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ERV9 is a class I family of human endogenous retroviral sequences. Somatic cell hybrid genomic hybridization experiments using a mono-chromosomal panel indicate the presence of approximately 120 ERV9 loci in the human genome distributed on most chromosomes. Fluorescence in situ hybridization (FISH) using an ERV9 cDNA probe containing gag , pol and env sequences, verified this observation and a consistent signal was found at the chromosome region 11q13.3→q13.5. By analysis of a panel of radiation hybrids, an ERV9 locus was mapped to within a 300-kbp region at the chromosome site 11q13. The marker cCLGW567 and the locus MAP3K11/D11S546 centromeric and telomeric flanked it, respectively. Northern blot analysis, using an ERV9 LTR probe, indicated that most normal tissues examined expressed low abundant ERV9 LTR driven mRNAs of various sizes. The most prominent expression was found in adrenal glands and testis. However, the level of expression varied in the same tissues among different individuals indicating that ERV9 mRNA expression probably is inducible in certain tissues or at various cell stages. Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The tentative amino acid sequence of pooled, papain-solubilized HLA antigen heavy chains has been determined. The amino acid sequence comprises 273 residues. As the structural analyses were performed on HLA antigen heavy chains comprising a mixture of several allelic forms derived from the A, B, and possibly C loci, multiple residues were encountered in several positions. However, a quantitatively dominating residue could always be easily identified. The present data suggest that the amino acid variability of the HLA-A, -B, and -C antigens is found in restricted regions of the molecule. The COOH-terminal third of the HLA antigen heavy chain appears to be less variable than other regions of the molecule. Previous work has shown that the HLA antigen heavy chain contains two immunoglobulin-like disulphide loops. The COOH-terminal third of the heavy chain was shown to be similar in primary structure to β2-microglobulin and the immunoglobulin G constant domains. [ABSTRACT FROM AUTHOR]

Purified intestine epithelial cells express la antigens as determined by immunoprecipitation and sodium dodecyl sulphate polyacrylamide-gel electropheresis. The intestimal la antigens react at least with alloantisera directed against the H-2 I-A subregion. [ABSTRACT FROM AUTHOR]

Deoxycholate-solubilized HLA antigens have been isolated from platelets and comprised a mixture of 43,000- and 39,000-dalton polypeptide chains associated with β2-microglobulin. Limited proteolysis experiments suggested that the 39,000-dalton chain is a fragment of the intact 43,000-dalton chain. Further proteolysis of the 39.000-dalton fragment yields a 33,000- dalton component. The 39,000-dalton molecule is more acidic than both the 43,000- and the 33,000-dalton chains. Differences in the amino acid compositions of the 43,000- and 39,000- dalton species demonstrate that the peptide(s) released on generation of the 39,000-dalton comcomponent are charged. The proteolytic split most probably occurs in the COOH-terminal end, which, owing to its content of charged amino acids, most probably is not integrated into the hydrocarbon matrix of the membrane. The 39,000- and 43,000-dalton components bind detergent in micellar form and can be incorporated into liposomes. The 33,000-dalton fragment has lost the ability to bind detergent micelles and is not incorporated into liposomes. [ABSTRACT FROM AUTHOR]

Papain-solubilized HLA-A,B,C antigen heavy chains have been cleaved by combined acid and CNBr treatment to yield three large fragments. A 14,000-dalton peptide comprises the NH2-terminal portion of the molecule, less a five-membered peptide. The 14,000-dalton fragment is followed in the linear sequence by a 9000-dalton peptide connected through an aspartyl-prolyl bond to the COOH-terminal 11,000-dalton fragment. The 9000- and 11,000-dalton fragments contain disulphide bridges that are immunoglobulin-like inasmuch as they encompass some fifty-five to sixty amino acid residues. The NH2-terminal portion of the HLA antigen heavy chain is devoid of cysteine. NH2-terminal amino acid sequence analyses do not reveal homologies between the 14,000- and 9000-dalton fragments, β2-microglobulin, and the constant immunoglobulin domains. However, the NH2-terminal sequence of the 11,000-dalton fragment is as homologous to β2-microglobulin and the constant immunoglobulin domains as they are to one another. [ABSTRACT FROM AUTHOR]

The genetic basis for the extensive polymorphism of major histocompatibility complex (MHC) class II molecules was investigated by statistical analysis. Nucleotide sequences of human DQA1, DQB1, DRB1, and DRB3 genes and routine Aα, Aβ, and Eβ genes were used. The results show that polymorphism is selected for in the antigen recognition site of class II molecules since replacement substitutions in this region were found to occur at a significantly higher frequency than expected in the absence of selection. In contrast, replacement substitutions are selected against in the remaining part of the first domain exon and in the second domain exon. Furthermore, comparing the sequence variability pattern among different class II α and sequences, using a variability index for each residue, showed that, with few exceptions, highly polymorphic residues occur in the antigen recognition site. There was a strong and highly significant correlation in the variability pattern in the homologous DRB/Eβ sequences but not for DQB/Aβ or DQA/Aα sequences. This difference may be related to the fact that both α and chains of DQ/A molecules are polymorphic, while only β chains of DR/E molecules vary. [ABSTRACT FROM AUTHOR]

