Identification of 33 candidate oncogenes by screening for base-specific mutations.

BACKGROUND: Genes with recurrent codon-specific somatic mutations are likely drivers of tumorigenesis and potential therapeutic targets. Hypermutable cancers may represent a sensitive system for generation and selection of oncogenic mutations. METHODS: We utilised exome-sequencing data on 25 sporadic microsatellite-instable (MSI) colorectal cancers (CRCs) and searched for base-specific somatic mutation hotspots. RESULTS: We identified novel mutation hotspots in 33 genes. Fourteen genes displayed mutations in the validation set of 254 MSI CRCs: ANTXR1, MORC2, CEP135, CRYBB1, GALNT9, KRT82, PI15, SLC36A1, CNTF, GLDC, MBTPS1, OR9Q2, R3HDM1 and TTPAL. A database search found examples of the hotspot mutations in multiple cancer types. CONCLUSIONS: This work reveals a variety of new recurrent candidate oncogene mutations to be further scrutinised as potential therapeutic targets.

Regulation of membrane-type matrix metalloproteinase 1 activity by dynamin-mediated endocytosis.

Membrane-type matrix metalloproteinase 1 (MT1-MMP) plays a critical role in extracellular matrix remodeling under both physiological and pathological conditions. However, the mechanisms controlling its activity on the cell surface remain poorly understood. In this study, we demonstrate that MT1-MMP is regulated by endocytosis. First, we determined that Con A induces proMMP-2 activation in HT1080 cells by shifting endogenous MT1-MMP from intracellular compartments to cell surface. This phenotype was mimicked by the cytoplasmic truncation mutant MT1 Delta C with more robust pro-MMP-2 activation and cell surface expression than wild-type MT1-MMP in transfected cells. MT1 Delta C was subsequently shown to be resistant to Con A treatment whereas MT1-MMP remains competent, suggesting that Con A regulates MT1-MMP activity through cytoplasmic domain-dependent trafficking. Indeed, MT1-MMP was colocalized with clathrin on the plasma membrane and with endosomal antigen 1 in endosomes. Internalization experiments revealed that MT1-MMP is internalized rapidly in clathrin-coated vesicles whereas MT1 Delta C remains on cell surface. Coexpression of a dominant negative mutant of dynamin, K44A, resulted in elevation of MT1-MMP activity by interfering with the endocytic process. Thus, MT1-MMP is regulated by dynamin-dependent endocytosis in clathrin-coated pits through its cytoplasmic domain.

Methodological issues in interviewing families in family nursing research.

AIM OF THE STUDY: The aim of this study is to discuss what methodological problems can be met in family research with one family member as an interviewee speaking on behalf of the whole family and, vice versa, what is the meaning of having multiple family members or the whole family unit as informants. BACKGROUND: Family nursing research is part of multidisciplinary research with families. It is a basis for family nursing and contributes to research, especially from the perspective of family welfare and its promotion. Family nursing research generates knowledge concerning families' and family members' wellbeing and experiences and expectations of nursing and health care. METHODS: The examination of methodological problems while pursuing family research is based on two studies conducted in Finland. FINDINGS: Quantitative methods add to the general knowledge of families. Qualitative methods are well suited to the study of family experiences. Family interviews performed for research purposes differ from interviews aiming at caring for families. They aim at obtaining knowledge of families on a general level so as to improve family nursing. Family research has to be looked at as a whole. It faces many challenges such as the definition of the family, gaining access, methods of data collection and data management. CONCLUSIONS: A family is a complex system and research with families need flexible, sensitive and practical methods. Family research should also aim at developing new methods for data collection and analysis.

Family dynamics of child-bearing families in Athens, Greece: a pilot study.

The purpose of this pilot study was to describe family dynamics in Greek families during the third trimester of a low-risk pregnancy with a first or second child. The description is based on Barnhill's framework for healthy family systems. The sample consisted of families expecting their first or second child. Both mothers (n = 160) and fathers (n = 47) participated in the study. The Family Dynamics Measure and a sociodemographic questionnaire were used in data collection. Data on 160 mothers and 47 couples were analyzed. Fathers perceived their families as having greater stability and role reciprocity than mothers. Fathers also reported clearer communication than mothers. There were no statistically significant relationships among family dynamics dimensions, maternal age and parity. Delayed first-time expectant mothers reported greater flexibility than normative first-time expectant mothers. The results provide some useful clues for prenatal care and also for further family dynamics research in Greece.

