Collagen-like peptide sequences inhibit bacterial invasion of root dentine.

AIM: To investigate the effects of peptides derived from the sequence of collagen to inhibit penetration of human or bovine dentine by species of streptococci and enterococci. METHODOLOGY: Blocks of human or bovine root dentine were infected for 14 days with bacterial cultures, in the presence or absence of various collagen-like peptide sequences. Invasion of dentinal tubules was determined from microscopic images of histochemically stained dentine thin sections. Extent of invasion was expressed as tubule invasion index (TI), or tubule invasion factor (TIF) which, in addition to the density of invasion, took into account the depth of invasion. Data were analysed by two-way anova. RESULTS: Streptococcus gordonii, Streptococcus mutans and Enterococcus faecalis were associated with heavy invasion (TI >2.5, TIF >4) of human or bovine root dentinal tubules, with E. faecalis being the most penetrative. Incorporation of peptides Gly-Pro-Ala or Gly-Pro-Hyp into the in vitro model system significantly reduced (P < 0.05) dentine invasion by the three species of highly invasive organisms. Inhibition of bacterial invasion by the peptides was dose dependent, and the peptides did not inhibit bacterial growth in culture. CONCLUSION: Specific collagen-like peptide sequences inhibited the invasion of dentine in vitro by a range of oral bacteria. The peptides likely act as competitive inhibitors blocking bacterial collagen receptors and could potentially allow for target-specific control of dentine infections.

A cohort study of the associations between udder conformation, milk somatic cell count, and lamb weight in suckler ewes.

A cohort study of 67 suckler ewes from 1 farm was carried out from January to May 2010 to investigate associations between udder conformation, udder half milk somatic cell count (SCC), and lamb weight. Ewes and lambs were observed at lambing. Ewe health and teat condition and lamb health and weight were recorded on 4 to 5 further occasions at 14-d intervals. At each observation, a milk sample was collected from each udder half for somatic cell counting. Two weeks after lambing, ewe udder conformation and teat placement were scored. Low lamb weight was associated with ewe SCC >400,000 cells/mL (-0.73 kg), a new teat lesion 14 d previously (-0.91 kg), suboptimal teat position (-1.38 kg), rearing in a multiple litter (-1.45 kg), presence of diarrhea at the examination (-1.19 kg), and rearing by a 9-yr-old ewe compared with a 6-yr-old ewe (-2.36 kg). High lamb weight was associated with increasing lamb age (0.21 kg/d), increasing birth weight (1.65 kg/kg at birth), and increasing number of days the ewe was given supplementary feed before lambing (0.06 kg/d). High udder half SCC was associated with pendulous udders (9.6% increase in SCC/cm of drop) and greater total cross-sectional area of the teats (7.2% increase of SCC/cm(2)). Low SCC were associated with a heavier mean litter weight (6.7% decrease in SCC/kg). Linear, quadratic, and cubic terms for days in lactation were also significant. We conclude that poor udder and teat conformation are associated with high levels of intramammary infection, as indicated by increased SCC and that both physical attributes of the udder and SCC are linked to lamb growth, suggesting that selection of suckler ewes with better udder and teat conformation would reduce intramammary infection and increase lamb growth rate.

Short communication: Preliminary investigation into the effect of freezing and a cryopreservant on the recovery of mastitis pathogens from ewe milk.

The objective of this study was to investigate the recovery of bacteria from ewe milk after freezing for 4 or 8 wk with and without the addition of glycerol as a cryopreservant. A total of 50 udder-half milk samples with a known range of bacterial species were selected, stored, and analyzed in 5 treatment groups: time zero; frozen for 4 wk with, and without, glycerol; and frozen for 8 wk with, and without, glycerol. A lower recovery was observed in all bacterial species studied after freezing. Samples containing fewer than 100 cfu/mL came from ewes with a lower somatic cell count and were more likely to be bacteriologically negative after freezing than those above this threshold. The addition of glycerol increased recovery of gram-negative bacteria after freezing, although this requires further study to draw strong conclusions. The effects on gram-positive species were inconsistent. We conclude that although the addition of glycerol had a small beneficial effect on the sensitivity of detection of bacteria from frozen sheep milk, sensitivity was highest in cultures from fresh milk.

Aedes aegypti in Jamaica, West Indies: container productivity profiles to inform control strategies.

