The behavior of dairy cattle in late gestation: Effects of parity and dystocia.

The aim of this study was to objectively assess, using an automated behavioral monitoring system, any behavioral differences between primiparous and multiparous cows before calving, and to quantify any behavioral differences between assisted (dystocic) and unassisted (eutocic) calvings. Data were collected from 32 multiparous and 12 primiparous Holstein dairy cattle to describe normal calving behavior and parity differences. To quantify behavior related to calving difficulty, the data from 14 animals that had dystocia at calving were matched to cows that had an eutocic calving based on parity, locomotion score, calf breed, calf sex, month, and year of calving. An IceQube (IceRobotics Ltd., South Queensferry, United Kingdom) was fitted to the right hind leg of cows 4 wk before their expected calving date. Data for lying time, standing time, number of steps, motion index (total motion), and the total number of standing and lying bouts (postural transitions) were automatically collected and summed into 15-min blocks. Behavioral variables were summarized into 2-h periods and 24-h periods before analyses. Mixed-effect models were used to analyze cow behavior in the last 4 d before calving (d -4 to -1), and on the day of calving. In the 4 d before calving, compared with multiparous cows, primiparous cows lay down an average 2.8 h/d less, had 9.1 more postural transitions/d (37.7 ± 1.2 vs. 27.6 ± 0.7), walked 172 more steps/d, and had a higher motion index (2,673.2 vs. 1,981.5 units/d). There was an effect of 2-h period on all behavioral variables on the day of calving. No indicator of calving difficulty was found on the day of calving, nor the days leading up to calving. These findings suggest that parity should be considered when predicting the day of calving, and changes in cow behavior on the day of calving could be used to identify calving cows, and to predict the time of calving.

The behavior of dairy cattle in the transition period: Effects of blood calcium status.

The aim of this study was to use an automated behavior-monitoring system to objectively assess the association between lying and activity behavior in the precalving, calving, and postcalving periods between multiparous and primiparous cows with (1) normocalcemia, (2) subclinical hypocalcemia, or (3) clinical hypocalcemia at calving. Behavioral data and blood serum samples were collected from 51 multiparous and 21 primiparous Holstein dairy cattle. Blood samples from the coccygeal vein were taken within 24 h of calving, and serum was analyzed to measure total calcium concentration. Cows were classified into one of 3 categories: normocalcemia (serum calcium concentration ≥ 2.0 mmol/L), subclinical hypocalcemia (serum calcium concentration < 2.0 mmol/L, absence of clinical signs), and clinical hypocalcemia (clinical signs and successful treatment). An activity sensor was fitted to the right hind leg of cows 3 wk before their expected calving date. Data for lying time, standing time, number of steps, and the total number of standing and lying bouts (postural transitions) were automatically collected and summed into 15-min blocks. Behavioral variables were summarized into 2-h and 24-h periods before analyses. Mixed effect models were used to analyze cow behavior in the entire 14 d before calving (d -14 to -1), on the day of calving, and the entire 21 d postcalving (d 1 to 21). In the precalving period, multiparous cows with normocalcemia had fewer postural transitions (18.5 ± 6.9 no./d) compared with cows with subclinical hypocalcemia (23.5 ± 8.0 no./d) and clinical hypocalcemia (23.5 ± 8.6 no./d). However, there was no association between blood calcium status on lying time (min/d) or step count (no./d) for multiparous cows. For primiparous cows, the step count of cows with subclinical hypocalcemia remained constant across the period, and the step count of cows with normocalcemia decreased from 842.8 steps/d on d -14 to 427.5 steps/d on d -1. Postpartum cows with clinical hypocalcemia were less active (fewer steps) and spent 88 min/d (1.5 h) and 125 min/d (2.1 h) more time lying down compared with cows with subclinical hypocalcemia and normocalcemia, respectively. This shows that clinical hypocalcemia is associated with significant long-lasting behavioral effects on cows during the critical postpartum period.

Aberrant expression of metastasis-inducing proteins in ectopic and matched eutopic endometrium of women with endometriosis: implications for the pathogenesis of endometriosis.