Cost--benefit analysis of team supervision: the development of an innovative model and its application as a case study in one Finnish university hospital.

AIM: To develop a model of costs and benefits of team supervision and a formula, which are examined more closely by means of an example. BACKGROUND: The popularity of clinical supervision (CS) as one of the methods of supporting health care practitioners' professional development (formative function), coping (restorative function) and quality improvement (normative function) has increased in the 1990s. CS may take the form of one-to-one or group supervision. Team supervision is a special form of group supervision. It means a group that has an interrelated work life outside the group. A host of literature and articles is available on CS. However, the costs and benefits of CS are less examined even though these have given rise to discussion particularly among decision-makers, because the monetary benefit of CS remains unsolved. METHOD: A nominal group technique was used to develop a model of costs and benefits of team supervision and a formula was derived on the basis of the model. The existing statistical data, for example a hospital ward's annual reports, data on sick days and reports on indemnities were utilized in the application of the formula. FINDINGS AND CONCLUSION: Team supervision was efficient in economic terms on the example ward. The model and the formula constitute a first attempt to ascertain the net present benefit of team supervision. Both the model and the formula need to be further tested, specified and refined.

Expression and purification of soluble and inactive mutant forms of membrane type 1 matrix metalloproteinase.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteinase and a cell-surface receptor and activator of gelatinase A in normal and neoplastic cells. We have expressed and purified a soluble deletion mutant of MT1-MMP lacking the transmembrane and cytoplasmic domains and an inactive mutant of the soluble MT1-MMP, where the active-site glutamic acid(240) was substituted by alanine (E240A). A baculovirus transfer vector coding for amino acids 21-539 of MT1-MMP (DeltaTM) and a similar vector coding for the mutation (E240ADeltaTM) were constructed for expression in insect cells. Both DeltaTM and E240ADeltaTM were secreted to the culture medium of infected High Five insect cells. They were then purified by cation-exchange followed by gel-filtration chromatography. DeltaTM was able to cleave denatured type I collagen and fibronectin and activate MMP-2/gelatinase-A, while E240ADeltaTM had only low proteolytic activity against denatured collagen I. The current expression and purification protocol should prove useful for the production of large amounts of enzymatically active soluble MT1-MMP.

Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic domain.

Membrane-type-1 matrix metalloproteinase (MT1-MMP) has transmembrane and cytoplasmic domains, which target it to invasive fronts. We analyzed the role of the cytoplasmic tail by expressing wild type MT1-MMP and three mutants with progressively truncated C termini in human Bowes melanoma cells. We examined gelatinase A activation and the localization and processing of recombinant proteins in stable cell clones using gelatin zymography, immunoblotting, and immunofluorescence. Cell invasion was analyzed in vitro by Matrigel invasion assays. Gelatinase A was activated in all cell clones. However, the localization of MT1-MMP to the leading edge of migrating cells and cell invasion through Matrigel were strongly enhanced only in cells expressing either wild type or truncated MT1-MMP lacking 6 C-terminal amino acid residues (Delta577). Truncations of 10 or 16 amino acid residues in the cytoplasmic domain (Delta567 and Delta573, respectively) disturbed MT1-MMP localization. The expression of wild type and Delta577 MT1-MMPs induced also their cleavage to 43-kDa cell surface forms and the release of soluble, approximately 20-kDa N-terminal fragments containing the catalytic center. A synthetic MMP inhibitor but not a gelatinase inhibitor prevented the processing, suggesting that autocatalytic cleavage occurs. Purified soluble MT1-MMP was also autoproteolytically processed to 43- and 20-kDa forms in vitro. Our results indicate that the cytoplasmic domain has an important role in cell invasion by controlling both the targeting and degradation/turnover of MT1-MMP.

Structural analysis and promoter characterization of the human membrane-type matrix metalloproteinase-1 (MT1-MMP) gene.

