Morphine exposure prevents up-regulation of MR and GR binding sites in the brain of adult male and female rats due to prenatal stress

Our previous work demonstrated that the hormone response to stress and the negative feedback inhibition to these hormones are sex-dependently altered by prenatal morphine exposure in adult rats. An alteration in the glucocorticoid negative feedback inhibition is mediated by glucocorticoid receptors (GR) that are distributed throughout the brain, and mineralocorticoid receptors (MR) localized mainly in the hippocampus and involved in a tonic influence of brain functions. Therefore, the present study examined the binding characteristics of MR and GR in young adult male and female rats exposed prenatally (E11-E18) to morphine (10 mg/kg/2 x /day), saline or no treatment at all (controls). At 60-90 days of age, animals were adrenalectomized (ADX) 24 h prior to decapitation. The hippocampus and hypothalamus were dissected for saturation binding assays. The data demonstrate that prenatal stress due to maternal saline injections up-regulates MR and GR binding in the hippocampus of adult male rats and this effect is prevented by prenatal morphine exposure. There is no effect of prenatal morphine exposure on GR binding in the hypothalamus of males. In female rats, prenatal morphine exposure does not affect the binding of MR and GR in the hippocampus or GR in the hypothalamus relative to controls; however, they are affected by ovarian hormone fluctuation. Moreover, prenatal stress decreases MR binding in the hippocampus of diestrous females and GR binding in the hypothalamus of estrous females. Both decreases are prevented by prenatal morphine exposure. Thus, the present study demonstrates that: (1) prenatal stress due to maternal saline injections alters MR and GR binding of adult male and female rats and is prevented by prenatal morphine exposure; (2) the MR and GR binding in adult female rats are affected by ovarian hormone fluctuations.     

Expression of the β‐adhesin part of HRgpA in Sprague Dawley rats induces a specific antibody response

The β‐adhesin part of the Porphyromonas gingivalis W50 (ATCC 53978) protease HRgpA was cloned in an eukaryotic expression vector and expressed in COS‐7 cells. The monoclonal antibody MAb (61BG1.3), specific for the hemagglutinating domain of β‐adhesin, recognized the expressed β‐adhesin in the transfected cells both by immunoblot and immunofluorescence. Sprague Dawley rats were immunized intramuscularly with β‐adhesin encoding expression plasmid and expression plasmid without β‐adhesin insert. Skeletal muscle tissue at the site of immunization in the β‐adhesin immunized animals was shown to express this protein. The immunization induced a β‐adhesin‐specific antibody response. Sera from the immunized animals were tested for hemagglutination inhibiting activity. Due to high natural inhibiting activity in all rat sera tested, no increased hemagglutination inhibition was detected in sera from the β‐adhesin immunized animals.     

Investigation of the polymerization of octanelactam initiated with hydrochloric acid

The polymerization of octanelactame ( 1 ) initiated by HCl takes place according to the characteristic kinetic curves. Both characteristics, i.e., the fast initial and the extremely slow further stages, and the kinetic anomaly (in certain cases under otherwise identical conditions less polymer is formed at higher than at lower temperatures) can be interpreted easily on the basis of a mechanism already suggested by the authors. The two relevant chain growth reactions, i.e., lactam addition on \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm R}\mathop {\rm N}\limits^ \oplus {\rm H}_{\rm 3} $\end{document}-groups and the reaction between \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm R}\mathop {\rm N}\limits^ \oplus {\rm H}_{\rm 3} $\end{document} and N-acylamide groups, proceed through a tetrahedral intermediate in two directions, one of them being the known chain growth, the other one an amidine (or acylamidine) formation, in which the active groups, ensuring chain growth, desactivate. Both kinetic characteristics could be interpreted on the basis of the suggested mechanism, by measuring the amounts of amino and amidine groups with the progress of polymerization.     

Essential and regulatory light chains of Placopecten striated and catch muscle myosins

Summary ATPase activities of molluscan adductor muscle myosins show both muscle and species specific differences: ATPase activity of catch muscle myosin is lower than that of phasic muscle myosin; a 4–5-fold difference exists between the activities of phasic striated muscle myosins from the bay scallop (Argopecten irradians) and sea scallop (Placopecten magellanicus). To characterize the light chains of these myosins we determined the cDNA sequences of the essential light chains and the regulatory light chains from Placopecten striated and catch muscle. The nucleotide sequences of the essential light chains from Placopecten striated and catch muscle myosins are identical and show 94% identity and 98% homology to the Argopecten essential light chain indicating that the tissue and species specific differences in ATPase activities are not due to the essential light chain. We identified three regulatory light chain isoforms, one from striated and two from catch muscle. Sequence differences were restricted to nucleotides encoding some of the N-terminal 52 amino acids. The three recombinant Placopecten regulatory light chain isoforms and the Argopecten regulatory light chain were incorporated into hybrid myosins that contained the essential light chain and heavy chain from Placopecten striated, Placopecten catch, or Argopecten striated muscle. Measurement of the ATPase activities of these hybrids indicates clearly that it is the myosin heavy chain and not the regulatory light chains that are responsible for the muscle and species specific differences in enzymatic activities. Analysis of genomic DNA indicated that these regulatory light chain isoforms are products of a single regulatory light chain gene that is alternatively spliced in the 5 region only.     

