Expression of lactate transporters MCT1, MCT2, MCT4 and the ancillary protein CD147 in horse muscle and red blood cells

Monocarboxylate transporters (MCTs) transport lactate and protons across cell membranes. During intense exercise, lactate and protons accumulate in the exercising muscle and are transported to the plasma. In the horse, MCTs are responsible for the majority of lactate and proton removal from exercising muscle, and are therefore also the main mechanism to hinder the decline in pH in muscle cells. Two isoforms, MCT1 and MCT4, which need an ancillary protein CD147, are expressed in equine muscle. In the horse, as in other species, MCT1 is predominantly expressed in oxidative fibres, where its likely role is to transport lactate into the fibre to be used as a fuel at rest and during light work, and to remove lactate during intensive exercise when anaerobic energy production is needed. The expression of CD147 follows the fibre type distribution of MCT1. These proteins were detected in both the cytoplasm and sarcolemma of muscle cells in the horse breeds studied: Standardbred and Coldblood trotters. In humans, training increases the expression of both MCT1 and MCT4. In this study, the proportion of oxidative fibres in the muscle of Norwegian-Swedish Coldblood trotters increased with training. Simultaneously, the expression of MCT1 and CD147, measured immunohistochemically, seemed to increase more in the cytoplasm of oxidative fibres than in the fast fibre type IIB. Horse MCT4 antibody failed to work in immunohistochemistry. In the future, a quantitative method should be introduced to examine the effect of training on muscle MCT expression in the horse. Lactate can be taken up from plasma by red blood cells (RBCs). In horses, two isoforms, MCT1 and MCT2, and the ancillary protein CD147 are expressed in RBC membranes. The horse is the only species studied in which RBCs have been found to express MCT2, and the physiological role of this protein in RBCs is unknown. The majority of horses express all three proteins, but 10-20% of horses express little or no MCT1 or CD147. This leads to large interindividual variation in the capacity to transport lactate into RBCs. Here, the expression level of MCT1 and CD147 was bimodally distributed in three studied horse breeds: Finnhorse, Standardbred and Thoroughbred. The level of MCT2 expression was distributed unimodally. The expression level of lactate transporters could not be linked to performance markers in Thoroughbred racehorses. In the future, better performance indexes should be developed to better enable the assessment of whether the level of MCT expression affects athletic performance. In human subjects, several mutations in MCT1 have been shown to cause decreased lactate transport activity in muscle and signs of myopathy. In the horse, two amino acid sequence variations, one of which was novel, were detected in MCT1 (V432I and K457Q). The mutations found in horses were in different areas compared to mutations found in humans. One mutation (M125V) was detected in CD147. The mutations found could not be linked with exercise-induced myopathy. MCT4 cDNA was sequenced for the first time in the horse, but no mutations could be detected in this protein.

Origins of the sex differences in handedness and mental rotation ability : genetic, environmental and hormonal effects

In humans, well-replicated and robust sex differences in cognitive functions exist for handedness and mental rotation ability. A common characteristic in human cognitive functions is the lateralization of language functions. Handedness is a common measure of laterality and is related to language lateralization. The prevalence of left-handedness is higher in males than in females, the male to female ratio being about 1.2. Among cognitive abilities, the largest sex difference is evident in the Vandenberg and Kuse Mental Rotation Test (MRT), which requires the ability to rotate objects in mental space. On average, males achieve scores one standard deviation higher than females in the MRT. The present thesis investigated the origins of the sex differences in laterality and spatial ability as represented by handedness and mental rotation ability, respectively. Two population-based Finnish twin cohorts were utilized in this study. Handedness was studied in 25 810 twins and 4068 singletons born before 1958 from the Older Finnish Twin Cohort, and in 4736 twins born in 1983-87 from the FinnTwin12. MRT was studied in a sub-sample of 804 young adult participants from the FinnTwin12 sample. The main findings of this study were: 1) the prevalence of left-handedness was higher among males than among females in both singletons and in twins; 2) males had significantly higher scores than females in MRT; 3) about one quarter of the variance in handedness and about half of the variance in MRT was explained by genetic effects, whereas the remainder of the variance in these traits was explained by environmental effects unique to each individual. The magnitude of the genetic effects was similar in both sexes; 4) left-handedness was significantly less common in female co-twins of a male than in female co-twins of a female, and female co-twins of a male scored significantly higher than did female co-twins of a female in the Mental Rotation Test. This dissertation discusses whether these differences between females from opposite- and same-sex twin pairs are due to the prenatal transfer of testosterone from the male fetus in females with male co-twins or whether they arise from postnatal socialization effects.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3

