New benzidine procedure for determination of manganese in biological samples

A simple and accurate method for the determination of 0.25 to 1.0 μg. of manganese (in samples containing 1 to 4 μg. of manganese) has been developed by modifying the conditions for the reaction between permanganate and benzidine. Manganese is oxidized with potassium periodate in orthophosphoric acid and selectively estimated in the presence of excess oxidant with benzidine in formic acid. The procedure is applicable for estimation of manganese in biological samples, with recoveries in the range 97.5 to 106.1%.

Interaction of heterocyclic thiols/thiones eliminated from cephalosporins with iodine and its biological implications

Hydrolysis of beta-lactam antibiotics by beta-lactamases (e. g., metallo-beta-lactamase, m beta l) is one of the major bacterial defense systems. These enzymes can catalyze the hydrolysis of a variety of antibiotics including the latest generation of cephalosporins, cephamycins and imipenem. It is shown in this paper that the thiol/thione moieties eliminated from certain cephalosporins by m beta l-mediated hydrolysis readily react with molecular iodine to produce ionic compounds having S-I bonds. While the reaction of MTT with iodine produced the corresponding disulfide, MDT and DMETT produced the charge-transfer complexes MDT-I-2 and DMETT-I-2, respectively. Addition of two equivalents of I-2 to MDT produced a novel cationic complex having an almost linear S-I+-S moiety and I-5(-) counter anion.However, this reaction appears to be highly solvent dependent. When the reaction of MDT with I2 was carried out in water, the reaction produced a monocation having I-5(-), indicating the reactivity of MDT toward I2 is very similar to that of the most commonly used antithyroid drug methimazole (MMI). In contrast to MMI, MDT and DMETT, the triazine-based compound MTDT acts as a weak donor toward iodine. (C)2010 Elsevier Ltd. All rights reserved.

Can biological motion be a biometric?

Biological motion has successfully been used for analysis of a person's mood and other psychological traits. Efforts are made to use human gait as a non-invasive mode of biometric. In this reported work, we try to study the effectiveness of biological gait motion of people as a cue to biometric based person recognition. The data is 3D in nature and, hence, has more information with itself than the cues obtained from video-based gait patterns. The high accuracies of person recognition using a simple linear model of data representation and simple neighborhood based classfiers, suggest that it is the nature of the data which is more important than the recognition scheme employed.

Glioblastoma-Specific Protein Interaction Network Identifies PP1A and CSK21 as Connecting Molecules between Cell Cycle-Associated Genes

Glioblastoma (GBM; grade IV astrocytoma) is a very aggressive form of brain cancer with a poor survival and few qualified predictive markers. This study integrates experimentally validated genes that showed specific upregulation in GBM along with their protein-protein interaction information. A system level analysis was used to construct GBM-specific network. Computation of topological parameters of networks showed scale-free pattern and hierarchical organization. From the large network involving 1,447 proteins, we synthesized subnetworks and annotated them with highly enriched biological processes. A careful dissection of the functional modules, important nodes, and their connections identified two novel intermediary molecules CSK21 and protein phosphatase 1 alpha (PP1A) connecting the two subnetworks CDC2-PTEN-TOP2A-CAV1-P53 and CDC2-CAV1-RB-P53-PTEN, respectively. Real-time quantitative reverse transcription-PCR analysis revealed CSK21 to be moderately upregulated and PP1A to be overexpressed by 20-fold in GBM tumor samples. Immunohistochemical staining revealed nuclear expression of PP1A only in GBM samples. Thus, CSK21 and PP1A, whose functions are intimately associated with cell cycle regulation, might play key role in gliomagenesis. Cancer Res; 70(16); 6437-47. (C)2010 AACR.