BACKGROUND: Endometriosis is a metastatic disease without obvious tumorigenesis. Expression of S100P, S100A4, osteopontin (OPN) or anterior gradient homologue 2 (AGR2) proteins can induce metastasis but fail to induce tumorigenesis per se. We now explore whether this group of metastasis-inducing proteins (MIPs) are associated with the pathogenesis of endometriosis. METHODS: Eutopic endometrial biopsies were taken from 73 women (35 fertile women without endometriosis and 38 women with surgically diagnosed endometriosis). Ectopic endometriotic lesions were collected from eight of the women with endometriosis. The expression of MIPs at the cellular level was evaluated by immunohistochemistry and the presence of these proteins in the endometrial tissues was verified by western blotting and their gene expression was confirmed by RT-PCR. RESULTS: All four MIPs were immunolocated in the endometrium of control women and S100P, AGR2 and OPN showed a cyclical variation. Proliferative phase eutopic endometrium of both groups showed a similar staining pattern for all MIPs, whereas secretory phase endometrium showed a differential expression between controls and cases. The secretory phase endometrial immunostaining of controls showed weak stromal and perivascular AGR2, and decreased stromal and glandular S100P. In contrast, immunostaining for all MIPs was increased in the late secretory endometrial samples of women with endometriosis and intense immunostaining was seen for S100A4 in the stroma (P< 0.05) and for S100P (P< 0.001) and AGR2 (P< 0.0001) in both glands and stroma (P< 0.001). All active peritoneal endometriotic lesions showed strong immunostaining for each of the MIPs studied. CONCLUSIONS: We propose that these MIPs enhance endometrial cell invasiveness and contribute to the establishment of ectopic endometriotic deposits after retrograde menstruation.

Impact of worker education on respiratory symptoms and sensitization in bakeries.

BACKGROUND: Flour exposure is known to cause significant respiratory problems. AIMS: To investigate the development of work-related sensitization, the period between first exposure and the development of symptoms (latent period) and the impact of workplace training programmes on respiratory health in plant bakers. METHODS: Two hundred and sixty-four bakers were investigated by assessing work-related respiratory symptoms and latent period before symptoms/sensitization, spirometry and testing for an array of workplace-specific IgE. RESULTS: There was a significant relationship between the presence of work-related respiratory symptoms and flour dust allergen-specific IgE. Latent periods varied widely: median for work-related nasal symptoms 36 months, cough 42 months and chest tightness 120 months. Latent periods were shorter for workers with evidence of flour sensitization (work-related wheeze: mean 13 months with sensitization, 97 months without, P < 0.05, work-related nasal symptoms, respectively; mean 19 months, 71 months, P < 0.01). Those warned of the health implications of flour dust had less work-related wheeze (warned; 1%, not warned 11%, P < 0.05). There was an excess of work-related symptoms and work-related-specific IgE combined in those who had not been warned of these health implications (12 versus 1%, P <0.01). CONCLUSIONS: Reporting of 'being warned' of potential health implications from breathing flour dust protected strongly against the reporting of important health end points. Latent periods for the development of work-related symptoms varied widely. Simple health messages, which may be overlooked in worker training programmes, can have significant benefits for worker health in the bakery population.

Comparison of various airflow measurements in symptomatic textile workers.

AIMS: To investigate the poorly understood relationship between work-related respiratory symptoms, airway reactivity, across working shift change in forced expiratory volume in 1 s (FEV(1)) and work-related changes in serial peak expiratory flow (sPEF) measures in a group of textile workers. METHODS: Fifty-three workers, 34 exposed to cotton dust and 19 to man-made fibre (MMF), were investigated using a standard respiratory questionnaire, sPEF, across-shift FEV(1) measurement and airway responsiveness. RESULTS: Thirty-four workers (64%) were male, and 9 workers (17%) had a >5% across-shift fall in FEV(1), and these falls were associated with the presence of work-related symptoms. Seven workers had a positive sPEF chart as judged by the software analysis (OASYS), although there was no relationship between work-related symptoms and sPEF. Six cotton workers (18%) and one MMF worker (5%) had airway hyperreactivity, which was associated strongly with work-related symptoms. Five of the 7 subjects with a positive sPEF had airway hyperreactivity compared with 12 of 46 with a negative sPEF. CONCLUSIONS: In this worker group, the presence of work-related respiratory symptoms was best associated with airway hyperresponsiveness and across-shift changes in FEV(1). While a positive sPEF chart was associated with increased airway responsiveness, it was not associated with work-related symptoms. sPEF measurements may not be the initial investigation of choice for such workers. As these findings also have relevance to developing evidence-based approaches to health surveillance, further work is needed to better define these relationships in other workers complaining of work-related respiratory symptoms.