OBJECTIVE: To describe the Aedes aegypti container profile in the three parishes of Portland, St. Anns and St. Catherine, Jamaica. METHOD: Traditional stegomyia and pupae per person indices. RESULTS: A total of 8855 containers were inspected. A. aegypti were breeding in 19.2% of the 4728 containers in Portland, in 6.7% of the 2639 containers in St. Ann, and in 27.2% of the 1488 containers in Tryhall Heights, St. Catherine. Container types differed between Portland (P > 0.02) on one hand and St. Ann and Tryhall Heights, St. Catherine on the other hand: there were with no vases or potted plants with water saucers in St. Ann and St. Catherine. A. aegypti were breeding in more containers in St. Catherine (38%) (38% in wet season and 21% in the dry season) than in Portland (19%) or St. Ann (6%), both of which had more containers but A. aegypti breeding in fewer: 17.7% and 11.2% in the wet and 20.4% and 3.5% in the dry seasons respectively. The daily production of adult mosquitoes in the three study sites was 1.51, 1.29 and 0.66 adult female mosquitoes per person in Portland, St. Ann and St. Catherine during the dry season and 1.12, 0.23 and 1.04 female mosquitoes per person in the wet season respectively. CONCLUSION: All three communities are at risk for dengue outbreaks and vector control should concentrate on reducing the mosquito populations from the most productive containers before a new dengue virus serotype is introduced into Jamaica.

Exploring the longitudinal dynamics of herd BVD antibody test results using model-based clustering.

Determining the Bovine Viral Diarrhoea (BVD) infection status of cattle herds is a challenge for control and eradication schemes. Given the changing dynamics of BVD  virus (BVDV) antibody responses in cattle, classifying herds based on longitudinal changes in the results of BVDV antibody tests could offer a novel, complementary approach to categorising herds that is less likely than the present system to result in a herd's status changing from year to year, as it is more likely to capture the true exposure dynamics of the farms. This paper describes the dynamics of BVDV antibody test values (measured as percentage positivity (PP)) obtained from 15,500 bovines between 2007 and 2010 from thirty nine cattle herds located in Scotland and Northern England. It explores approaches of classifying herds based on trend, magnitude and shape of their antibody PP trajectories and investigates the epidemiological similarities between farms within the same cluster. Gaussian mixture models were used for the magnitude and shape clustering. Epidemiologically meaningful clusters were obtained. Farm cluster membership depends on clustering approach used. Moderate concordance was found between the shape and magnitude clusters. These methods hold potential for application to enhance control efforts for BVD and other infectious livestock diseases.

Quasi-Isotropic and Pseudo-Ductile Highly Aligned Discontinuous Fibre Composites Manufactured with the HiPerDiF (High Performance Discontinuous Fibre) Technology.

Conventional composite materials reinforced with continuous fibres display high specific strength but have a number of drawbacks including: the elastic-brittle behaviour, difficulties in producing defect-free components of complex shape with high-volume automated manufacturing processes, and inherent lack of recyclability. Highly aligned, discontinuous fibre-reinforced composites (ADFRCs) are truly beneficial for mass production applications, with the potential to offer better formability and comparable mechanical properties with continuous fibre-reinforced composites. In previous publications, the High Performance Discontinuous Fibre (HiPerDiF) technology has been shown to offer the possibility to intimately hybridise different types of fibres, to achieve pseudo-ductile tensile behaviour, and remanufacture reclaimed fibres into high-performance recycled composites. However, to date, the work has been conducted with unidirectional (UD) laminates, which is of limited interest in engineering applications with mechanical stresses acting across many directions; this paper reports, for the first time, the mechanical behaviour of quasi-isotropic (QI) ADFRCs. When compared with randomly-oriented discontinuous fibre composites (RODFRCs), QI ADFRCs offer enhanced stiffness (+26%) and strength (+77%) with higher consistency, i.e., a reduction of the coefficient of variance from the 25% of RODFRCs to the 6% of ADFRCs. Furthermore, hybrid QI ADFRCs retain the pseudo-ductility tensile behaviour previously observed in unidirectional (UD) lay-up.

Survey of the prevalence, diagnosis and treatment of dermatological conditions in small animals in general practice.