Uniparental DNA markers and forensic genetics in Finland

Over the years, a wide range of methods to verify identity have been developed. Molecular markers have been used for identification since the 1920s, commencing with blood types and culminating with the advent of DNA techniques in the 1980s. Identification is required by authorities in many occasions, e.g. in disputed paternity cases, identification of deceased, or crime investigation. To clarify maternal and paternal lineages, uniparental DNA markers in mtDNA and Y-chromosome can be utilized. These markers have several advantages: male specific Y-chromosome can be used to identify a male from a mixture of male and female cells, e.g. in rape cases. MtDNA is durable and has a high copy number, allowing analyses even from old or degraded samples. However, both markers are lineage-specific, not individualizing, and susceptible to genetic drift. Prior to the application of any DNA marker in forensic casework, it is of utmost importance to investigate its qualities and peculiarities in the target population. Earlier studies on the Finnish population have shown reduced variation in the Y-chromosome, but in mtDNA results have been ambiguous. The obtained results confirmed the low diversity in Y-chromosome in Finland. Detailed population analysis revealed large regional differences, and extremely reduced diversity especially in East Finland. Analysis of the qualities affecting Y-chromosomal short tandem repeat (Y-STR) variation and mutation frequencies, and search of new polymorphic markers resulted a set of Y-STRs with especially high diversity in Finland. Contrary to Y-chromosome, neither reduced diversity nor regional differences were found in mtDNA within Finland. In fact, mtDNA diversity was found similar to other European populations. The revealed peculiarities in the uniparental markers are a legacy of the Finnish population history. The obtained results challenge the traditional explanation which emphasizes relatively recent founder effects creating the observed east-west patterns. Uniparentally inherited markers, both mtDNA and Y-chromosome, are applicable for identification purposes in Finland. By adjusting the analysed Y marker set to meet the characteristics of Finnish population, Y-chromosomal diversity increases and the regional differentiation decreases, resulting increase in discrimination power and thus usefulness of Y-chromosomal analysis in forensic casework.

Crosstalk between the sympathetic nervous system, inflammation and coagulation in gestational diabetes; a therapeutic approach in postmenopausal hypertension

The occurrence of gestational diabetes (GDM) during pregnancy is a powerful sign of a risk of later type 2 diabetes (T2D) and cardiovascular diseases (CVDs). The physiological basis for this disease progression is not yet fully understood, but increasing evidence exists on interplay of insulin resistance, subclinical inflammation, and more recently, on unbalance of the autonomic nervous system. Since the delay in development of T2D and CVD after GDM ranges from years to decades, better understanding of the pathophysiology of GDM could give us new tools for primary prevention. The present study was aimed at investigating the role of the sympathetic nervous system (SNS) in GDM and its associations with insulin and a variety of inflammatory cytokines and coagulation and fibrinolysis markers. This thesis covers two separate study lines. Firstly, we investigated 41 women with GDM and 22 healthy pregnant and 14 non-pregnant controls during the night in hospital. Blood samples were drawn at 24:00, 4:00 and 7:00 h to determine the concentrations of plasma glucose, insulin, noradrenaline (NA) and adrenomedullin, markers of subclinical inflammation, coagulation and fibrinolysis variables and platelet function. Overnight holter ECG recording was performed for analysis of heart rate variability (HRV). Secondly, we studied 87 overweight hypertensive women with natural menopause. They were randomised to use a central sympatholytic agent, moxonidine (0.3mg twice daily), the β-blocking agent atenolol (50 mg once daily+blacebo once daily) for 8 weeks. Inflammatory markers and adiponectin were analysed at the beginning and after 8 weeks. Activation of the SNS (increase in NA, decreased HRV) was seen in pregnant vs. non-pregnant women, but no difference existed between GDM and normal pregnancy. However, modulation (internal rhythm) of HRV was attenuated in GDM. Insulin and inflammatory cytokine levels were comparable in all pregnant women but nocturnal variation of concentrations of C-reactive protein, serum amyloid A and insulin were reduced in GDM. Levels of coagulation factor VIII were lower in GDM compared with normal pregnancy, whereas no other differences were seen in coagulation and fibrinolysis markers. No significant associations were seen between NA and the studied parameters. In the study of postmenopausal women, moxonidine treatment was associated with favourable changes in the inflammatory profile, seen as a decrease in TNFα concentrations (increase in atenolol group) and preservation of adiponectin levels (decrease in atenolol group). In conclusion, our results did not support our hypotheses of increased SNS activity in GDM or a marked association between NA and inflammatory and coagulation markers. Reduced biological variation of HRV, insulin and inflammatory cytokines suggests disturbance of autonomic and hormonal regulatory mechanisms in GDM. This is a novel finding. Further understanding of the regulatory mechanisms could allow earlier detection of risk women and the possibility of prevention. In addition, our results support consideration of the SNS as one of the therapeutic targets in the battle against metabolic diseases, including T2D and CVD.