Membrane type-1 matrix metalloproteinase (MT1-MMP) degrades extracellular matrix components directly and indirectly by activation of other matrix metalloproteinases (MMPs). In the present study, we have isolated and characterized the human MT1-MMP gene and its promoter. The gene consists of 10 exons and nine introns spanning more than 10 kilobases (kb). The locations of two exon-intron splicing sites are distinct from the preserved positions among other known MMP genes. Primer extension and RNAse and S1 nuclease protection analyses indicated that there are four major and several minor transcription start sites. The 5'-flanking sequence of the gene contains putative regulatory elements, including one Sp-1 site and four CCAAT-boxes, whereas there is no TATA-box. The Sp-1 binding site was functional, as shown by gel shift and supershift analyses. Transfection studies with promoter constructs containing 0.1 to 7.2 kb of 5'-flanking sequence coupled to a luciferase reporter gene indicated that the promoter contains additional positive and negative regulatory sequences. Deletion of the Sp-1 binding site by site-directed mutagenesis reduced luciferase activity by about 90%, demonstrating the crucial role of this element in maintaining MT1-MMP transcription. Our findings indicate that the human MT1-MMP promoter has distinctive structural and functional features compared with other MMP genes, which may lead to a unique expression pattern and regulation during physiological and pathological processes.

MMP2 activation by collagen I and concanavalin A in cultured human hepatic stellate cells.

Fibrosis occurs in most chronic liver injuries and results from changes in the balance between synthesis and degradation of extracellular matrix components. In fibrotic livers, there is a markedly increased activity of matrix metalloproteinase 2 (MMP2), a major enzyme involved in extracellular matrix remodeling. We have previously shown that hepatic stellate cells secrete latent MMP2 and that MMP2 activation occurs in coculture of hepatic stellate cells and hepatocytes concomitantly with matrix deposition. In the present work we investigated the effects of various extracellular matrix components and concanavalin A, an inducer of immune-mediated liver injuries, on MMP2 activation in cultured human hepatic stellate cells. Collagen I induced a dose-dependent MMP2 activation, which was not blocked by both actinomycin and cycloheximide. Collagen VI, laminin, and a reconstituted basement membrane (matrigel) were ineffective in inducing activation. Specific antibodies against the subunits of alpha2beta1 integrins, the major collagen I receptor, induced partial inhibition of MMP2 activation. Treatment of cells with concanavalin A resulted in a marked activation of MMP2 that correlated with the proteolytic processing of MT1-MMP, the MMP2 activator, from a Mr=60 kd toward a Mr=43 kd polypeptide. Actinomycin and cycloheximide inhibited the MMP2 activation induced by concanavalin A. Recombinant tissue inhibitor of metalloproteinase-2 and the MMP inhibitor BB-3103, but not PMSF, blocked MMP2 activation induced by collagen I or concanavalin A, and MT1-MMP processing to its Mr-43 kd form. These results suggest that the accumulation of collagen I may specifically contribute to the remodeling of extracellular matrix in fibrotic livers by inducing MMP2 activation.

Expression of collagenases-1 and -3 and their inhibitors TIMP-1 and -3 correlates with the level of invasion in malignant melanomas.

Since proteolysis of the dermal collagenous matrix and basement membranes is required for local invasive growth and early metastasis formation of cutaneous melanomas, we have analysed the activities/expression levels of certain metalloproteinases in melanomas and cultured melanoma cells by in situ hybridization and Northern analysis. In addition to collagenases-1 and -3 that have been implicated in invasive growth behaviour of various malignant tumours, we analysed the levels of 72-kDa gelatinase and its activators MT1-MMP and TIMP-2 in cultured melanoma cells. The lesions examined included three cases of lentigo maligna and 28 cases of Clark grade I-V melanomas. The premalignant as well as the grade I tumours were consistently negative for collagenase-1 and -3 and TIMP-1 and -3. The collagenases were predominantly expressed in the cancer cells of Clark grade III and IV tumours. TIMP-1 and -3 were abundantly expressed in the cancer and/or stromal cells of grade III and IV melanomas, while TIMP-2 protein was detected also in melanomas representing lower invasive potential. Northern analysis of seven melanoma cell lines showed that the expression of collagenase-1 and TIMPs-1 and -3 was associated with 72-kDa gelatinase positivity. All melanoma cell lines were positive for MT1-MMP and TIMP-2 mRNAs. Our results suggest that overexpression of collagenases-1 and -3 and TIMPs-1 and -3 is induced during melanoma progression. Expression of TIMPs may reflect host response to tumour invasion in an effort to control MMP activity and preserve extracellular matrix integrity.