A truncated form of the class II antigen DRα chain of the human major histoeompatibility complex was produced in bacteria. A cDNA clone encoding the intact chain was modified so that the segment encoding the signal sequence was replaced by an ATG codon and the 3′ region downstream to the part corresponding to the third exon was replaced by a stop codon. The new construct was put under the control of the Tae promoter in a bacterial expression vector. The distance between the Shine-Delgarno sequence and the initiation codon was randomized so that clones with optimal expression of the truncated DRα chain could be obtained after induced expression and immunoscreening. The truncated DR a chain was subjected to limited proteolysis with chymotrypsin, and the resulting cleavage products were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Two fragments were visualized by western blotting. Electrophoresis in the absence and presence of reducing agents suggested that one of the proteolytic fragments contained a disulphide bridge. It is concluded that the extracellular portion of the DRα chain is composed of two compactly folded domains connected by an extended stretch of the polypeptide chain. [ABSTRACT FROM AUTHOR]

Thirty-six consecutive paediatric patients (0-16 years old) with recently contracted juvenile diabetes (IDDM) during 1982-84 were included in the study. Sera were assayed for recent or current Coxsackie B virus (CBV) infection using a specific and sensitive IgM RIA, Eighteen patients (50%) had IgM against CBV 1-5, The patients were also assayed for restriction fragment length polymorphism (RFLP) patterns with DNA probes coding for HLA-DR and DQ beta chains. The CBV-positive patients (n=18) had either RFLP patterns associated with HLA-DR 3 or 4 or HLA-DQ patterns III or IV beta. Two of the CBV negative patients had neither HLA-DR 3 nor DR 4 and four of them had neither DQ patterns III nor IV, Eleven out of 18 CBV-positive patients had HLA-DQ III and DR 3 (61%) versus 5 out of 18 (28%) of the CBV-negative patients. All 11 patients with serology positive for CBV 2,3, and 5 had HLA-DR 4 and DQ IV patterns. This was significantly (P<0.01) different from all five CBV 4-positive patients, who in contrast all had HLA-DR 3 or HLA-DQ III patterns, CBV 1-positive patients (n=2) all had HLA-DR 3, 4, and HLA-DQ III, IV patterns. Thus CBV 4 seems to be significantly associated with a different host genetic constitution from at any rate CBV 2,3, and 5, and possibly CBV 1. [ABSTRACT FROM AUTHOR]

The signal sequences of two HLA-DR β chains and the DR α chain were determined. In addition, the major part of a DC β-chain signal sequence was also elucidated. The data were obtained by combining amino acid sequence analyses of isolated α and β chains with nucleotide sequencing of four cDNA clones. All signal sequences comprise 25 amino acids or more. The two HLA-DR β-chain signal sequences are identical and exhibit only marginal homology to the DC β-chain signal sequence. No homology is apparent between α- and β-chain signal sequences. The differences in the signal sequences of the DR and DC β chains suggest that these sequences may be used to assign β chains to different loci of the human major histocompatibility complex. [ABSTRACT FROM AUTHOR]

Membrane-bound mRNA was isolated from Raji cells and enriched for message coding for the HLA-DR transplantation antigen α-chain by sucrose gradient centrifugation. Double-stranded cDNA was constructed from this mRNA fraction, ligated to plasmid pBR322, and cloned into Escherichia coli. By hybrid selection, a plasmid, pDR-α-1, able to hybridize with mRNA coding for the HLA-DR α-chain was identified. From the nucleotide sequence of one end of the insert an amino acid sequence was predicted which is identical to part of the amino-terminal sequence of an HLA-DR α-chain preparation isolated from Raji cells. This clearly shows that pDR-α-1 carries almost the complete message for an HLA-DR α-chain. From the nucleotide sequence of this plasmid it will be possible to predict the primary structure of an HLA-DR α-chain. [ABSTRACT FROM AUTHOR]

cDNA for a Βchain of HLA-DR antigens was cloned and the partial nueleotide sequence was determined. The data suggest that the fl-chain consists of approximately 230 amino acids. ot which about 200 are exposed on the cell surface. The 6-chain appears to be composed of Two exposed disulphide-containing domains. The arrangement of the disulphide loops suggests that the 5-chain is similar in structure to the HLA-A, B, C antigen subunits and the immunoglobulin chains. for the Βchain domain closest to the membrane this similarity was verified at the level of primary structure. The partial amino acid sequence of the NH2>terminal domain did not display any apparent homology to HLA-A, B, C antigens and immunoglobulins. However, the similarity established here between the two types of major histocompatibility antigen subunits and the immunoglobulin chains suggests a common ancestral origin for at least some regions of these molecules. [ABSTRACT FROM AUTHOR]