The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05 M cadmium sulfate hydrate, 0.1 M HEPES buffer pH 7.5 and 1.0 M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 95.62, c = 149.13 angstrom. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (V-M) of 2.3 angstrom(3) Da(-1), corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides.

Japanese Encephalitis Virus Utilizes the Canonical Pathway To Activate NF-kappa B but It Utilizes the Type I Interferon Pathway To Induce Major Histocompatibility Complex Class I Expression in Mouse Embryonic Fibroblasts

Flaviviruses have been shown to induce cell surface expression of major histocompatibility complex class I (MHC-I) through the activation of NF-kappa B. Using IKK1(-/-), IKK2(-/-), NEMO-/-, and IKK1-/- IKK2-/- double mutant as well as p50(-/-) RelA(-/-) cRel(-/-) triple mutant mouse embryonic fibroblasts infected with Japanese encephalitis virus (JEV), we show that this flavivirus utilizes the canonical pathway to activate NF-kappa B in an IKK2- and NEMO-, but not IKK1-, dependent manner. NF-kappa B DNA binding activity induced upon virus infection was shown to be composed of RelA: p50 dimers in these fibroblasts. Type I interferon (IFN) production was significantly decreased but not completely abolished upon virus infection in cells defective in NF-kappa B activation. In contrast, induction of classical MHC-I (class 1a) genes and their cell surface expression remained unaffected in these NF-kappa B-defective cells. However, MHC-I induction was impaired in IFNAR(-/-) cells that lack the alpha/beta IFN receptor, indicating a dominant role of type I IFNs but not NF-kappa B for the induction of MHC-I molecules by Japanese encephalitis virus. Our further analysis revealed that the residual type I IFN signaling in NF-kappa B-deficient cells is sufficient to drive MHC-I gene expression upon virus infection in mouse embryonic fibroblasts. However, NF-kappa B could indirectly regulate MHC-I expression, since JEV-induced type I IFN expression was found to be critically dependent on it.

ErbB-2 expression and its association with other biological parameters of breast cancer among Indian women

Overexpression of the epidermal growth factor receptor family genes, which include ErbB-1, 2, 3 and 4, has been implicated in a number of cancers. We have studied the extent of ErbB-2 overexpression among Indian women with sporadic breast cancer. Methods: Immmunohistochemistry and genomic polymerase chain reaction (PCR) were used to study the ErbB2 overexpression. ErbB2 status was correlated with other clinico-pathological parameters, including patient survival. Results: ErbB-2 overexpression was detected in 43.2% (159/368) of the cases by immunohistochemistry. For a sub-set of patients (n = 55) for whom total DNA was available, ErbB-2 gene amplification was detected in 25.5% (14/55) of the cases by genomic PCR. While the ErbB2 overexpression was significantly higher in patients with lymphnode (χ2 = 12.06, P≤ 0.001), larger tumor size (χ2 = 8.22, P = 0.042) and ductal carcinoma (χ2 = 15.42, P ≤ 0.001), it was lower in patients with disease-free survival (χ2 = 22.13, P ≤ 0.001). Survival analysis on a sub-set of patients for whom survival data were available (n = 179) revealed that ErbB-2 status (χ2 =25.94, P ≤ 0.001), lymphnode status (χ2 = 12.68, P ≤ 0.001), distant metastasis (χ2 = 19.49, P ≤ 0.001) and stage of the disease (χ2 = 28.04, P ≤0.001) were markers of poor prognosis. Conclusions: ErbB-2 overexpression was significantly greater compared with the Western literature, but comparable to other Indian studies. Significant correlation was found between ErbB-2 status and lymphnode status, tumor size and ductal carcinoma. ErbB-2 status, lymph node status, distant metastasis and stage of the disease were found to be prognostic indicators.