New species of haematozoa from the avian families Campephagidae and Apodidae.

Leucocytozoon coracinae sp. nov. is described from the avian family Campephagidae and Hepatozoon apodis sp. nov. from the Apodidae. The distribution of these parasites within their respective families is discussed.

Increased expression of anterior gradient-2 is significantly associated with poor survival of prostate cancer patients.

Anterior gradient-2 (AGR2) expression was examined in a series of prostate cell lines and in an archival set of prostate tissues. The relative levels of AGR2 expression in the malignant cell lines PC-3 and PC-3M were, respectively, 5.3+/-0.1 and 3.8+/-0.2 times that detected in the benign cell line PNT-2. Immunohistochemical staining in 106 cases showed that amongst seven normal cases, one (14.3%) was unstained, five (71.4%) stained weakly positive and one (14.3%) stained moderately positive. Amongst 34 benign prostate hyperplastic (BPH) cases, 12 (35.3%) were unstained, 18 (52.9%) stained weakly positive and four (11.8%) stained moderately positive. Amongst 65 carcinomas, three (4.6%) were unstained, 14 (21.5%) stained weakly positive, 19 (29.2%) stained moderately positive and 29 (44.9%) stained strongly positive. AGR2 expression in carcinomas was significantly higher than that in BPH (chi(2)-test, P<0.001). Kaplan-Meier survival analysis showed that increased AGR2 expression was significantly (log rank test, P=0.007) associated with reduced patient-survival time. Increased joint Gleason score (GS) was significantly (log rank test, P=0.001) associated with poor patient survival. However, neither prostate specific antigen (PSA) level, nor androgen receptor (AR) index, was significantly associated with patient-survival time. Increased AGR2 expression was significantly correlated with high GS (two-sided Fisher's exact test, P<0.001) and PSA levels (Mann-Whitney U-test, P=0.047), but not significantly related to the level of AR (Mann-Whitney U-test, P=0.286). These results suggest that increased AGR2 expression is a valuable prognostic factor to predict the clinical outcome of the prostate cancer patients.

Gene Expression Meta-Analysis of Potential Metastatic Breast Cancer Markers.

BACKGROUND: Breast cancer metastasis is a highly prevalent cause of death for European females. DNA microarray analysis has established that primary tumors, which remain localized, differ in gene expression from those that metastasize. Crossanalysis of these studies allow to revile the differences that may be used as predictive in the disease prognosis and therapy. OBJECTIVE: The aim of the project was to validate suggested prognostic and therapeutic markers using meta-analysis of data on gene expression in metastatic and primary breast cancer tumors. METHOD: Data on relative gene expression values from 12 studies on primary breast cancer and breast cancer metastasis were retrieved from Genevestigator (Nebion) database. The results of the data meta-analysis were compared with results of literature mining for suggested metastatic breast cancer markers and vectors and consistency of their reported differential expression. RESULTS: Our analysis suggested that transcriptional expression of the COX2 gene is significantly downregulated in metastatic tissue compared to normal breast tissue, but is not downregulated in primary tumors compared with normal breast tissue and may be used as a differential marker in metastatic breast cancer diagnostics. RRM2 gene expression decreases in metastases when compared to primary breast cancer and could be suggested as a marker to trace breast cancer evolution. Our study also supports MMP1, VCAM1, FZD3, VEGFC, FOXM1 and MUC1 as breast cancer onset markers, as these genes demonstrate significant differential expression in breast neoplasms compared with normal breast tissue. CONCLUSION: COX2 and RRM2 are suggested to be prominent markers for breast cancer metastasis. The crosstalk between upstream regulators of genes differentially expressed in primary breast tumors and metastasis also suggests pathways involving p53, ER1, ERB-B2, TNF and WNT, as the most promising regulators that may be considered for new complex drug therapeutic interventions in breast cancer metastatic progression.