Significance of wet autoclave pretreatment in immunohistochemistry

Until recently the only way to rescue masked epitopes in routinely processed surgical pathological material was enzymatic digestion. The use of heat for antigen retrieval, first by microwave irradiation, represents an important breakthrough in immunohistochemistry. With the acceptance of microwave oven pretreatment, various modified techniques and alternative heating methods have also been proposed. Wet autoclave pretreatment for tissue proteolysis is a highly reliable alternative to the microwave antigen retrieval technique. It provides uniform heating of the slides, hence an even enhancement of staining intensity in a variety of formalin-sensitive antigens, and it also offers consistent interlaboratory results. The method has been introduced in routine diagnostic immunohistochemistry for the detection of estrogen-and progesterone receptors, L26-, Ki-67- and bcl-2 antigens and variable types of cytokeratins (1/5/10/11, 8, 13, 19). Experimentally, wet autoclaving can be used very successfully for the immunophenotyping of p53 and mdm2 expression, for the detection of adhesion molecules (CD44, integrins) and some anti-inflammatory molecules (annexins), among others. It has produced a substantial improvement in the visualisation of silver-stained nucleolar organizer regionsassociated proteins (AgNORs) in routine paraffin sections and along with modified silver staining and standardized AgNOR parameters assessed by image analysis. Wet autoclaving-based AgNOR staining has been proposed by a European multicentric study group as the standardized method for AgNOR analysis in archival material.     

Proliferative (mib1, mdm2) versus anti-proliferative (p53) markers in head and neck cancer. An Immunohistochemical study

Formalin fixed and paraffin embedded samples from 36 squamous cell carcinomas of the larynx and the oral cavity (pT₂N₀M₀, R₀) surrounded by non-tumorous mucosa were studied immunohistochemically using a panel of four different anti-p53 antibodies (CM1, PAbl801, D07, PAb240), a monoclonal anti-mdm2 antibody and MIB1, following wet autoclave antigen retrieval. P53 immunoreactivity was detected in 11/14 laryngeal and in 9/22 oral carcinomas. All p53 positive oral, and all but one laryngeal tumors revealed mdm2 positivity as well, whereas in p53 negative tumors 4/12 and 1/3 mdm2 immunopositive cases were demonstrated, respectively. MIB1 labeling indices of the tumors ranged between 18% – 64% in p53 positive cases, and 10% – 53% in p53 negative ones. The difference was not statistically significant. Close spatial coexpression of p53, mdm2 and MIB1 immunoreactivity was observed at the invasive front of the carcinomas and in the basal and suprabasal layers of the non-tumorous epithelium in all p53 positive cases. However, the MIB1 expression was similarly increased at the invasive margins in carcinomas lacking immunohistochemically detectable p53 alterations. Our results strongly suggest that p53 overexpression does not necessarily correspond to increased rate of proliferation, but rather to mdm2 overexpression and is largely dependent on the anatomical site in case of small and localized squamous cell carcinomas of the head and neck region.     

A case of cutaneous angiolipoleiomyoma (angiomyolipoma) in a budgerigar (Melopsittacus undulatus)

We report a case of cutaneous angiolipoleiomyoma (angiomyolipoma) found on the anterior wall of the ventral part of the abdomen of a three-year-old female budgerigar (Melopsittacus undulatus). Histologic examination of the well-circumscribed, surgically removed tumour (1.5 cm in diameter) showed a benign admixed proliferation of blood vessels of different size, smooth muscle bundles, and mature adipose tissue, without evidence of malignancy. Endothelial cells of the haemangioma component were positive for claudin-5 endothelium-specific immunohistochemical marker, and the leiomyoma component was positive for α-smooth muscle actin. The differentiated lipocytes showed S-100 protein positivity. The Ki-67 labelling index was 2 to 3%. The mesenchymal tumour was negative for HMB45 melanocytic immunhistochemical marker. To the best of our knowledge, this is the first report describing a cutaneous angiolipoleiomyoma in a budgerigar with histological and immunohistochemical analyses.     

Effectiveness of intratympanic dexamethasone for the treatment of vertigo attacks in patients with Ménière’s disease compared with betahistine pharmacotherapy

ObjectiveThis study analyzed the possible effects of intratympanic steroid (ITS) therapy in the symptomatic treatment of vertigo attacks in patients with Ménière’s disease.MethodsThirty-five patients treated with ITS (dexamethasone) plus betahistine (Group A) and 35 patients treated with betahistine alone (Group B) were enrolled in this investigation. Complaints were analyzed using medical records and vertigo diaries. Statistical analysis was conducted using IBM SPSS V24 software.ResultsBased on the analysis, there were no significant differences in baseline features between the two groups. When the occurrence of vertigo attacks was compared using the Kaplan–Meier method, no significant difference was detected between Groups A and B (odds ratio [OR] = 1.051, 95% confidence interval [CI] = 0.965–1.067; p = 0.972). In addition, no difference in the incidence of vertigo attacks was noted in group A between the periods of treatment with betahistine alone and betahistine plus ITS when the groups were analyzed via logistic regression (OR = 1.07, 95% CI = 0.065–1.467; p = 0.614).ConclusionIt can be concluded that the addition of ITS therapy to betahistine did not improve outcomes in patients with Ménière’s disease. Further prospective studies should be conducted to analyze the results in a more detailed manner.     

Electron Microscopic Characteristics of β2-Microglobulin Amyloid Deposits in Long-Term Haemodialysis

The electron microscopic features of β2-microglobulin amyloid, deposited in the synovial membrane, are presented and discussed. The patient, a 69-year-old woman underwent chronic hemodialysis for 3 years. Because of constant pain and destructive arthropathy, endoprosthesis of the hip joints were implanted. Extra- and intracellular filamentous–fibrillar amyloid deposits have been demonstrated in ultrathin sections. The extracellular amyloid deposits showed a loose, filamentous or fibrillar structure at the periphery and a dense central core. The loose, filamentous structure may represent an early stage of fresh, newly deposited β2-microglobulin amyloid, while the condensed and fragmented amyloid filaments may be an advanced “mature” stage of amyloid deposition.