Expression of drug resistance markers by spheroplasts ofMycobacterium smegmatis after fusion

Mycobacterial spheroplasts were prepared by treatment of the glycinesensitized cells with a combination of lipase and lysozyme. They were stable for several hours at room temperature but were lysed on treatment with 0.1% sodium dodecyl sulfate. The spheroplasts could be regenerated on a suitable medium. Fusion and regeneration of the spheroplasts were attempted using drug resistant mutant strains ofM. smegmalis. Recombinants were obtained from spheroplast fusion mediated by polyethylene glycol and dimethyl sulfoxide. Simultaneous expression of rccombinant properties was observed only after an initial lag in the isolated clones. This has been explained as due to “chromosome inactivation” in the fused product.

Identification of molecules relevant for the invasiveness of fibrosarcomas and melanomas

Cancer is becoming the leading cause of deaths in the world. As 90% of all deaths from cancer are caused by metastasis, discovery of the mechanisms behind cancer cell invasion and metastasis is of utmost importance. Only new effective therapies targeting cancer progression can reduce cancer mortality rates. The aim of this study was to identify molecules that are relevant for tumor cell invasion and spreading in fibrosarcomas and melanomas, and to analyze their potential for cancer biomarkers or therapeutic targets. First, the gene expression changes of normal cells and transformed cells showing high invasiveness, S-adenosylmethionine decarboxylase (AdoMetDC)-transfected murine fibroblasts and human melanoma cells, were studied by microarray analyses. The function of the identified candidate molecules were then studied in detail in these cell lines. Finally, the physiological relevance of the identified changes was studied by immunohistochemical analyses of human sarcoma and melanoma specimens or by a mouse xenograft model. In fibrosarcoma cells, the most remarkable change detected was a dramatic up-regulation of the actin-sequestering molecule thymosin beta 4 (TB4), which was shown to be important for the transformed phenotype of the AdoMetDC-transfected cells (Amdc-s and -as). A sponge toxin latrunculin A, inhibiting the binding of TB4 to actin, was found to selectively inhibit the migration and invasion of these cells. Further, Amdc-s-induced mouse tumors and human high-grade sarcomas were found to show intense TB4 immunostaining. In addition to TB4, integrin subunits alfa 6 and beta 7 (ItgA6 and ItgB7) were found to be up-regulated in Amdc-s and -as cells. ItgA6 was shown to dimerize mainly with ItgB1 in Amdc-s. Inhibition of ItgA6 or ItgB1 function with neutralizing antibodies fully blocked the invasiveness of Amdc-s cells, and importantly also human HT-1080 fibrosarcoma cells, in three-dimensional (3D)-Matrigel mimicking tumor extracellular matrix (ECM). By immunohistochemical analyses, strong staining for ITGA6 was detected in human high-grade fibrosarcomas and other sarcomas, especially at the invasion fronts of the tumors. In the studied melanoma cell lines, the expression levels of the adhesion-related ECM proteins tenascin-C (TN-C), fibronectin (FN), and transforming growth factor beta-induced (TGFBI) were found to be highly up-regulated. By immunohistochemistry, intense TN-C and FN staining was detected in invasive and metastatic melanoma tumors, showing co-localization (together with procollagen-I) in tubular meshworks and channels around the invading melanoma cells. In vitro, TN-C and FN were further found to directly stimulate the migration of melanoma cells in 3D-collagen-I matrix. The third candidate protein, TGFBI, was found to be an anti-adhesive molecule for melanoma cells, and knockdown of its expression in metastatic melanoma cells (TGFBI-KD cells) led to dramatically impaired tumor growth in immunocompromized mice. Interestingly, the control tumors showed intense TGFBI immunostaining in the invasion fronts, showing partial co-localization with the fibrillar FN staining, whereas the small TGFBI-KD cell-induced tumors displayed amorphous, non-fibrillar FN staining. These data suggest an important role for TGFBI in FN fibrillogenesis and melanoma progression. In conclusion, we have identified several invasion-related molecules, which show potential for cancer diagnostic or prognostic markers, or therapeutic targets. Based on our previous and present fibrosarcoma studies, we propose the possibility of using ITGA6 antagonists (affecting tumor cell adhesion) in combination with TB4 inhibitors (affecting tumor cell migration) and cathepsin L inhibitors (affecting the degradation of basement membrane and ECM proteins) for the treatment of fibrosarcomas and other tumors overexpressing these molecules. With melanoma cells, in turn, we point to the importance of three secreted ECM proteins, TN-C, FN, and TGFBI, in melanoma progression. Of these, especially the potential of TN-C as a prognostic melanoma biomarker and TGFBI as a promising therapeutic target molecule are clearly worth additional studies.