Induction of metastatic ability in a stably diploid benign rat mammary epithelial cell line by transfection with DNA from human malignant breast carcinoma cell lines.

Transfection of the stably diploid rat mammary epithelial cell line, Rama 37, which yields nonmetastasizing, adenomatous tumors in syngeneic rats with HindIII-fragmented DNA from malignant or nonmalignant human breast epithelial cell lines and the drug-resistance plasmid pSV2neo, yields transformants with a frequency of 10(-4) to 10(-5). The resultant cell lines form tumors with varying frequencies when injected s.c. into the mammary fat pads of syngeneic rats. Cells transfected with DNA from the malignant human breast carcinoma cell line, Ca2-83, or DNA from the human pleural effusion-derived cell lines, MCF-7 or ZR-75-1, yield transformants which metastasize to lungs and/or lymph nodes at high frequency, whereas transfection of HindIII-fragmented DNA from nonmetastatic human mammary epithelial cell lines, transfection of the drug-resistance plasmid pSV2neo alone, or nonspecific DNA such as salmon sperm DNA fails to yield transformants expressing the metastatic phenotype. Transfectants which metastasized were reestablished in culture and reinjected into syngeneic rats to confirm their metastatic properties. These transfectants yield rapidly growing tumors with reduced latent periods, which give rise to significant numbers of metastases. The karyotype of selected transfectants after passage in vivo remains stably diploid. Hybridization of a 32P-labeled oligonucleotide probe specific for the human Alu family of sequences to DNA from these transfectants reveals the presence of human-specific DNA sequences integrated into the genome. It is suggested that transfection of specific genomic DNA sequences from the malignant human cell lines can induce the metastatic phenotype in the nonmetastatic Rama 37 cell line in a genetically dominant manner, whereas genomic DNA from the nonmetastatic cells cannot confer metastatic properties to the Rama 37 cell line.

Metastasis-inducing dna regulates the expression of the osteopontin gene by binding the transcription factor Tcf-4.

Small 1,000-bp fragments of genomic DNA obtained from human malignant breast cancer cell lines when transfected into a benign rat mammary cell line enhance transcription of the osteopontin gene and thereby cause the cells to metastasize in syngeneic rats. To identify the molecular events underlying this process, transient cotransfections of an osteopontin promoter-reporter construct and fragments of one metastasis-inducing DNA (Met-DNA) have identified the active components in the Met-DNA as the binding sites for the T-cell factor (Tcf) family of transcription factors. Incubation of cell extracts with active DNA fragments containing the sequence CAAAG caused retardation of their mobilities on polyacrylamide gels, and Western blotting identified Tcf-4, beta-catenin, and E-cadherin in the relevant DNA complexes in vitro. Transfection of an expression vector for Tcf-4 inhibited the stimulated activity of the osteopontin promoter-reporter construct caused by transiently transfected active fragments of Met-DNA or permanently transfected Met-DNA. This stimulated activity of the osteopontin promoter-reporter construct is accompanied by an increase in endogenous osteopontin mRNA but not in fos or actin mRNAs in the transfected cells. Permanent transfection of the benign rat mammary cell line with a 20-bp fragment from the Met-DNA containing the Tcf recognition sequence CAAAG caused an enhanced permanent production of endogenous osteopontin protein in vitro and induced the cells to metastasize in syngeneic rats in vivo. The corresponding fragment without the CAAAG sequence was without either effect. Therefore, the regulatory effect of the C9-Met-DNA is exerted, at least in part, by a CAAAG sequence that can sequester the endogenous inhibitory Tcf-4 and thereby promote transcription of osteopontin, the direct effector of metastasis in this system.

Prognostic significance of the metastasis-inducing protein S100A4 (p9Ka) in human breast cancer.