A nucleoside diphosphate kinase has been highly purified from baker's yeast, and has been obtained in a crystalline form from an ethanol‐containing medium. During short‐time incubation with [32P]ATP, the enzyme was phosphorylated to an extent of about 3–4 phosphoryl groups per mole of enzyme assuming a molecular weight of 105. This indicates an intermediate phosphorylation of the enzyme. 1‐[32P]Phosphohistidine was shown to be the dominating radioactive degradation product in an alkaline hydrolysate of the 32P‐labelled enzyme. In addition, 3‐[32P]‐phosphohistidine and Ne‐[32P]phospholysine were obtained in small amounts. The results indicate that the amino acid sequence around the reactive 1‐phosphohistidine of the yeast enzyme is different from that of human erythrocytic and bovine liver nucleoside diphosphate kinase. [ABSTRACT FROM AUTHOR]

Vitamin A has, apart from its function in the visual pigments, general effects on several organs. Early signs of vitamin A deficiency include keratinization of epithelia and hyperkeratosis of the skin. To elucidate a generalized function for vitamin A, we have taken the approach of tracing the vitamin from its storage site in the liver via its blood transport by the retinol-binding protein (RBP) to its uptake by susceptible cells. We have also examined the intracellular occurrence of vitamin A as regards its binding to specific receptor proteins. Here we summarize data on the amino acid sequences of several vitamin A-binding proteins. The finding that CRBP and CRABP, the two intracellular proteins, are homologous to each other, to a myelin protein, and to a fatty acid-binding protein may shed light on the functions of these proteins. Retinoic acid, which binds to CRABP but not CRBP, induces differentiation of teratocarcinoma cells, This is accompanied by a lowering of the CRABP concentration, an increase of the CRBP level, and an increase in the uptake of retinol from RBP. The epidermis contains both CREP and CRABP, and their distributions are rather similar, However, in contrast to CRBP, CRABP is most abundant in cells lining the hair follicles. CRBP occurs in greatest relative amounts in the outer layers of the epidermis. Since techniques have been developed to measure CRBP and CRABP, normal and disease-affected skin may now be explored as to quantity and cellular distribution of the retinoid-binding proteins. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium tuberculosis is the main causative agent of tuberculosis. BCG, the only licensed vaccine, provides inadequate protection against pulmonary tuberculosis. Controlled human infection models are useful tools for vaccine development. We aimed to determine a safe dose of aerosol-inhaled live-attenuated Mycobacterium bovis BCG as a surrogate for M tuberculosis infection, then compare the safety and tolerability of infection models established using aerosol-inhaled and intradermally administered BCG., Methods: This phase 1 controlled human infection trial was conducted at two clinical research facilities in the UK. Healthy, immunocompetent adults aged 18-50 years, who were both M tuberculosis-naive and BCG-naive and had no history of asthma or other respiratory diseases, were eligible for the trial. Participants were initially enrolled into group 1 (receiving the BCG Danish strain); the trial was subsequently paused because of a worldwide shortage of BCG Danish and, after protocol amendment, was restarted using the BCG Bulgaria strain (group 2). After a dose-escalation study, during which participants were sequentially allocated to receive either 1 × 10 3 , 1 × 10 4 , 1 × 10 5 , 1 × 10 6 , or 1 × 10 7 colony-forming units (CFU) of aerosol BCG, the maximum tolerated dose was selected for the randomised controlled trial. Participants in this trial were randomly assigned (9:12), by variable block randomisation and using sequentially numbered sealed envelopes, to receive aerosol BCG (1 × 10 7 CFU) and intradermal saline or intradermal BCG (1 × 10 6 CFU) and aerosol saline. Participants were masked to treatment allocation until day 14. The primary outcome was to compare the safety of a controlled human infection model based on aerosol-inhaled BCG versus one based on intradermally administered BCG, and the secondary outcome was to evaluate BCG recovery in the airways of participants who received aerosol BCG or skin biopsies of participants who received intradermal BCG. BCG was detected by culture and by PCR. The trial is registered at ClinicalTrials.gov, NCT02709278, and is complete., Findings: Participants were assessed for eligibility between April 7, 2016, and Sept 29, 2018. For group 1, 15 participants were screened, of whom 13 were enrolled and ten completed the study; for group 2, 60 were screened and 33 enrolled, all of whom completed the study. Doses up to 1 × 10 7 CFU aerosol-inhaled BCG were sufficiently well tolerated. No significant difference was observed in the frequency of adverse events between aerosol and intradermal groups (median percentage of solicited adverse events per participant, post-aerosol vs post-intradermal BCG: systemic 7% [IQR 2-11] vs 4% [1-13], p=0·62; respiratory 7% [1-19] vs 4% [1-9], p=0·56). More severe systemic adverse events occurred in the 2 weeks after aerosol BCG (15 [12%] of 122 reported systemic adverse events) than after intradermal BCG (one [1%] of 94; difference 11% [95% CI 