The Organisation of Policy Meets the Commercialisation of Sex - Global Linkages, Policies, Technologies

During recent years the commercialisation of sex has increased and intensified both locally and globally. This thesis explores the commercialisation of bodies, sex and sexualities, particularly the sex trade, and the impact of rapidly evolving information and communication technologies on this globalising trade. The main focus of this work is on the policies, discourses and policy developments in the area especially in the Finnish context. The study is based on multidisciplinary theoretical sources through which the framework of multiple linkages relevant to the commercialisation of sex is conceptualised. The sex trade functions through a web of intersecting linkages of a substantive, economic, organisational, temporal, spatial, cultural, technological, as well as of legislative and policy nature. This framework of linkages forms the basis for the analysis of the main empirical data, namely qualitative interviews with thirty key managers and professionals, who are responsible for the preparation and implementation of policies on commercial sex. In addition, the thesis addresses the policies and policy practices on the sex trade through the analysis of national and international policy instruments. In addition to analysing the processes of the commercialisation of sex and its effects, the study also discusses their further implications for organisational policy-making, research, and society more generally. The thesis thus seeks to contribute to practice, research and theory on gender, management and organisations.

Expression of cyclin E in endomitotic silk-gland cells from mulberry silkworm

The silk glands of mulberry silkworm Bombyx mori are endoreplicating tissues in which the genomic DNA undergoes multiple rounds of replication without mitosis and nuclear division. In the absence of normal mitotic division, the cell cycle essentially alternates between the G1 and S phases. Cyclin E is crucial for the G1/S transition in both mitotic and endoreplicating cycles. We have cloned and characterized cyclin E (cyclin box) from B. mori, which is nearly identical to the Drosophila cyclin E box except for an insertion of 21 amino acids. Two distinct cyclin E transcripts (1.7 and 2.1 kb) were detected in the silk-gland cells of B. mori and in the B. mori-derived embryonic cell line, BmN. Using anti Cyclin E antibodies two protein bands of 52 and 44 kDa were detected in silk glands and BmN cells at Comparable levels. Both BmN- and the silk-gland cells showed the presence of the interacting kinase Cdk2. Transcripts of the mitotic cyclin, cyclin B, were barely detectable in the endoreplicating silk-gland cells and amounted to only 4-7% of that seen in the mitotically dividing BmN cells. The near absence of cyclin B transcripts and the abundant expression of cyclin E in the silk glands correlate well with the alternation of only G1 and S phases without the intervening mitosis in these cells. (C) 2000 Elsevier Science B.V. All rights reserved.

Expression of GC-C, a receptor-guanylate cyclase, and its endogenous ligands uroguanylin and guanylin along the rostrocaudal axis of the intestine

Members of the receptor-guanylate cyclase (rGC) family possess an intracellular catalytic domain that is regulated by an extracellular receptor domain. GC-C, an intestinally expressed rGC, was initially cloned by homology as an orphan receptor. The search for its Ligands has yielded three candidates: STa (a bacterial toxin that causes traveler's diarrhea) and the endogenous peptides uroguanylin and guanylin. Here, by performing Northern and Western blots, and by measuring [I-125]STa binding and STa-dependent elevation of cGMP levels, we investigate whether the distribution of GC-C matches that of its endogenous ligands in the rat intestine. We establish that 1) uroguanylin is essentially restricted to small bowel; 2) guanylin is very low in proximal small bowel, increasing to prominent levels in distal small bowel and throughout colon; 3) GC-C messenger RNA and STa-binding sites are uniformly expressed throughout the intestine; and 4) GC-C-mediated cGMP synthesis peaks at the proximal and distal extremes of the intestine (duodenum and colon), but is nearly absent in the middle (ileum). These observations suggest that GC-C's activity may be posttranslationally regulated, demonstrate that the distribution of GC-C is appropriate to mediate the actions of both uroguanylin and guanylin, and help to refine current hypotheses about the physiological role(s) of these peptides.