A survey was made of the prevalence, diagnosis and treatment of dermatological conditions in small animals in general practice in the UK. Out of 3707 small animal consultations in general practice that were observed and recorded, 795 (21.4 per cent) involved animals that had a dermatological problem. In dogs and exotic species, pruritus was the most common presenting sign, accounting for 30 to 40 per cent of the dermatological consultations. In cats, cutaneous swellings were the most common presentation (36 per cent). A diagnosis or recommendation for treatment was made on the basis of the presenting clinical signs and physical examination alone in 576 (72 per cent) of the cases, and various diagnostic tests were performed in the other cases. In dogs, parasitic infestations, bacterial infections and neoplasia accounted for the majority of the diagnoses. In cats, parasites and bacterial infections were the most common. In exotic species, parasites accounted for over 80 per cent of the dermatological diagnoses. In dogs, the most common final diagnoses were otitis, pyoderma, anal sac impaction, flea infestation and atopic dermatitis. In cats, abscesses, flea infestation, and otitis were the most common diagnoses. In exotic species, the most common diagnosis was an unspecified mite infestation. Systemic antibiotics were prescribed in 196 cases (25 per cent), systemic glucocorticoids were prescribed in 162 cases (20 per cent) and treatment with an ectoparasiticide was prescribed in 167 cases (21 per cent).

A cross-sectional study of 329 farms in England to identify risk factors for ovine clinical mastitis.

The aims of this study were to estimate the incidence rate of clinical mastitis (IRCM) and identify risk factors for clinical mastitis in suckler ewes to generate hypotheses for future study. A postal questionnaire was sent to 999 randomly selected English sheep farmers in 2010 to gather data on farmer reported IRCM and flock management practices for the calendar year 2009, of which 329 provided usable information. The mean IRCM per flock was 1.2/100 ewes/year (CI:1.10:1.35). The IRCM was 2.0, 0.9 and 1.3/100 ewes/year for flocks that lambed indoors, outdoors and a combination of both, respectively. Farmers ran a variety of managements before, during and after lambing that were not comparable within one model, therefore six mixed effects over-dispersed Poisson regression models were developed. Factors significantly associated with increased IRCM were increasing percentage of the flock with poor udder conformation, increasing mean number of lambs reared/ewe and when some or all ewes lambed in barns compared with outdoors (Model 1). For ewes housed in barns before lambing (Model 2), concrete, earth and other materials were associated with an increase in IRCM compared with hardcore floors (an aggregate of broken bricks and stones). For ewes in barns during lambing (Model 3), an increase in IRCM was associated with concrete compared with hardcore flooring and where bedding was stored covered outdoors or in a building compared with bedding stored outdoors uncovered. For ewes in barns after lambing (Model 4), increased IRCM was associated with earth compared with hardcore floors, and when fresh bedding was added once per week compared with at a frequency of ≤2 days or twice/week. The IRCM was lower for flocks where some or all ewes remained in the same fields before, during and after lambing compared with flocks that did not (Model 5). Where ewes and lambs were turned outdoors after lambing (Model 6), the IRCM increased as the age of the oldest lambs at turnout increased. We conclude that the reported IRCM is low but highly variable and that the complexity of management of sheep around lambing limits the insight into generating hypotheses at flock level for risks for clinical mastitis across the whole industry. Whilst indoor production was generally associated with an increased IRCM, for ewes with large litter size indoor lambing was protective, we hypothesise that this is possibly because of better nutrition or reduced exposure to poor weather and factors associated with hygiene.

Destabilization and denaturation of cellular protein by glutathione depletion.

This investigation tested the hypothesis that depletion of intracellular glutathione, in contrast to its oxidation could lead to non-native oxidation of protein thiols, thereby trapping proteins in an unstable conformation. Chinese hamster cells were exposed to the alpha, beta-unsaturated dicarboxylic acid diethylmaleate in order to produce rapid glutathione (GSH) depletion without oxidation. Measurement of the fluorescence of oxidized 2',7'-dichlorofluorescein diacetate indicated that reactive oxygen species accumulated in GSH depleted cells. Glutathione depletion was found to alter protein thiol/disulfide exchange ratios such that 17 to 23 nmol of protein SH/mg protein underwent oxidation. Differential scanning calorimetry (DSC) of glutathione depleted cells yielded a profile of specific heat capacity versus temperature that was characteristic of cells containing destabilized and denatured protein. In addition, cells depleted of glutathione exhibited a two-fold increase in NP-40 insoluble protein. These results indicate that depletion of intracellular glutathione caused oxidation of protein thiols, protein denaturation and aggregation and provide a mechanism to explain how GSH depletion can initiate stress responses.