5-17]; p=0·0013), but no difference was observed in the severity of respiratory adverse events (two [1%] of 144 vs zero [0%] of 97; 1% [-1 to 3]; p=0·52). All adverse events after aerosol BCG resolved spontaneously. One serious adverse event was reported-a participant in group 2 was admitted to hospital to receive analgesia for a pre-existing ovarian cyst, which was deemed unrelated to BCG infection. On day 14, BCG was cultured from bronchoalveolar lavage samples after aerosol infection and from skin biopsy samples after intradermal infection., Interpretation: This first-in-human aerosol BCG controlled human infection model was sufficiently well tolerated. Further work will evaluate the utility of this model in assessing vaccine efficacy and identifying potential correlates of protection., Funding: Bill & Melinda Gates Foundation, Wellcome Trust, National Institute for Health Research Oxford Biomedical Research Centre, Thames Valley Clinical Research Network, and TBVAC2020., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background/aim: New markers for ovarian cancer are needed. This study aimed to examine the expression of tumour cell p53 and endothelial cell CD31 proteins and correlate them to clinicopathological factors., Patients and Methods: Expression of proteins was immunohistochemically assessed using tissue sections from 585-599 ovarian cancer patients from the Danish MALOVA study., Results: High CD31 expression was found in poorly differentiated tumours (p=0.0006), and high p53 expression was found in poorly differentiated cancers (p<0.0001), high clinical stage (p<0.0001), non-radical surgery (p<0.0001) and high serum CA-125 values (p<0.0001). CD31 expression showed no prognostic survival value, but high hazard ratios were found for patients with high p53 expression (HR=2.313, p<0.0001). An interaction was found between p53 and stage of cancer, suggesting a prognostic impact of p53 in low-stage, but not in advanced-stage cancer., Conclusion: More than 5% of p53 tissue expression may predict shorter survival of ovarian cancer patients and may be useful for predicting the risk of disease progression in low-stage patients following primary surgery. CD31 has no strong prognostic value., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Visual short-term memory (vSTM) is a cognitive resource that declines with age. This study investigated whether electroencephalography (EEG) correlates of vSTM vary with cognitive development over individuals' lifespan. We measured vSTM performance and EEG in a lateralized whole-report task in a healthy birth cohort, whose cognitive function (intelligence quotient) was assessed in youth and late-middle age. Higher vSTM capacity (K; measured by Bundesen's theory of visual attention) was associated with higher amplitudes of the contralateral delay activity (CDA) and the central positivity (CP). In addition, rightward hemifield asymmetry of vSTM (K λ ) was associated with lower CDA amplitudes. Furthermore, more severe cognitive decline from young adulthood to late-middle age predicted higher CDA amplitudes, and the relationship between K and the CDA was less reliable in individuals who show higher levels of cognitive decline compared to individuals with preserved abilities. By contrast, there was no significant effect of lifespan cognitive changes on the CP or the relationship between behavioral measures of vSTM and the CP. Neither the CDA, nor the CP, nor the relationships between K or K λ and the event-related potentials were predicted by individuals' current cognitive status. Together, our findings indicate complex age-related changes in processes underlying behavioral and EEG measures of vSTM and suggest that the K-CDA relationship might be a marker of cognitive lifespan trajectories., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cell-mediated contraction of collagenous matrices is modulated by various growth factors and cytokines, such as platelet-derived growth factor-BB (PDGF-BB). Here we used a genetic cell model to delineate defined signaling pathways that enhance collagen gel contraction downstream of ligand-stimulated platelet-derived growth factor receptor-β (PDGF-Rβ). Our data show that PDGF BB-enhanced activations of phosphatidylinositol 3'-kinase (PI3K) and phospholipase Cγ (PLCγ) were necessary for PDGF-enhanced collagen gel contraction. Importantly, other defined signaling pathways down-stream of PDGF-Rβ were, however, dispensable. The decisive roles for PI3K and PLCγ were corroborated by experiments using selective inhibitors. Furthermore, we show that de-phosphorylation and thereby activation of cofilin that is important for the turnover of actin filaments, is depended on PI3K and PLCγ down-stream of PDGF-Rβ. Moreover, inhibition of protein kinase C (PKC) by GÖ6976 and bisindolylmaleimide-II abolished cofilin de-phosphorylation, as well as PDGF-enhanced contraction. In contrast, activation of the PKC protein family by 4β-phorbol 12-myristate 13-acetate (PMA) did not accelerate collagen gel contraction although it induced long-term cofilin de-phosphorylation, showing the need of a dynamic control of cofilin de-phosphorylation for PDGF-enhanced collagen gel contraction. Taken together, our data point to the involvement of a PI3K/PLCγ-PKC-cofilin pathway in both PDGF-enhanced cofilin de-phosphorylation and PDGF-enhanced collagen gel contraction.