Pre-eclampsia : The role of soluble VEGF receptor-1 and related anti-angiogenic factors beyond

Pre-eclampsia is a pregnancy complication that affects about 5% of all pregnancies. It is known to be associated with alterations in angiogenesis -related factors, such as vascular endothelial growth factor (VEGF). An excess of antiangiogenic substances, especially the soluble receptor-1 of VEGF (sVEGFR-1), has been observed in maternal circulation after the onset of the disease, probably reflecting their increased placental production. Smoking reduces circulating concentrations of sVEGFR-1 in non-pregnant women, and in pregnant women it reduces the risk of pre-eclampsia. Soluble VEGFR-1 acts as a natural antagonist of VEGF and placental growth factor (PlGF) in human circulation, holding a promise for potential therapeutic use. In fact, it has been used as a model to generate a fusion protein, VEGF Trap , which has been found effective in anti-angiogenic treatment of certain tumors and ocular diseases. In the present study, we evaluated the potential use of maternal serum sVEGFR-1, Angiopoietin-2 (Ang-2) and endostatin, three central anti-angiogenic markers, in early prediction of subsequent pre-eclampsia. We also studied whether smoking affects circulating sVEGFR-1 concentrations in pregnant women or their first trimester placental secretion and expression in vitro. Last, in order to allow future discussion on the potential therapy based on sVEGFR-1, we determined the biological half-life of endogenous sVEGFR-1 in human circulation, and measured the concomitant changes in free VEGF concentrations. Blood or placental samples were collected from a total of 268 pregnant women between the years 2001 2007 in Helsinki University Central Hospital for the purposes above. The biomarkers were measured using commercially available enzyme-linked immunosorbent assays (ELISA). For the analyses of sVEGFR-1, Ang-2 and endostatin, a total of 3 240 pregnant women in the Helsinki area were admitted to blood sample collection during two routine ultrasoundscreening visits at 13.7 ± 0.5 (mean ± SD) and 19.2 ± 0.6 weeks of gestation. Of them, 49 women later developing pre-eclampsia were included in the study. Their disease was further classified as mild in 29 and severe in 20 patients. Isolated early-onset intrauterine growth retardation (IUGR) was diagnosed in 16 women with otherwise normal medical histories and uncomplicated pregnancies. Fifty-nine women remaining normotensive, non-proteinuric and finally giving birth to normal-weight infants were picked to serve as the control population of the study. Maternal serum concentrations of Ang-2, endostatin and sVEGFR-1, were increased already at 16 20 weeks of pregnancy, about 13 weeks before the clinical manifestation of preeclampsia. In addition, these biomarkers could be used to identify women at risk with a moderate precision. However, larger patient series are needed to determine whether these markers could be applied for clinical use to predict preeclampsia. Intrauterine growth retardation (IUGR), especially if noted at early stages of pregnancy and not secondary to any other pregnancy complication, has been suggested to be a form of preeclampsia compromising only the placental sufficiency and the fetus, but not affecting the maternal endothelium. In fact, IUGR and preeclampsia have been proposed to share a common vascular etiology in which factors regulating early placental angiogenesis are likely to play a central role. Thus, these factors have been suggested to be involved in the pathogenesis of IUGR. However, circulating sVEGFR-1, Ang-2 and endostatin concentrations were unaffected by subsequent IUGR at early second trimester. Furthermore, smoking was not associated with alterations in maternal circulating sVEGFR-1 or its placental production. The elimination of endogenous sVEGFR-1 after pregnancy was calculated from serial samples of eight pregnant women undergoing elective Caesarean section. As typical for proteins in human compartments, the elimination of sVEGFR-1 was biphasic, containing a rapid halflife of 3.4 h and a slow one of 29 h. The decline in sVEGFR-1 concentrations after mid-trimester legal termination of pregnancy was accompanied with a simultaneous increase in the serum levels of free VEGF so that within a few days after pregnancy VEGF dominated in the maternal circulation. Our study provides novel information on the kinetics of endogenous sVEGFR-1, which serves as a potential tool in the development of new strategies against diseases associated with angiogenic imbalance and alterations in VEGF signaling.