The calcium-binding protein S100A4 is capable of inducing metastasis in rodent models for breast cancer. We now show that rabbit antibodies to recombinant rat S100A4 recognize specifically human S100A4 using Western blotting techniques and use them to assess the prognostic significance of S100A4 in primary tumors from a group of 349 patients treated between 1976 and 1982 for stage I and stage II breast cancer. The antibody stains normal breast tissue heterogeneously, but stains positively 41% of the carcinomas, leaving the remaining 59% as negatively stained. In addition to the carcinoma cells, some host stromal cells and lymphocytes are also stained, but these have been discounted in subsequent analyses. There is an association of staining of carcinomas for S100A4 with some tumor variables considered to be associated with poor prognosis for patients: tumor present in axillary lymph nodes (borderline P = 0.058), staining for c-erbB-3 (P = 0.002), cathepsin D (P = 0.024), and c-erbB-2 (P = 0.048). The association of staining for S100A4 with patient survival has been evaluated using life tables and analyzed using generalized Wilcoxon statistics. Eighty percent of the S100A4-negative patients but only 11% of the S100A4-positive patients are alive after 19 years of follow-up, and this association is highly significant (P < 0.0001); the former have a median survival of >228 months and the latter 47 months. The other tumor variables that show significant association with survival time are nodal status (P < 0.0001), tumor size (P = 0.0035), histological grade (P = 0.013), staining for c-erbB-2 (P = 0.0015), estrogen receptor (P = 0.028), and p53 (P = 0.032). Analysis of the association of patients with carcinomas staining for S100A4 and their survival in subgroups defined by these other tumor variables shows that in each subgroup, staining for S100A4 is associated with poorer survival. Patients whose tumors stain for S100A4 and possess involved lymph nodes (P < 0.0001), which are fixed to the chest wall (P = 0.015) or which stain for c-erbB-2 (P = 0.050), show a significant reduction in survival times over those with only S100A4-staining tumors. Patients with involved lymph nodes, or staining for c-erbB-2 in the S100A4-negative group fail to show any significant reduction in survival times. Multivariate regression analysis for 137 patients shows that staining for S100A4 is most highly correlated with patient deaths (P < 0.0001), but involved lymph nodes (P = 0.001), fixed tumors (P = 0.0002), and high histological grade (P = 0.022) are also significant independent prognostic variables. These results suggest that in this group of patients, the metastasis-inducing protein S100A4 is most tightly correlated with patient demise.

The identification of osteopontin as a metastasis-related gene product in a rodent mammary tumour model.

The rat mammary epithelial cell line, Rama 37, yields benign, non-metastasizing adenomatous tumours in syngeneic Furth-Wistar rats. Transfection of this stably diploid cell line with genomic DNA fragments from a human metastasizing breast cancer cell line yields cells which, when injected subcutaneously in syngeneic rats, give rise to secondary tumours in a number of the animals. From one such secondary lung tumour, a cell line was established designated Ca2-5-LT1. This cell line, when introduced into the syngeneic rat host, also showed the ability to metastasise. To determine key changes in gene expression that occur during the progression from Rama 37, the benign tumour-inducing cell line, to the metastatic derivative Ca2-5-LT1, a general method of subtractive hybridization has been employed. This procedure in conjunction with Northern blotting and nucleic acid sequencing has been used to identify mRNAs expressed differentially between the metastatic and nonmetastatic cell lines described above. So far, of the subtracted cDNAs that have been identified which represent differentially expressed mRNAs, a large proportion of these cDNAs corresponded to the mRNA for rat osteopontin (OPN). The mRNA for OPN was expressed at a ninefold higher level in the metastatic Ca2-5-LT1 cell line when compared to the nonmetastatic parental Rama 37 cell line. Rama 37 cells transfected with DNA from a human benign cell line failed to show elevated levels of OPN mRNA. Following transfection of Rama 37 cells with an expression-construct producing elevated levels of OPN, the newly-transfected cells, when introduced into the rat host, developed metastases in 55% of the animals that produced primary tumours. These experiments show that increasing the expression of OPN in a previously benign cell tine is sufficient to produce a metastatic phenotype in this particular rat mammary model.