Neocortical gamma activity is crucial for sensory perception and cognition. This study examines the value of using non-task stimulation-induced EEG oscillations to predict cognitive status in a birth cohort of healthy Danish males (Metropolit) with varying cognitive ability. In particular, we examine the steady-state VEP power response (SSVEP-PR) in the alpha (8Hz) and gamma (36Hz) bands in 54 males (avg. age: 62.0 years) and compare these with 10 young healthy participants (avg. age 27.6 years). Furthermore, we correlate the individual alpha-to-gamma difference in relative visual-area power (ΔRV) with cognitive scores for the older adults. We find that ΔRV decrease with age by just over one standard deviation when comparing young with old participants (p<0.01). Furthermore, intelligence is significantly negatively correlated with ΔRV in the older adult cohort, even when processing speed, global cognition, executive function, memory, and education (p<0.05). In our preferred specification, an increase in ΔRV of one standard deviation is associated with a reduction in intelligence of 48% of a standard deviation (p<0.01). Finally, we conclude that the difference in cerebral rhythmic activity between the alpha and gamma bands is associated with age and cognitive status, and that ΔRV therefore provide a non-subjective clinical tool with which to examine cognitive status in old age.

Importance: Cognitive skills are known to decline through the lifespan with large individual differences. The molecular mechanisms for this decline are incompletely understood. Although leukocyte telomere length provides an index of cellular age that predicts the incidence of age-related diseases, it is unclear whether there is an association between cognitive decline and leukocyte telomere length. Objective: To examine the association between changes in cognitive function during adult life and leukocyte telomere length after adjusting for confounding factors such as education, mental health and life style. Design, Setting, and Participants: Two groups of men with negative ( n = 97) and positive ( n = 93) change in cognitive performance were selected from a birth cohort of 1985 Danish men born in 1953. Cognitive performance of each individual was assessed at age ~20 and 56 years. Leukocyte telomere length at age ~58 was measured using qPCR. Linear regression models were used to investigate the association between cognitive function and leukocyte telomere length. Results: Men with negative change in cognitive performance during adult life had significantly shorter mean leukocyte telomere length than men with positive change in cognitive performance (unadjusted difference β = -0.09, 95% CI -0.16 to -0.02, p = 0.02). This association remained significant after adjusting for smoking, alcohol consumption, leisure time activity, body mass index (BMI) and cholesterol (adjusted difference β = -0.09, 95% CI -0.17 to -0.01, p = 0.02) but was non-significant after adjusting for smoking, alcohol consumption, leisure time activity, BMI, cholesterol, current cognitive function, depression and education (adjusted difference β = -0.07, 95% CI -0.16 to -0.01, p = 0.08). Conclusion and Relevance: Preclinical cognitive changes may be associated with leukocyte telomere length.

Exposure to psychosocial stress is associated with increased risk of a number of somatic and mental disorders with relation to immune system functioning. We aimed to explore whether stressful events in early and recent life was associated with leucocyte telomere length (TL), which is assumed to reflect the accumulated burden of inflammation and oxidative stress occurring during the life course. We specifically aimed to address whether childhood constitutes a sensitive period and how much of the relation between stressful life events and TL is mediated through somatic and mental health, lifestyle, and markers of low-grade inflammation. A cohort of Danish men born in 1953 has been followed since birth in the Metropolit Cohort. These men underwent a health examination including blood sampling in 2010 and a subset of 324 also had a quantitative PCR-based measurement of TL. The relation between stressful life events and TL was analysed using structural equation modelling, which also provided an estimate of the proportion of the total effect mediated by somatic and mental health (cardiovascular disease, body mass and depressive mood), lifestyle factors, and low grade inflammation (C-reactive protein (CRP), interleukin (IL)-6 and IL-10). Total number of stressful events experienced during the life course was not associated with TL. In terms of sensitive periods, we found that number of stressful events in childhood was associated with shorter TL (β per number stressful events in childhood =-0.02(SE=-0.02); P=0.05). This relation was particularly strong for being placed away from home (β=-0.16; P<0.000). Thirty percent of the total effect of stressful events in childhood on TL was mediated by the included variables, with the largest proportion being mediated through depressive mood (16%) and CRP (9%). This study suggests that stressful events in childhood are associated with shorter TL in middle-aged men and that part of this relation is explained by depressive mood and low grade inflammation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system.

The primary objective of this study was to analyse Tetranectin (TN) expression in tumour tissues and TN serum concentration in 758 women with epithelial ovarian tumours. The second was to evaluate, whether TN tissue expression levels correlate with clinico-pathological parameters and prognosis of the disease. Using tissue arrays we analysed the expression levels in tissues from 166 women with borderline ovarian tumours (BOTs) and 592 women with ovarian cancer (OC). A panel of three antibodies was used for immunohistochemistry: a polyclonal and two monoclonal antibodies. Serum TN was measured using the polyclonal antibody A-371. Univariate survival analyses stratified for chemotherapy showed that positive tissue TN as demonstrated by the polyclonal antibody indicated a significantly longer overall survival (OS) (p = 0.0001) as well as cancer specific survival (CSS) (p < 0.0001). High serum TN was likewise found to imply longer OS (p < 0.0001) and CSS (p < 0.0001), whereas tissue staining with the two monoclonal antibodies failed to demonstrate any significant correlation with either survival type. Univariate Kaplan-Meier survival analysis performed on all OC cases showed a significantly longer OS (p = 0.0009) and CSS (p = 0.0006) for women with TN positive tumour tissue and in women with high serum TN levels (p < 0.0001 for both). However, in the multivariate Cox regression analysis, only serum TN was found to be an independent prognostic factor for OS (p = 0.01) and not for CSS (p = 0.08). In conclusion, our results predict that a positive TN expression of both tumour tissue and serum points to a more favourable outcome for OC patients., (© 2015 APMIS. Published by John Wiley & Sons Ltd.)