Evaluation of bupivacaine nerve blocks in the modification of pain and pulmonary function changes after thoracotomy.

Thirty-one patients undergoing thoracotomy were prospectively randomized to receive (1) no nerve blocks (n = 12), (2) placement of percutaneous catheters for intermittent nerve blocks with bupivacaine (Marcaine) (n = 10), or (3) bupivacaine nerve blocks intraoperatively (n = 8). One patient refused postoperative evaluation and was not included in this study. All patients received similar preoperative, intraoperative, and postoperative medications. Comparison of preoperative and postoperative arterial blood gases, assessments of pain and alertness, and chest roentgenograms showed no statistical advantage for any group. Analgesic requirements and pulmonary functions (functional residual capacity, tidal volume, minute ventilation peak flow, or forced expiratory volume) did not differ among the groups. Statistically significant differences were seen in mean respiratory rate and forced vital capacity. These differences, however, indicate that bupivacaine either by intraoperative use or by intermittent percutaneous administration did not improve postoperative increases in respiratory rate or decreases in forced vital capacity.

PCB and dioxin re-entry criteria for building surfaces and air.

A number of fires involving polychlorinated biphenyl (PCB)-containing transformers and capacitors have occurred in the United States. PCB fires generate by-products such as polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzodioxins (PCDDs) and, when the transformer is in a building, contaminate the interior. Considerable concern exists over the potential human health effects associated with exposure by inhabitants to residual levels of PCBs, PCDFs and PCDDs. Office workers, for example, may be exposed to these chlorinated compounds via inhalation of contaminated particulates and vapors, dermal contact with contaminated surfaces, and incidental ingestion of dusts. A wide range of re-entry or cleanup levels have been developed for PCDDs and PCBs to protect workers who re-occupy a building following a PCB fire. Re-entry criteria have been used by property owners and regulatory agencies to determine whether the building is safe to re-occupy or to determine the extent of needed remediation. This paper presents a mass balance approach to deriving risk-based re-entry surface and air criteria for PCBs and PCDD/PCDFs. These criteria were based on a lifetime risk level of 10(-5), recent toxicological data on PCDDs and PCBs, and plausible exposure scenarios. Our analysis suggests that 125 ng/m2 2,3,7,8-TCDD TEQ for surfaces and 10 pg/m3 for air are acceptable. Based on Aroclor 1260, risk-based re-entry criteria for PCBs on surfaces and in air were 750 micrograms/m2 and 0.1 microgram/m3, respectively. In comparison to most previous guidelines, these risk-based criteria are less stringent, but can still be considered conservative. The surface criteria are 5 to up to 125 fold higher than previous guidelines. Air criteria range up to 5 times higher than criteria used at past PCB fire sites. Air concentrations associated with these were modeled and were negligible. For PCBs in air, the NIOSH guideline of 1 microgram/m3 is also appropriate for occupational settings.

Distribution of MUC1 in the normal human oral cavity is localized to the ducts of minor salivary glands.

The change in expression of MUC1 from health to disease forms the basis of its use as a potential disease marker. Previous attempts at isolating MUC1 from normal, healthy human oral mucosa have, however, drawn conflicting conclusions as to its presence. Furthermore, when MUC1 was detected in the oral glycocalyx, it was not clear which cells were synthesising it. We examined human oral glycocalyx using pooled buccal smears from 50 normal individuals. Following isopycnic density centrifugation and membrane extraction with octyl glucoside and saponin, MUC1 was detected with the polyclonal antibody CT1. Immunohistochemistry using antibodies CT1 and BC2 was performed on sections from eight labial, seven palatal, four buccal, three retromolar pad, three dorsum of tongue and two ventral surface of tongue biopsies. In-situ hybridisation using MUC1 and cytoplasmic tail oligoprobes on sections from four palatal, seven labial and two retromolar pad biopsies was also carried out. MUC1 mRNA could only be detected in the minor salivary mucous glands. MUC1 has already been identified in the ducts of normal parotid and submandibular gland, and our findings demonstrate a similar distribution in minor salivary glands. We conclude that when present in the normal oral glycocalyx, the only oral source of MUC1 is from cell membranes of the minor salivary glands.

