Internalizing disposition and preschool children's cortisol fluctuations.

BACKGROUND: Research on the impact of childcare suggested that young children's cortisol tends to increase or remain 'flat' across the day while their cortisol levels follow the typical circadian decrease between mid-morning and mid-afternoon at home. However, studies are needed to investigate what is happening to cortisol levels and whether individual variation exists in the cortisol rhythm in childcare. METHODS: Internalizing disposition was examined as a possible moderator of cortisol-linked stress response of young children in full-time, centre-based childcare. Ambulatory salivary sampling for cortisol was performed on 37 preschoolers at four different times for 10 consecutive school days. In order to test the interaction effect of internalizing disposition on cortisol levels, children were divided into four groups--the most internalizing group, the least internalizing group, and two groups in between, based on teacher-rated 'approach' scores. RESULTS: Repeated measure analyses of variance with polynomial contrast for time indicated that (1) a linear trend of significant increase between mid-morning and mid-afternoon exists for all children without group interactions; (2) quadratic and cubic trends exist and the interaction effects among the groups are significant, meaning the four groups are different in fluctuation pattern. Further analyses demonstrated that (1) the least internalizing children showed significant decreases in cortisol levels from morning to noon and after nap, although there is a considerable increase after lunch, (2) cortisol levels of the other three groups across the day fit the pattern of upward curve. CONCLUSION: The tradition of the field in comparing mid-morning and mid-afternoon levels of cortisol as an indicator of childcare effect may not reflect individual variation in the fluctuation patterns of cortisol. Moderating effect of child characteristics needs to be considered in future research of childcare effect.

High-potentiality preliminary selection criteria and transformation time-dependent factors analysis for establishing Epstein-Barr virus transformed human lymphoblastoid cell lines.

Infection of freshly isolated and cryopreserved lymphocytes with Epstein-Barr virus (EBV) leads to the establishment of human B lymphoblastoid cell lines (LCLs). Techniques for optimal infection of the lymphocytes are vital for the establishment of a human biobank. The present study found that more than half (58-86%) of such established LCLs had transport times of less than 48 h, cell densities exceeding 10(6) cells/ml and cell viabilities greater than 90%. After EBV infection, 3306 freshly isolated lymphocytes required 30.0 +/- 0.1 days to become LCLs. Conversely, 1210 cryopreserved lymphocytes required 36.2 +/- 0.4 days. Cell density and viability of the culture affected transformation time in freshly isolated lymphocytes. On the other hand, blood transport time, cryopreservation time and initial cell viability were major factors in cryopreserved specimens. These results contribute to general information concerning the establishment of a human biobank for EBV infected cells.

Molecular cloning of ras and rap genes from Entamoeba histolytica.

To better understand growth regulation in the protozoan parasite Entamoeba histolytica, ameba genes homologous to the ras oncogene and rap (Krev-1) anti-oncogene were cloned. Two putative ameba ras genes (Ehras1 and Ehras2) were identified, which contain 205 and 203 amino acid (aa) open reading frames (ORFs), respectively. The Ehras1 ORF shows an 91% positional identity with that of Ehras2, a 55% identity with Dictyostelium discoideum (Dd) ras, and a 47% identity with human (Hs) ras. Two ameba rap genes (Ehrap1 and Ehrap2) were identified, both of which contain 184-aa ORFs. The Ehrap1 ORF shows a 93% positional identity with that of Ehrap2, a 60% identity with Dd rap, a 61% identity with Hs Krev-1, and a 45% identity with that of Ehras1. Conserved aa in each ameba ras and rap ORF include GTP-binding sites, effector site, site of ADP-ribosylation by Pseudomonas exoenzyme S, and COOH-terminus CAAX. As all Xs = Leu or Phe, ameba ras and rap proteins may be gerenylgerenylated and not farnesylated. Both ras and rap genes are transcribed by trophozoites. A single 21-kDa ameba ras protein reacts with the rat Y13-259 anti-ras monoclonal antibody, which is located on the cytosolic side of the plasma membrane. These are the first ras and rap genes identified from a protozoan parasite.

P-glycoprotein genes of Entamoeba histolytica.

Six different P-glycoprotein gene segments were identified from an emetine-resistant E. histolytica mutant, which overexpresses mRNAs homologous to segments of the human mdr1 (P-glycoprotein) gene. The open reading frames of two completely sequenced genes EhPgp1 and EhPgp2 were 1,302 and 1,310 amino acids long, respectively, and showed a 67% positional identity with each other and 41 and 40% positional identities, respectively, with human mdr1 gene. Within each ameba P-glycoprotein were the ATP-binding sites found twice in eukaryotic P-glycoproteins and once in prokaryotic transport proteins. A phylogenetic tree showed that Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins than to the Plasmodium and Leishmania P-glycoproteins. In addition, there were two P-glycoprotein pseudogenes, each with a frame shift and stop codons in identical places within the amino ATP-binding site.

Actin associated proteins of Entamoeba histolytica.