We established a cell line (HEK-hMel) expressing melanopsin in a tetracycline dependent manner to elucidate new aspects of melanopsin's light response. Different light stimuli were evaluated using FOS expression as response parameter. Immunoblotting was used to evaluate expression of melanopsin and FOS and qPCR to quantify FOS mRNA responses. The magnitude of the FOS response was found to correlate with the amount of melanopsin expressed by the cells, and a transient FOS mRNA induction followed by FOS protein still elevated after 24 h of illumination was revealed. Exposing the cells to darkness after light resulted in reduction of the response compared to exposure to light solely showing dependency on continuous light. Increasing irradiances of blue light (480 nm) up to 10(11) quanta cm(-2)  s(-1) elicited steep increases in FOS mRNA, while increases between 10(12) and 5 × 10(13) quanta  cm(-2)  s(-1) resulted in equally high FOS expression. The HEK-hMel cells were used to characterize facets of melanopsin's light-induced FOS response not approachable in vivo. Novel information such as dependency of the FOS response on both melanopsin amount and light intensity in addition to a detailed time-course of both FOS mRNA and protein were revealed., (© 2014 The American Society of Photobiology.)

The functional significance of fibrin deposits typically seen in inflammatory lesions, carcinomas and in healing wounds is not fully understood. In the present study, we demonstrate that fibrinogen/fibrin specifically bound to native Col I (collagen type I) and used the Col I fibre network as a base to provide a functional interface matrix that connects cells to the Col I fibres through αVβ3 integrins. This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via αVβ3 integrin. We show that fibrinogen specifically bound to immobilized native Col I at the site known to bind matrix metalloproteinase-1, discoidin domain receptor-2 and fibronectin, and that binding had no effect on Col I fibrillation. A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels. Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects αVβ3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.

Purpose: Therapeutic decisions in breast cancer are increasingly guided by prognostic and predictive biomarkers. Non-protein-coding microRNAs (miRNAs) have recently been found to be deregulated in breast cancers and, in addition, to be correlated with several clinico-pathological features. One of the most consistently up-regulated miRNAs is miR-21. Here, we specifically searched for differentially expressed miRNAs in high-risk breast cancer patients as compared to low-risk breast cancer patients. In the same patients, we also compared miR-21 expression with the expression of its presumed target PTEN., Methods: Both microarray and RT-qPCR techniques were used to assess miRNA expression levels in lymph node-positive and -negative human invasive ductal carcinoma tissues. Simultaneously, PTEN protein expression levels were assessed using immunohistochemistry., Results: miR-486-5p and miR-139-5p were found to be down-regulated in patients with lymph node metastases, whereas miR-21 was found to be up-regulated in patients with a positive lymph node status. miR-21 expression levels were found to significantly correlate with tumour size (r = 0.403, p = 0.009; Spearman's rank), whereas no relation was found between miR-21 and PTEN expression levels (Kruskal-Wallis test)., Conclusion: Down-regulation of miR-486-5p and miR-139-5p, in conjunction with up-regulation of miR-21, may represent a useful signature for the identification of high-risk breast cancer patients.

Cancer metastasis is the foremost cause of death in cancer patients. A series of observable pathological changes takes place during progression and metastasis of cancer, but the underlying genetic changes remain unclear. Therefore, new approaches are required, including insights from cancer mouse models. To examine the mechanisms involved in tumor metastasis, we first generated a stably transfected Lewis Lung carcinoma cell line expressing a far-red fluorescent protein, called Katushka. After in vivo growth in syngeneic mice, two fluorescent Lewis Lung cancer subpopulations were isolated from primary tumors and lung metastases. The metastasis-derived cells exhibited a significant improvement in in vitro invasive activity compared to the primary tumor-derived cells, using a quantitative invasion chamber assay. Moreover, expression levels of 84 tumor metastasis-related mRNAs, 88 cancer-related microRNAs as well as Dicer and Drosha were determined using RT-qPCR. Compared to the primary Lewis Lung carcinoma subculture, the metastasis-derived cells exhibited statistically significantly increased mRNA levels for several matrix metalloproteinases as well as hepatocyte growth factor (HGF) and spleen tyrosine kinase (SYK). A modest decrease in Drosha and Dicer mRNA levels was accompanied by significant downregulation of ten microRNAs, including miR-9 and miR-203, in the lung metastatic Lewis Lung carcinoma cell culture. Thus, a tool for cancer metastasis studies has been established and the model is well suited for the identification of novel microRNAs and mRNAs involved in malignant progression. Our results suggest that increases in metalloproteinase expression and impairment of microRNA processing are involved in the acquirement of metastatic ability., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Multidrug resistance (MDR) poses a major obstacle to successful chemotherapeutic treatment of cancer, and often involves multiple genes, which may be regulated post-transcriptionally by microRNAs (miRNAs). The purpose of the present study was therefore to identify any resistance-associated changes in miRNA expression in a sensitive and five increasingly drug-resistant Ehrlich ascites tumor (EAT) cell lines, representing different steps in the development of resistance. We used an LNA-enhanced microarray platform to study the global miRNA expression profiles in the six murine EAT cell lines, and identified growth-, hypoxia-, and resistance-specific miRNA patterns. Among the differentially expressed miRNAs, we found the two clusters miR-183∼miR-96∼miR-182 and miR-200b∼miR-200a∼miR-429 as well as miR-141 to be consistently upregulated in the MDR cell lines, while miR-125b-5p and the two clusters miR-30d∼miR-30b and miR-23b∼miR-27b∼miR-24-1 were downregulated in most of the resistant EAT cells. Several of the target genes for these miRNAs-including Zeb1/Zeb2 and members of the Fox gene family-could contribute to the drug-resistant phenotype, although we did not find that the degree of resistance was directly correlated to any specific changes in miRNA expression. Probably, the observed miRNA expression patterns reflect the underlying genomic instability of the tumor cells, and further studies are needed to explore how the highly complex regulatory miRNA networks contribute to the development of MDR.