Population genetics of the invasive plant species Impatiens glandulifera in Southern Finland

Biological invasions affect biodiversity worldwide, and, consequently, the invaded ecosystems may suffer from significant losses in economic and cultural values. Impatiens glandulifera Royle (Balsaminaceae) is an invasive annual herb, native to the western Himalayas and introduced into Europe in the 19th century as a garden ornamental plant. The massive invasion of I. glandulifera is due to its high reproductive output, rapid growth and its ability to outcompete native species. In Finland, the first observations regarding the presence of I. glandulifera date from the year 1947, and today it is considered a serious problem in riparian habitats. The aim of this master’s thesis research is to reveal the population genetic structure of I. glandulifera in Finland and to find out whether there have been one or multiple invasions in Finland. The study focuses on investigating the origin of I. glandulifera in Southern Finland, by comparing plant samples from the Helsinki region with those from its native region and other regions of invasion. Samples from four populations in Helsinki and from the United Kingdom, Canada, India and Pakistan were collected and genotyped using 11 microsatellite markers. The genetic analyses were evaluated using the programs Arlequin and Structure. The results of the genetic analyses suggested that I. glandulifera has been introduced to Finland more than once. Multiple introductions are supported by the higher level of genetic diversity detected within and among Finnish populations than would be expected for a single introduction. Results of the Bayesian Structure analysis divided the four Finnish populations into four clusters. This geographical structure was further supported by pairwise Fst values among populations. The causes and potential consequences of such multiple introductions of I. glandulifera in Finland and further perspectives are discussed.

Functions of human papillomavirus E5 oncogene in epithelial cells and in the onset of cervical cancer

Human papillomaviruses (HPVs) are the causal agents of cervical cancer, which is the second most common cancer among women worldwide. Cellular transformation and carcinogenesis depend on the activities of viral E5, E6 and E7 proteins. Alterations in cell-cell contacts and in communication between epithelial cells take place during cervical carcinogenesis, leading to changes in cell morphology, increased cell motility and finally invasion. The aim of this thesis was to study genome-wide effects of the HPV type 16 (HPV-16) E5 protein on the expression of host cell messenger RNAs (mRNAs) and microRNAs by applying microarray technology. The results showed that the HPV-16 E5 protein alters several cellular pathways involved in cellular adhesion, motility and proliferation as well as in the extracellular matrix. The E5 protein was observed to enhance wound healing of epithelial cell monolayers by increasing cell motility in vivo. HPV-16 E5-induced alterations in the expression of cellular microRNAs and their target genes seem to favour increased proliferation and tumorigenesis. E5 was also shown to affect the expression of adherens junction proteins in HaCaT epithelial keratinocytes. In addition, a study of a membrane cytoskeletal cross-linker protein, ezrin, revealed that when activated, it localizes to adherens junctions. The results suggest that ezrin distribution to forming adherens junctions is due to Rac1 activity in epithelial cells. These studies reveal for the first time the holistic effects of HPV-16 E5 protein in promoting precancerous events in epithelial cells. The results contribute to identifyinging novel markers for cervical precancerous stages and to predicting disease behaviour.

The role of Acinetobacter sp. in biological phosphorus removal from forest industry wastewaters

Yhteenveto: Acinetobacter sp. metsäteollisuuden jätevesien biologisessa fosforinpoistossa