Treatment of E. histolytica HM1-IMSS trophozoite extracts to conditions that produce gels of actin and associated cytoskeletal proteins in other ameboid cells caused formation of macroscopic actin rich complexes (ARCs). The one-dimensional PAGE protein profile of this ARC was similar to those of Dictyostelium and Acanthamoeba actin gels. Formation of the E. histolytica ARCs was enhanced by added lipids. In addition to actin, the ARC was enriched with proteins that showed cross-reactivity to antibodies to alpha-actinin and the 50K actin binding protein (elongation factor 1 alpha) from Dictyostelium. E. histolytica ARCs appear to be comprised of a number of actin cytoskeleton proteins and provide a source for their isolation and characterization.

Primary structures of alcohol and aldehyde dehydrogenase genes of Entamoeba histolytica.

Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite E. histolytica under the anaerobic conditions found in the lumen of the colon. With the goal of finding new targets for anti-amebic drugs, the E. histolytica NADP(+)-dependent alcohol dehydrogenase gene (EhADH1; EC 1.1.1.2) and an aldehyde dehydrogenase gene (EhALDH1; EC 1.3.2) were cloned. The EhADH1 alcohol dehydrogenase gene encoded -39 kDa protein with 62 and 60% amino acid identities, respectively, with NADP(+)-dependent alcohol dehydrogenases of anaerobic bacteria Thermoanaerobium brockii and Clostridia beijerinckii. In contrast, EhADH1 showed a 15% amino acid identity with the closest human alcohol dehydrogenase. An EhADH1-glutathione-S-transferase fusion protein showed the expected NADP(+)-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADH1 fusion protein were inhibited by pyrazole and 4-methyl pyrazole. The E. histolytica aldehyde dehydrogenase EhALDH1 gene encoded a 60 kDa protein, which showed a 36% amino acid identity over a 451 amino acid overlap with the human stomach aldehyde dehydrogenase (ALDH3).

Immunofluorescent, immunogold, and electrophoretic studies for desmin in embryonic hearts of normal and cardiac mutant Mexican axolotls, Ambystoma mexicanum.

Recessive mutant gene c for "cardiac nonfunction" in axolotls results in an absence of normal heart contractions in affected embryos due to a failure of myofibril formation. In the present study, the intermediate filament protein, desmin, is compared in developing normal and mutant hearts by means of two-dimensional gel electrophoresis, immunofluorescent microscopy, and immunoelectron microscopy. Tissues were fixed in periodate-lysine-paraformaldehyde or paraformaldehyde-glutaraldehyde solutions and rapidly frozen or embedded in Lowicryl resin. Frozen sections stained with FITC-conjugated antibodies by an indirect approach revealed that desmin is localized in the I-band regions of adult cardiac myofibrils. In normal embryonic hearts at stage 32 (preheartbeat) desmin is localized as "spots" or amorphous collections in the cells. As development progresses to stage 35, staining for desmin in normal hearts becomes more intense with localization being most pronounced at the cell peripheries. By stage 41 most of the desmin in normal hearts is localized in the I band areas of the organized myofibrils and the staining of amorphous areas is much less prominent. During early development, the distribution of desmin in mutant hearts is similar to normal. However, while most of the desmin in normal organs at stage 41 is associated with myofibrils, the staining remains diffuse in mutants. Two-dimensional gel electrophoresis reveals comparable patterns for desmin from normal and mutant hearts. Immunogold staining shows desmin localization to be between the myofibrils and around the I-band regions in adult cardiac muscle and in stage 41 normal embryonic hearts. Immunogold staining confirms a diffuse distribution of desmin in mutant hearts.

Analysis of two sling procedures using polypropylene mesh for treatment of stress urinary incontinence.

OBJECTIVES: To evaluate and compare the surgical outcome between the innovative tension-free vaginal tape (TVT) and conventional pubovaginal sling (PVS) procedures using polypropylene mesh. METHODS: Eighty consecutive women with urodynamic stress urinary incontinence (SUI), who chose to undergo either a TVT (n=23) or a PVS (n=57) procedure using polypropylene mesh based on financial consideration, were recruited for this study. The surgical results were analyzed and compared subjectively and objectively. RESULTS: The mean follow-up interval was 23 months for the TVT and 20 months for the PVS procedure (P=0.062). Postoperatively, SUI (91.3% vs. 93.0%), concomitant urge symptoms (85.0% vs. 85.3%) and the negative impact of incontinence and urogenital distress on patients' quality of life (79.8% vs. 77.8%) (77.4% vs. 68.8%) had improved markedly. After a multivariable logistic regression analysis, the treatment outcome of SUI was found to be independent of the main effects of patient age, parity, concurrent gynecological surgeries, intrinsic sphincter deficiency, previous failed incontinence surgeries, and concomitant urge symptoms. However, it was significantly related to treatment procedures (TVT vs. PVS) and their interaction with patient body mass index (BMI). Based on the fitted logistic model, we see that TVT performs better than PVS when BMI is less than 27.27 kg/m2, and the advantage of TVT decreases as BMI increases. CONCLUSION: Both TVT and PVS procedures using polypropylene mesh are effective treatment modalities for female SUI. However, TVT was not as effective in treating overweight or obese women as PVS.