Low-risk and high-risk breast cancer patients are stratified primarily according to their lymph node (LN) status and grading. However, some low-risk patients relapse, and some high-risk patients have a favorable clinical outcome, implying a need for better prognostic and predictive tests. Micro RNAs are often aberrantly expressed in cancer and microRNA-21 is upregulated in a variety of cancers, including breast cancer. High miR-21 levels have been associated with poor prognosis. To determine the cellular localization of miR-21 and to compare its expression levels with histopathological features, we performed in situ hybridization and semi-quantitative assessment of the miR-21 signal on 12 LN negative grade I (assumed low risk), and 12 LN positive grade II (high risk) breast cancers. miR-21 was predominantly seen in cancer associated fibroblast-like cells, with no difference in expression levels between grade I and grade II carcinomas. Immunohistochemical scoring of the prognostic proliferation marker Ki-67 and tumor suppressor p53 showed that the miR-21 expression levels significantly correlated with the Ki-67 score (p = 0.043), whereas no correlation between p53 and miR-21 was found. Our results indicate that miR-21 may contribute to improve clinical stratification according to growth rate and facilitate tailored treatment of breast cancer patients., (© 2011 The Authors. APMIS © 2011 APMIS.)

In orthodox seeds, the transcriptional activator ABI3 regulates two major stages in embryo maturation: a mid-maturation (MAT) stage leading to accumulation of storage compounds, and a late maturation (LEA) stage leading to quiescence and desiccation tolerance. Our aim was to elucidate mechanisms for transcriptional shutdown of MAT genes during late maturation, to better understand phase transition between MAT and LEA stages. Using transgenic and transient approaches in Nicotiana, we examined activities of two ABI3-dependent reporter genes driven by multimeric RY and abscisic acid response elements (ABREs) from a Brassica napus napin gene, termed RY and ABRE, where the RY reporter requires ABI3 DNA binding. Expression of RY peaks during mid-maturation and drops during late maturation, mimicking the MAT gene program, and in Arabidopsis thaliana RY elements are over-represented in MAT, but not in LEA, genes. The ABI3 transactivation of RY is inhibited by staurosporine, by a PP2C phosphatase, and by a repressor of maturation genes, VAL1/HSI2. The RY element mediates repression of MAT genes, and we propose that transcriptional shutdown of the MAT program during late maturation involves inhibition of ABI3 DNA binding by dephosphorylation. Later, during seedling growth, VAL1/HSI2 family repressors silence MAT genes by binding RY elements.

Myrosinases (EC 3.2.1.147) are beta-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1-TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS-PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation-inhibition responses towards ascorbic acid with maximal activity around 0.7-1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-beta-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.

Melanopsin is an opsin expressed in the plasma membrane of retinal ganglion cells that mainly project to the circadian clock and thus is important for nonvisual responses to light. Rat melanopsin contains two potential sites (Asn31 and Asn35) for N-linked glycosylation in the N-terminal extracellular part. To investigate if melanopsin is N-linked glycosylated and whether N-bound glycans influence the response of melanopsin to light as evidenced by Fos mRNA induction, we transfected PC12 cells to stably express rat wild-type melanopsin or mutant melanopsin lacking both N-linked glycosylation sites. Immunoblotting for membrane-bound melanopsin from the PC12 cells transfected to express wild-type melanopsin disclosed two immunoreactive bands of 62 and 49 kDa. Removal of N-linked glycosylation by tunicamycin or PNGase F changed the 62 kDa band to a 55 kDa band, while the 49 kDa band corresponding to the core melanopsin protein was unaffected. Likewise, mutation of the two extracellular N-linked glycosylation sites gave a melanopsin size comparable to that of PNGase F or tunicamycin treatment (55 kDa). Further in vitro O-linked deglycosylation of wild-type or mutant melanopsin with O-glycosidase and neuraminidase converted the 55 kDa band to a 49 kDa band. Finally, neither in vivo N-linked deglycosylation nor mutations of the two N-linked glycosylation sites significantly affected melanopsin function measured by Fos induction after light stimulation. In conclusion, we have shown that heterologously expressed rat melanopsin is both N-linked and O-linked glycosylated and that N-linked glycosylation is not crucial for the melanopsin response to light.