Functional significance of MMP-2 and MMP-9 expression by human malignant oral keratinocyte cell lines.

This study examined the expression of MMP-2 and MMP-9 in normal and human malignant oral keratinocytes. The expression of pro-MMP-2 and pro-MMP-9 was heterogeneous in the malignant cell lines. Normal oral keratinocytes expressed less pro-MMP-2 and more pro-MMP-9 than their malignant counterparts. Cells that expressed high levels of both MMP-2 and MMP-9 showed the greatest degree of invasion through Matrigel in vitro compared to cells with either low or variable levels of these enzymes; normal keratinocytes were non-invasive in these conditions. The degree to which the cells invaded through Matrigel was similar to their motility in the absence of Matrigel and was not influenced by the activation of the pro-enzymes or the inhibition of enzyme activity using a chemical inhibitor of gelatinases. Cells were transplanted orthotopically to athymic mice and demonstrated a variable capacity not only to form tumours at the site of inoculation but, also, to metastasise; normal oral keratinocytes were non-tumorigenic. There was no correlation between the expression of either MMP-2 or MMP-9 and the tumorigenic/metastatic phenotype. The results emphasise the limitations of correlating in vitro and in vivo assays of tumour cell behaviour and suggest that invasion/motility in vitro may be a distinct phenotype from tumorigenicity/metastasis in vivo.

A longitudinal study of risk factors for teat lesions in 67 suckler ewes in a single flock in England.

A longitudinal study of 67 suckler ewes on a commercial sheep farm near Wolverhampton, England was carried out from March to July 2010. Data on ewe teat lesions and udder and teat conformation were collected together with data on lamb health and weight at lambing and at 14-day intervals until lambs were 8-10 weeks old. Each ewe was examined on 4-5 occasions. Teat lesions were categorised as traumatic or non-traumatic based on appearance. Risk factors for the development of each type of teat lesion were investigated using mixed effect binomial regression models. The incidence of traumatic teat lesions was greatest 3-4 weeks after lambing with 27.9% new cases by udder half. There was an increased risk of traumatic teat lesions in 2 year old ewes (OR 3.5, CI 1.2, 10.5) compared with 6 year old ewes and in ewes with abnormal compared with normal teat placement, a wide or narrow udder width compared with a normal udder width, a heavy total litter weight, body condition score (BCS)<2.5, compared with BCS of 2.5 14-days previously, and in ewes with abnormal udder skin (waxy, dry or chapped) compared with normal udder skin. The incidence of non-traumatic teat lesions was greatest 7-10 weeks after lambing with 21.4% new cases. Non-traumatic lesions were more likely to occur in ewes with BCS=3 compared with BCS of 2.5. The risk factors for traumatic and non-traumatic teat lesions differ. Traumatic lesions are associated with teat and udder morphology, ewe age and BCS, and lamb growth rate, whereas non-traumatic lesions are associated with ewe BCS.

Computer-aided real-time acquisition of physiological data: technical design and selection constraints.

For many years, physiological investigations have utilized the power and speed of analogue, digital and hybrid computers. Complex protocols often require the recording of large amounts of data in short periods, typically with simultaneous fine control of multiple experimental variables. Current systems often include data analysis in the same program which controls data acquisition. Although this is convenient from a package point of view, acquisition and analysis routines have different and sometimes conflicting purposes and requirements. This paper examines acquisition of continuous physiological signals by small dedicated computers in an attempt to separate these processes and to provide guidelines for computer selection, design and development.

Aedes aegypti in Jamaica, West Indies: container productivity profiles to inform control strategies