Oxidative stress in nonallergic nasal polyps associated with bronchial hyperresponsiveness.

BACKGROUND: Nasal polyposis (NP) is a chronic inflammatory disease of upper airway with unknown etiology. NP is frequently associated with asthma; the interaction between these comorbidities remains interesting. Oxidative stress has been implicated in the pathophysiology of NP and asthma. The aim of this study is to investigate the significance of oxidative stress in sinonasal microenvironments by evaluating its association with clinopathological parameters and its impacts on the pathogenesis of bronchial hyperresponsiveness (BHR) in NP. METHODS: Polyp biopsy specimens were obtained from 20 nonallergic patients; control mucosas were obtained from 20 volunteers. The levels of free radicals in the tissues and in blood were determined by a sensitive chemiluminescence (CL) method. NP patients were substratified into three subgroups, NP without BHR, NP with asymptomatic BHR, and NP with BHR and asthma by the results of provocative testing. Four histological characteristics of NP, inflammatory cells, eosinophil infiltration, edema and fibrosis were estimated and applied to correlate with the tissue-CL. RESULTS: The mean CL level in polyp-tissues, but not in blood, was higher than in the control specimens. In NP patients, tissue-CL was associated with endoscopy score; high tissue-CL levels were positively correlated with the abundance of inflammatory cells and eosinophils. Tissue-CL and endoscopy score were associated with BHR/asthma phenotype. CONCLUSION: These results suggest an important role for oxidative stress in the pathophysiology of NP and a causal relation between oxidative stress and inflammatory cells, especially the eosinophils. Free radical levels in polyp-tissues associated with NP severity and with BHR/asthma phenotype in nonallergic NP patients.

Cloning and expression of an NADP(+)-dependent alcohol dehydrogenase gene of Entamoeba histolytica.

Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite Entamoeba histolytica under the anaerobic conditions found in the lumen of the colon. Here an internal peptide sequence determined from a major 39-kDa amoeba protein isolated by isoelectric focusing followed by SDS/PAGE was used to clone the gene for the E. histolytica NADP(+)-dependent alcohol dehydrogenase (EhADH1; EC 1.1.1.2). The EhADH1 clone had an open reading frame that was 360 amino acids long and encoded a protein of approximately 39 kDa (calculated size). EhADH1 showed a 62% amino acid identity with the tetrameric NADP(+)-dependent alcohol dehydrogenase of Thermoanaerobium brockii. In contrast, EhADH1 showed a 15% amino acid identity with the closest human alcohol dehydrogenase. EhADH1 contained 18 of the 22 amino acids conserved in other alcohol dehydrogenases, including glycines involved in binding NAD(P)+ as well as histidine and cysteine residues involved in binding the catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, EhADH1 lacked a 23-amino acid stretch present in other alcohol dehydrogenases that includes four cysteines that bind a second noncatalytic zinc ion. An EhADH1-glutathione-S-transferase fusion protein showed the expected NADP(+)-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADH1 fusion protein were inhibited by pyrazole and 4-methylpyrazole.

Factors that affect recurrence after anterior colporrhaphy procedure reinforced with four-corner anchored polypropylene mesh.

The purpose of this study was to evaluate the effectiveness of the anterior colporrhaphy procedure reinforced with four-corner anchored polypropylene mesh in patients with severe (stage III or IV) anterior vaginal prolapse. Thirty-eight consecutive women were enlisted for this prospective study. The procedure consisted of an extensive vaginal dissection to join the vesicovaginal and retropubic space and an anchoring of a polypropylene mesh patch between the two Arcus Tendineus Fasciae Pelvis in a tension-free manner. The mean age of the study group was 63 (33-80) years. The success rate was 87% (33/38) at a mean follow-up interval of 21 (12-29) months. A total of eight (100%) patients were also cured of concomitant stress incontinence (five overt and three occult type) with an additional tension-free vaginal tape (TVT) operation. During follow-up, there were five de-novo stress incontinence cases (16.7%) and four vaginal erosions of mesh (10.5%). Four clinical variables--diabetes mellitus, recurrent anterior vaginal prolapse, chronic cough and vaginal erosions of mesh--were found to have a significant correlation with an unsatisfactory surgical result with large values of hazard ratios found by survival analysis. We concluded that the anterior colporrhaphy procedure reinforced with four-corner anchored polypropylene mesh was effective for most, but failed in some patients who had specific risk factors within short convalescence periods. Concomitant stress incontinence can be successfully treated by a TVT operation in combination with the anterior colporrhaphy procedure reinforced with four-corner anchored polypropylene mesh. However, the anterior colporrhaphy procedure may itself have adverse effects on urethral sphincter function.