Family member as a hospital patient: sentiments and functioning of the family.

The aim of this pilot study was to find out how families experience the hospitalization of one family member and to chart the participation of the family in the treatment of the hospitalized family member. A questionnaire was used to gather data for the study and the three open-ended questions in the questionnaire were interpreted using content analysis. The study population (n = 70) was the family members of patients in the neurological wards of Tampere University Hospital. The study demonstrated a variety of negative sentiments in the families, such as worry, fear, shock, anxiety and depression at the hospitalization of their family member. The families also expressed neutral and positive sentiments, such as approval, relief and faith in the help given. Nearly 80% of the families' statements dealt with emotional responses. Changes in the everyday life of the family caused by the hospitalization of a family member were also reported, with most changes affecting the immediate family. Hospital visits gave a rhythm to family life. There were changes in the sharing of housework and taking care of affairs, as well as in relationships within the family. Family members spoke of their loneliness, fear and longing. External changes in family life were present in 13% of statements. Helping the patient in hospital involved functions like participation in nursing care, taking the patient to the cafeteria and rehabilitation. Only 20% of statements dealt with emotional support for the patient. Future research could broaden the perspective to include the views of patients, nurses and doctors on the reality of family nursing.

Family dynamics of families with cancer in Finland.

The purpose of this study was to describe family dynamics of families with cancer on the basis of Barnhill's framework for healthy family systems. The sample consisted of families in which one member had cancer. Both the patients (n = 96) and their relatives (n = 96) participated in the study (n = 192). The data for the study were collected using the Family Dynamics Questionnaire and the Family Dynamics Measure. The results indicated that the cancer of a single family member did not impair family functioning, but that family dynamics were considered quite good. There were no statistically significant differences between cancer patients and relatives on any of the family dynamics dimensions. However, an examination of sociodemographic characteristics did reveal some differences. Older relatives reported more enmeshment and rigidity than did younger relatives, whereas the latter reported more role conflict than older relatives. Older patients reported more rigidity than younger patients. Relatives who were men reported more enmeshment than women, whereas women reported more role conflict. Relatives of two-member families reported more rigidity than relatives with a larger family. Patients who reported a serious illness in the family described more mutuality, better flexibility, and clearer communication than patients who did not report such an illness. Also, relatives who mentioned a serious illness reported more mutuality and flexibility.

Proteolytic processing of membrane-type-1 matrix metalloproteinase is associated with gelatinase A activation at the cell surface.

Human fibroblasts and HT-1080 fibrosarcoma cells express membrane-type-1 matrix metalloproteinase (MT1-MMP), the cell surface activator of gelatinase A, in separate forms of 63 kDa, 60 kDa and in some cases 43 kDa. In the present work the interrelationships between MT1-MMP processing and gelatinase A activation were analysed using HT-1080 fibrosarcoma cells as a model. It was found that MT1-MMP was synthesized as a 63 kDa protein, which was constitutively processed to a 60 kDa active enzyme with N-terminal Tyr112, as shown by immunoprecipitation, immunoblotting and sequence analyses. Co-immunoprecipitation results indicated that only the active 60 kDa form of MT1-MMP bound gelatinase A at the cell surface. Both the activation of pro-MT1-MMP and the membrane binding of the tissue inhibitor of metalloproteinases type 2 (TIMP-2) and gelatinase A, and subsequent activation of gelatinase A, were inhibited by calcium ionophores. Although the active MT1-MMP was required for cell surface binding and activation of gelatinase A, it was inefficient in activating gelatinase A in fibroblasts or in control HT-1080 cells alone. Low expression levels of TIMP-2 and rapid synthesis of MT1-MMP were found to be critical for gelatinase A activation. In HT-1080 cells, MT1-MMP was further processed to an inactive, 43 kDa cell surface form when overexpressed, or when the cells were treated with PMA. Under these conditions, the activated gelatinase A was detected in the culture medium, in cell membrane extracts and in MT1-MMP-containing complexes. These results indicate that proteolytic processing (activation and degradation/inactivation) of MT1-MMP and MT1-MMP/TIMP-2 relationships at the cell surface are important regulatory levels in the control of gelatinolytic activity.

