to control human immunodeficiency virus (HIV) infections. We have previously shown that prime-boost immunizations with influenza virus Env and vaccinia virus (VV) WR Env recombinants induced an enhanced systemic CD8(+) T-cell response against HIV-1 Env antigen. In this report, we analyzed in BALB/c mice after priming with influenza virus Env the ability of two VV recombinants expressing HIV-1 Env B (VV WR Env and the highly attenuated modified VV Ankara [MVA] Env) to boost cellular immune responses in the spleen and in the lymph nodes draining the genital and rectal tracts. Groups of mice were primed by the intranasal route with 10(4) PFU of influenza virus Env and boosted 14 days later by the intraperitoneal or intranasal route with 10(7) PFU of MVA Env or VV WR Env, while the control group received two immunizations with influenza virus Env. We found that the combined immunization (Flu/VV) increased more than 60 times the number of gamma interferon-specific CD8(+) T cells compared to the Flu/Flu scheme. Significantly, boosting with MVA Env by the intraperitoneal route induced a response 1.25 or 2.5 times (spleen or genital lymph nodes) higher with respect to that found after the boost with VV WR Env. Mice with an enhanced CD8(+) T-cell response also had an increased Th1/Th2 ratio, evaluated by the cytokine pattern secreted following in vitro restimulation with gp160 protein and by the specific immunoglobulin G2a (IgG2a)/IgG1 ratio in serum. By the intranasal route recombinant WR Env booster gave a more efficient immune response (10 and 1.3 times in spleen and genital lymph nodes, respectively) than recombinant MVA Env. However, the scheme influenza virus Env/MVA Env increased four times the response in the spleen, giving a low but significant response in the genital lymph nodes compared with a single intranasal immunization with MVA Env. These results demonstrate that the combination Flu/MVA in prime-booster immunization regimens is an effective vaccination approach to generate cellular immune responses to HIV antigens at sites critical for protective responses.