Human filamin A is a 280 kDa protein involved in actin-filament cross-linking. It is structurally divided into an actin-binding headpiece (ABD) and a rod domain containing 24 immunoglobulin-like (Ig) repeats. A fragment of human filamin A (Ig repeats 14-16) was cloned and expressed in Escherichia coli and the purified protein was crystallized in 1.6 M ammonium sulfate, 2% PEG 1000 and 100 mM HEPES pH 7.5. The crystals diffracted to 1.95 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.63, b = 52.10, c = 98.46 A, alpha = beta = gamma = 90 degrees.

Lipoprotein receptor-related protein-2 (LRP2)/megalin is a member of the low density lipoprotein receptor (LDLR) family, and is essential in absorptive epithelia for endocytosis of lipoproteins, low molecular weight proteins, cholesterol and vitamins, as well as in cellular signaling. Previous studies have shown megalin expression in ependymal cells and choroid plexus. We have investigated megalin expression in the spinal cord of postnatal mice with immunohistochemistry and immunoblot. Antibodies recognizing either the cytoplasmic tail (MM6) or the extracellular domain (E11) of megalin labeled oligodendrocytes in the spinal cord white matter, in parallel with myelination. MM6 antibodies, predominantly labeled the nuclei, whereas E11 antibodies labeled the cytoplasm of these cells. MM6 antibodies labeled also nuclei of oligodendrocytes cultured from embryonic mouse spinal cord. Immunoblots of spinal cord showed intact megalin, as well as its carboxyterminal fragment, the part remaining after shedding of the extracellular domain of megalin. Megalin-immunoreactive oligodendrocytes also expressed presenilin 1, an enzyme responsible for gamma-secretase mediated endodomain cleavage. These findings show that spinal cord oligodendrocytes are phenotypically different from those in the brain, and indicate that megalin translocates signals from the cell membrane to the nucleus of oligodendrocytes during the formation and maintenance of myelin of long spinal cord pathways.

The lipoprotein receptor LRP2/megalin is expressed by absorptive epithelia and involved in receptor-mediated endocytosis of a wide range of ligands. Megalin is expressed in the neuroepithelium during central nervous system (CNS) development. Mice with homozygous deletions of the megalin gene show severe forebrain abnormalities. The possible role of megalin in the developing spinal cord, however, is unknown. Here we examined the spatial and temporal expression pattern of megalin in the embryonic mouse spinal cord using an antibody that specifically recognizes the cytoplasmic part of the megalin molecule. In line with published data, we show expression of megalin in ependymal cells of the central canal from embryonic day (E)11 until birth. In addition, from E11 until E15 a population of cells was found in the dorsal part of the developing spinal cord strongly immunoreactive against megalin. Double labeling showed that most of these cells express vimentin, a marker for immature astrocytes and radial glia, but not brain lipid binding protein (BLBP), a marker for radial glial cells, or glial fibrillary acidic protein (GFAP), a marker for mature astrocytes. These findings indicate that the majority of the megalin-positive cells are astroglial precursors. Megalin immunoreactivity was mainly localized in the nuclei of these cells, suggesting that the cytoplasmic part of the megalin molecule can be cleaved following ligand binding and translocated to the nucleus to act as a transcription factor or regulate other transcription factors. These findings suggest that megalin has a crucial role in the development of astrocytes of the spinal cord.

Brassica napus complementary deoxyribonucleic acid (cDNA) clones encoding a DNA-binding protein, BnPEND, were isolated by Southwestern screening. A distinctive feature of the protein was a bZIP-like sequence in the amino-terminal portion, which, after expression in Escherichia coli, bound DNA. BnPEND transcripts were present in B. napus roots and flower buds, and to a lesser extent in stems, flowers and young leaves. Treatment in the dark for 72 h markedly increased the amount of BnPEND transcript in leaves of all ages. Sequence comparison showed that BnPEND was similar to a presumed transcription factor from B. napus, GSBF1, a protein deduced from an Arabidopsis thaliana cDNA (BX825084) and the PEND protein from Pisum sativum, believed to anchor the plastid DNA to the envelope early during plastid development. Homology to expressed sequence tag (EST) sequences from additional species suggested that BnPEND homologues are widespread among the angiosperms. Transient expression of BnPEND fused with green fluorescent protein (GFP) in Nicotiana benthamiana epidermal cells showed that BnPEND is a plastid protein, and that the 15 amino acids at the amino-terminal contain information about plastid targeting. Expression of BnPEND in Nicotiana tabacum from the Cauliflower Mosaic Virus 35S promoter gave stable transformants with different extents of white to light-green areas in the leaves, and even albino plants. In the white areas, but not in adjacent green tissue, the development of palisade cells and chloroplasts was disrupted. Our data demonstrate that the BnPEND protein, when over-expressed at an inappropriate stage, functionally blocks the development of plastids and leads to altered leaf anatomy, possibly by preventing the release of plastid DNA from the envelope.