Family members' experiences of their role in a hospital: a pilot study.

The purpose of the study is to explore the experiences of family members in their role of relative in a situation where a next of kin has been admitted to hospital. The questionnaire was used in data collection. The data reported here are from a pilot study. The sample consisted of 70 family members of patients receiving treatment on the neurological wards. It was found that family members spent a lot of time at their relative's bedside, most of them up to several hours a day. The daily routines of families and way of life were also very much affected. The most important way in which the hospital supported families was to keep them informed about the patient's care and treatment. However, this was not possible without an active interest and involvement on the part of family members themselves. There were obvious problems and shortcomings in terms of family orientation: only one third of the family members felt that the nursing staff were seriously interested in the family's well-being, and only one quarter had been told what they could do in hospital. About half of the family members needed to meet nurses to get support from nurses and over one third from doctors. The oldest respondents and women needed more help than did others. Men preferred to turn to doctors rather than nurses for help. In general family members had good experiences of visiting their relative in hospital. They believed that they were expected and that they were of help to their relative. Over half of the family members said they were actively involved in caring for their relative. Only four per cent of the family members reported bad experiences of their visits to hospital, in spite of the obvious shortcomings in family nursing. For this reason it is important that nurses facilitate the involvement and integration of relatives in the process of nursing.

Family dynamics of families with mental health problems in Finland.

This study forms part of the International Family Dynamics Project. Its purpose was describe the family functioning of families with mental health problems on the basis of Barnhill's framework for healthy family systems. The sample consisted of 160 families in which one family member had mental health problems. Both the patients and their relatives took part. The data were collected by questionnaires, i.e. The Family Dynamics Measure and The Family Dynamics Questionnaire. According to the results, mental health patients described family functioning as fairly poor, while relatives described it as fairly good. However, patients' and relatives' perceptions of family functioning did not differ significantly. There were some statistically significant differences between patients' and relatives' perceptions of different family dynamics dimensions. Relatives reported more mutuality (P = 0.006) and clearer communication (P = 0.009) than patients. Older mental health patients reported more isolation than patients under 30. Relatives who mentioned some serious illness in the family reported more role conflict than those who didn't. No differences were found by gender, family structure or education. The results indicated that the mental health problems of a single family member did not impair family dynamics. The study showed that the resources and functioning of families are fairly good in spite of the illness in the family.

Regulation of membrane-type matrix metalloproteinase-1 expression by growth factors and phorbol 12-myristate 13-acetate.

Overexpression of membrane-type matrix metalloproteinase (MT-MMP-1) results in the activation of both endogenous and exogenous 72-kDa gelatinase. To understand the effects of MT-MMP-1 on 72-kDa gelatinase activation, we analyzed its expression in human fibroblasts and HT-1080 fibrosarcoma cells. Both cell types expressed the MT-MMP-1 mRNA constitutively at a considerable level and treatment of cells with PMA enhanced the expression about 2-3-fold. Concanavalin A treatment increased MT-MMP-1 mRNA levels in fibroblasts about 4-fold. Induction of MT-MMP-1 by phorbol 12-myristate 13-acetate (PMA) required protein synthesis as shown by cycloheximide inhibition. The induction was also inhibited by dexamethasone. Analysis of MT-MMP-1 mRNA stability using actinomycin D indicated that the half-life was rather long and not affected by PMA, suggesting transcriptional regulation. Only HT-1080 cells had significant 72-kDa gelatinase processing activity after treatment with PMA or concanavalin A, while fibroblasts were virtually negative. Immunoblotting analysis of fibroblast lysates indicated that MT-MMP-1 was present mainly in a 60-kDa form. PMA and concanavalin A caused 2-4-fold increases in its protein levels, while in HT-1080 cells PMA, concanavalin A, or overexpression of MT-MMP-1 did not significantly enhance the level of the 60-kDa protein. Instead, an immunoreactive, proteolytically processed 43-kDa form was observed, and its appearance correlated to 72-kDa gelatinase processing activity. Thus 72-kDa gelatinase activation, while enhanced by MT-MMP-1 expression, needs additional co-operating factors.