Isolation of and effector for metastasis-inducing DNAs from a human metastatic carcinoma cell line.

Benign rat mammary epithelial cells transfected with restriction enzyme-fragmented DNA from a human malignant metastatic cell line (Ca2-83) produces transfectants that yield metastatic tumours in syngeneic rats. The six metastasis-inducing DNAs (Met-DNAs) that have been isolated from such transfectants are subgene in size and do not code for any expressed mRNAs, but correspond to potential regulatory regions of human DNA from malignant, metastatic cells. In pilot studies the one Met-DNA tested is detectable in some human breast tumours but not in normal tissue. Transfection of all six Met-DNAs singly into the benign mammary epithelial cells causes enhanced expression of osteopontin, whilst transfection of cDNA for osteopontin also induces the metastatic state. These results show that short regulatory DNAs exist in human cancer cells that can induce metastatic spread via a common effector gene, osteopontin, in model rat mammary cell lines.

Human S100A4 (p9Ka) induces the metastatic phenotype upon benign tumour cells.

The rodent S100-related calcium-binding protein, S100A4 induces metastasis in non-metastatic rat and mouse benign mammary cells and co-operates with benign-tumour-inducing changes in two transgenic mouse models, to yield metastatic mammary tumours. Co-transfection of the human gene for S100A4 with pSV2neo into the benign rat mammary cell line, Rama 37, yielded cells which expressed a low level of the endogenous S100A4 mRNA, and either high or undetectable levels of human S100A4 mRNA. The cells which expressed a high level of human S100A4 mRNA induced metastasis in the benign rat mammary cell line Rama 37 in an in vivo assay, whereas the cells which expressed an undetectable level of human S100A4 did not induce any detectable metastases. The primary tumours arising from the S100A4-expressing cells contained high levels of immunocytochemically-detected S100A4 and this high level of S100A4 and the metastatic potential were maintained when cells from a metastasis were re-injected into syngeneic rats. The results show that the human S100A4 possesses metastasis-inducing capabilities.

Regulatory region of metastasis-inducing DNA is the binding site for T cell factor-4.

Small 1000 bp fragments of DNA derived from human malignant breast cancer cells have been isolated which, when transfected into a benign rat mammary cell line induce the production of osteopontin and thereby endow those cells with the capability to metastasize in syngeneic rats. Using transient transfections of an osteopontin promoter-reporter construct, we have now identified the active moiety in the metastasis-inducing DNA as the binding site for the T cell factor (Tcf) family of transcription factors and located Tcf-4, beta-catenin and E-cadherin in the relevant DNA complex in vitro. The regulatory effects of the metastasis-inducing DNAs are therefore exerted, at least in part, by a CAAAG sequence which can sequester Tcf-4, thereby promoting transcription of the direct effector for metastasis in this system, osteopontin.

Induction of the metastatic phenotype by transfection of a benign rat mammary epithelial cell line with the gene for p9Ka, a rat calcium-binding protein, but not with the oncogene EJ-ras-1.

The rat mammary epithelial cell line, Rama 37, yields benign, non-metastasizing adenomatous tumours in syngeneic Wistar-Furth rats. Transfection of this line with a drug resistance plasmid containing both the gene for resistance to Geneticin (neo) and the gene for p9Ka (pSV2neo-p9Ka), a rat calcium-binding protein, or with a similar plasmid containing neo and the oncogene EJ-ras-1 (pSV2neo-ras) yields drug-resistant transformants that express high levels of the p9Ka or EJ-ras-1 mRNAs and proteins. These transfected cells all produce tumours when injected at subcutaneous sites with a shorter median latent period than the tumours produced by the parental untransfected Rama 37 cells in syngeneic hosts. Cells transfected with pSV2neo-p9Ka yield a higher incidence of tumours than untransfected Rama 37 cells, many of which metastasize to lungs and/or lymph nodes in syngeneic rats. However, cells transfected with pSV2-neo-ras or pSV2neo plasmid alone yield tumours that fail to metastasize. Immunofluorescent studies suggest an association of p9Ka with the cytoskeleton, as depicted by F-actin staining with the reagent phalloidin. It is suggested that the transfection of copies of the gene for the rat calcium-binding protein p9Ka can enhance the tumorigenic potential and induce the metastatic phenotype in this rat mammary model, whereas transfection of control plasmid DNA or the oncogene EJ-ras-1 fails to induce the metastatic phenotype, although EJ-ras-1 transfectants, like those containing p9Ka, possess increased growth properties in vivo.