Regulation of cytochrome P-450b/e gene expression by a heme- and phenobarbitone-modulated transcription factor

The cloned DNA fragment of the cytochrome P-450b/e gene containing the upstream region from position -179 through part of the first exon is faithfully transcribed in freeze-thawed rat liver nuclei. Phenobarbitone treatment of the animal strikingly increases this transcription, and the increase is blocked by cycloheximide (protein synthesis inhibitor) or CoCl2 (heme biosynthetic inhibitor) treatment of animals. This picture correlates very well with the reported cytochrome P-450b/e mRNA levels in vivo and run-on transcription rates in vitro under these conditions. The upstream region (from position -179) was assessed for protein binding with nuclear extracts by nitrocellulose filter binding, gel retardation, DNase I treatment ("footprinting"), and Western blot analysis. Phenobarbitone treatment dramatically increases protein binding to the upstream region, an increase once again blocked by cycloheximide or CoCl2 treatments. Addition of heme in vitro to heme-deficient nuclei and nuclear extracts restores the induced levels of transcription and protein binding to the upstream fragment, respectively. Thus, drug-mediated synthesis and heme-modulated binding of a transcription factor(s) appear involved in the transcriptional activation of the cytochrome P-450b/e genes, and an 85-kDa protein may be a major factor in this regard.

Consanguinity, twinning and secondary sex ratio in the population of Karnataka, South India

Consanguineous marriages are strongly favoured in the state of Karnataka. Of 65492 marriages studied 33·07% were consanguineous, equivalent to a coefficient of inbreeding (F) of 0·0298. The twinning rate was low, 6·9 per thousand, whereas the secondary sex ratio, 0·5221, was higher than in comparable major human populations. Consanguinity exerted no significant effect on either parameter. The results also indicate that consanguinity is not associated with excess antenatal losses and suggest the possibility of enhanced selection against mutations at X chromosome loci.

Gene expression profile of epithelial cells and mesenchymal cells derived from limbal explant culture

Purpose: Limbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out.Methods: RNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4x44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction. Results: The microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM.Conclusions: This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.

Mihin ryhmään itse haluat kuulua? : Kahdessa yläkoulun terveystiedon oppikirjassa rakentuvien terveysdiskurssien ja subjektipositioiden diskurssianalyyttistä tarkastelua

In the study, two upper elementary school health education textbooks were investigated. The purpose of the study was to examine the health discourses and subject- and reader positions constructed in text. Theoretically, the study is based on poststructuralist thought and critical sociology of health promotion. Methodologically, it draws mainly on critical lingvistics and new rhetorics. Textbooks were understood as informative, argumentative and persuasive texts in which different lexical and grammatical methods to secure the readers´ responsiveness were utilized. Also, the relations of the text to wider genres, social situations, structures, institutions and practices were investigated. The interpersonal and ideational dimensions of the texts were analysed with the aim of finding out the kinds of identities for and relations between the speaker and the ideal reader were constructed and the kinds of representations of health and the world around were produced in the textbooks. Multiple discourses of health, and genres and styles characteristic for many kinds of contexts and situations were found. The identities of and the relationships between the speaker and the ideal reader of the text were also multiple and changing. The text echoes both biomedical health discourse emphasizing prevention of illness and holistic discourse emphasizing personal welfare, fulfillment and happiness. Furthermore, traces for example from development psychological, ecological and civilization critical discourses were perceived. Formal scientific genre was found to be mixed with informal chatting imitating close and equal relationship between participants characteristic for advertisements and other persuasive texts, and obliging and ordering expression typical for school context and other situations where the relationship between participants is unequal and distant. The ideal reader of the text can be characterized as adolescent living in the world saturated by advertising and media. He or she is interested in the life of the celebrities, and is interested rather in her or his appearance, image and short-term enjoyment than health and long-term welfare. In the textbooks, healthy way of life is attempted to create a product which appeals to the values and interests of the imaginary public, the ideal reader of the text. Marketing healthy choices tend to reproduce stereotyped ideas of happiness, good life, youth and sex. Furthermore, individualizing approach mixed with wide definition of health legitimizes easily an erraneous impression of health, beauty and success being personal achievements dependent only on attitudes and competences.