Stillbirth rates and folic acid and zinc status in low-socioeconomic pregnant women of Brazilian Amazon.

Twenty-five women in their first trimester and 25 women in their third trimester of pregnancy were randomly chosen from the slum areas of eight towns in the Brazilian Amazon (Manaus, Sao Luis, Boa Vista, Porto Velho, Sao Gabriel, Santarem, Tefe, and Benjamin Constant). Blood samples were removed from the subjects for folic acid and zinc analyses, and the women were asked questions about whether they had suffered from complications during previous pregnancies. It was found that the stillbirth rates reported ranged from 0 to 20/1000 pregnancies. The town that had the highest stillbirth rate (Santarem) also had the highest percentage of women with deficient erythrocyte (red blood cell, RBC) folate status. All of the women in Santarem also had deficient serum folate levels in both trimesters of pregnancy, as well as the lowest average RBC folate value in the third trimester of pregnancy. The same town was also one of two in which the pregnant women had the lowest mean serum zinc levels in the third trimester.

Breast milk folic acid and zinc concentrations of lactating, low socioeconomic, Amazonian women and the effect of age and parity on the same two nutrients.

The levels of folic acid and zinc in breast milk of about 25 women in their first trimester of lactation from each of eight towns in the Brazilian Amazon basin were determined. In order to compare the effects of age and parity on the breast milk folate and zinc levels, the results were analysed together. Thirty-one (60 per cent) of the women in their first month of lactation and 35 (51.5 per cent) in their second month had milk total folate levels below the calculated lower limit of adequacy; only 21 (47 per cent) of the women in their third month of lactation had milk total folate concentrations below the same limit. The breast milk zinc levels, however, were worse, with 35 (95 per cent) and 61 (97 per cent) of the women in their first and second months of lactation and all the women in their third month of lactation having values below the calculated lower limit of adequacy. Both in the case of total folate and zinc, the calculated lower limits of adequacy were obtained from the respective Recommended Dietary Allowances (RDAs) of a 0-6 month old infant and the average milk production of lactating women. While the mean breast milk folate levels (both total and free) showed no significant changes during the months of lactation, the average milk zinc levels showed a significant decrease between the first and second and first and third months of lactation. The age of the women did not significantly influence either the breast milk folate or zinc levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Iron, folic acid and zinc intakes and status of low socio-economic pregnant and lactating Amazonian women.

Dietary intakes were obtained by 24-h recall from 25 women in the first and third trimesters of pregnancy and 25 women in the first 3 months of lactation from eight towns in the Amazon valley. No consistent differences were found between the towns, so the results have been analysed together. Intakes of iron, free and total folate and zinc were nearly all very low compared with current recommendations. Despite their low intakes, the majority of the women had acceptable values of haemoglobin, haematocrit and MCHC. Serum folate concentrations were almost all extremely low (less than 2.5 ng/ml). The levels of RBC folate were also low, but in general not as severely so as those for serum folate. Serum zinc concentrations ranged from 0.2 to 0.7 microgram/ml, whereas the lower limit of acceptability has been put by various authors as 0.59-0.69 microgram/ml according to the stage of pregnancy. In breast milk, total folate in the majority of women ranged from 25 to 50 ng/ml, the greater part of it being in the free form. Zinc levels in breast milk were within the range 0-2 microgram/ml.

PIP: Dietary intakes were obtained by 24-hour recall from 25 women in the 1st and 3rd trimesters of pregnancy and from 25 women during their 1st 3 months of lactation from 8 towns in the Amazon valley. No consistent differences were found between the towns and therefore the results have been analyzed together. Intakes of iron, free and total folate, and zinc were nearly all very low intakes, the majority of the women had acceptable values of hemoglobin, hematocrit, and MCHC. Serum folate concentrations were almost all extremely low ( 2.5 ng/ml). The levels of RBC folate were also low, but in general not as severely as those for serum folate. Serum zinc concentrations ranged from 0.2-0.7 mcg/ml, whereas the lower limit of acceptability has been put by various authors as 0.59-0.69 mcg/ml according to the stage of pregnancy. In breastmilk, total folate in the majority of women ranged from 25-50 ng/ml, the greater portion of it being in its free form. Zinc levels in breastmilk were within the 0-2 mcg/ml range.