Expression of the calcium-binding protein S100A4 (p9Ka) in MMTV-neu transgenic mice induces metastasis of mammary tumours.

Increased levels of S100A4 (p9Ka) confer metastatic ability on a normally non-metastatic epithelial cell line. To find out whether S100A4 can induce metastasis in vivo, transgenic mice expressing high levels of S100A4, but which show no phenotypic effect, have been mated with MMTV-neu transgenic mice which succumb to stochastic mammary neoplasia related to expression of the MMTV-neu transgene. Resultant bitransgenic, multiparous, female progeny expressing both S100A4 and Neu have a slightly earlier incidence of palpable mammary tumours than the MMTV-neu offspring and specifically exhibit macroscopic metastatic lesions in the lungs. The S100A4 transgene is expressed in primary and secondary lesions of bitransgenic offspring and its expression is particularly associated with regions of invasion of primary lesions and metastases.

Calcium-binding protein S100A4 in health and disease.

The S100 proteins contain two EF-hand motifs and are of generally unknown function. One of these proteins, S100A4, is an intracellular calcium-binding protein that is present in normal rodent and human cells. In cultured rodent mammary cells, S100A4 is expressed at a higher level in some metastatic epithelial cells than in non-metastatic counterparts. Similarly, in human breast cell lines, S100A4 is present at a higher level in cultured cells from the more malignant, than in those from the more benign tumours. Gene transfer experiments have shown that rodent or human S100A4 is able to induce metastatic capability in otherwise non-metastatic breast tumour cells. Furthermore, expression of rodent S100A4 transgenes can induce metastasis of benign tumours arising in transgenic model systems. Possible mechanisms for the metastasis-inducing effect of S100A4 and the relevance of these observations to human cancer are discussed.

Protein synthesis in chloroplasts. IX. Assembly of newly-synthesized large subunits into ribulose bisphosphate carboxylase in isolated intact pea chloroplasts.

Isolated pea (Pisum sativum) chloroplasts incorporate [35S]methionine into the large subunit of the chloroplast enzyme ribulose bisphosphate carboxylase. When chloroplasts are incubated in a medium containing KCl as osmoticum, newly-synthesised large subunits are not incorporated into the holoenzyme but can be separated from pre-existing enzyme by gel electrophoresis under non-denaturating conditions. Furthermore, newly-synthesised large subunits are not precipitated by antibodies which precipitate pre-existing holoenzyme and large subunit prepared from holoenzyme. When chloroplasts are incubated in a medium containing sorbitol as osmoticum, some of the newly-synthesised large subunits comigrate with holoenzyme on both 3% and 5% polyacrylamide non-denaturing gels. Such comigrating large subunits are precipitated by antibodies raised against the holoenzyme. These results indicate assembly of large subunits into ribulose bisphosphate carboxylase in the sorbitol medium. Time course experiments indicate that there is a time-lag of several minutes between onset of synthesis of large subunits and the onset of assembly. Newly-synthesised large subunits which do not comigrate with holoenzyme on both 3% and 5% polyacrylamide non-denaturing gels are associated with a protein of subunit molecular weight 60 000. This protein may be specifically combined with newly-synthesised large subunits, and the resulting aggregate be involved in the assembly of complete molecules of ribulose bisphosphate carboxylase.

A rapid procedure for production of human basic fibroblast growth factor in Escherichia coli cells.

Human basic fibroblast growth factor has been expressed in Escherichia coli cells at a level of 2-3 mg/l culture, using a rapid procedure which requires only simple DNA manipulative work. The recombinant material has the same potency as natural basic fibroblast growth factor from bovine pituitary glands.