OBJECTIVE: To describe the Aedes aegypti container profile in the three parishes of Portland, St. Anns and St. Catherine, Jamaica.METHOD: Traditional stegomyia and pupae per person indices.RESULTS: A total of 8855 containers were inspected. A. aegypti were breeding in 19.2% of the 4728 containers in Portland, in 6.7% of the 2639 containers in St. Ann, and in 27.2% of the 1488 containers in Tryhall Heights, St. Catherine. Container types differed between Portland (P > 0.02) on one hand and St. Ann and Tryhall Heights, St. Catherine on the other hand: there were with no vases or potted plants with water saucers in St. Ann and St. Catherine. A. aegypti were breeding in more containers in St. Catherine (38%) (38% in wet season and 21% in the dry season) than in Portland (19%) or St. Ann (6%), both of which had more containers but A. aegypti breeding in fewer: 17.7% and 11.2% in the wet and 20.4% and 3.5% in the dry seasons respectively. The daily production of adult mosquitoes in the three study sites was 1.51, 1.29 and 0.66 adult female mosquitoes per person in Portland, St. Ann and St. Catherine during the dry season and 1.12, 0.23 and 1.04 female mosquitoes per person in the wet season respectively.CONCLUSION: All three communities are at risk for dengue outbreaks and vector control should concentrate on reducing the mosquito populations from the most productive containers before a new dengue virus serotype is introduced into Jamaica.

Endogenous TGF-beta1 inhibits the growth and metastatic dissemination of rat oral carcinoma cell lines but enhances local bone resorption.

This study examined the effect of stable transfection of latent transforming growth factor-beta1 (TGF-beta1) cDNA into a predominantly polygonal, 4 nitroquinoline N-oxide (4NQO)-induced rat oral keratinocyte cell line. Seven polygonal and five spindle clonal populations were isolated that overexpressed TGF-beta1 protein by approximately two- to four-fold compared to vector-only transfected controls. Neutralisation experiments indicated that the majority of protein was in the latent form. There was no change in the proportion of polygonal and spindle cells in vitro after transfection with TGF-beta1 cDNA. Polygonal and spindle cells that overexpressed TGF-beta1 produced similar amounts of protein and grew more slowly in vitro than controls. The parent cell line and all transfected cells were growth inhibited (60-75%) by exogenous TGF-beta1. Orthotopic transplantation of the parent and the vector-only control cell lines resulted in primary tumours in the floor of the mouth in almost 100% (20/21) of athymic mice, with no evidence of bone resorption at the site of the primary tumour and pulmonary metastatic tumour deposits in some 40% (7/20) of these animals. The polygonal and spindle cells that overexpressed TGF-beta1 behaved similarly following orthotopic transplantation. A 96% (23/24) primary tumour take was evident following transplantation of cells that overexpressed TGF-beta1, with a significantly (P<0.02) higher number of animals showing bone resorption at the site of the primary tumour (35%; 8/23) compared to controls. By contrast, there was a significant (P<0.03) decrease in the number of animals with pulmonary metastases (4%; 1/23) following transplantation of TGF-beta1 overexpressing cells compared to controls. Overexpression of TGF-beta1 did not alter tumour cell differentiation in vivo. The results demonstrate that endogenous TGF-beta1 functions as a tumour suppressor in the rat-4NQO model of oral carcinogenesis without altering tumour cell morphology or differentiation but can also act to promote local bone resorption.

Destabilization of the Ca2+-ATPase of sarcoplasmic reticulum by thiol-specific, heat shock inducers results in thermal denaturation at 37 degrees C.

A number of protein reactive compounds, including the thiol reagents diamide and arsenite, are known inducers of heat shock protein (HSP) synthesis and thermotolerance. These compounds are thought to damage cellular protein, which has been proposed to serve as the signal for induction. The specific mechanism of protein damage and its relation to thermal denaturation are unknown. The Ca2+-ATPase of sarcoplasmic reticulum, a membrane protein that contains 24 cys residues, was used to determine the effect of diamide, arsenite, N-ethylmaleimide (NEM), and the cys-specific probes Br-DMC and IAEDANS, which label one or two specific cys residues, respectively, on protein conformation and stability. The Ca2+-ATPase was chosen because diamide has been shown to affect the thermal properties of a class of membrane proteins of CHO cells (Freeman et al., 1995). The labeling of one or two thiols has no effect on activity or conformation, while more extensive reaction (but with less than approximately five to eight groups titrated) results in destabilization of the Ca2+-ATPase such that it denatures thermally at 37 degrees C. Higher levels of titration result in greater destabilization such that the protein is no longer stable at room temperature, with the production of a state similar to the thermally denatured state as assayed by activity, differential scanning calorimetry, ANS binding, and light scattering. The fractional denaturation induced by these thiol reagents, determined by the decrease in the heat absorbed during thermal denaturation, is directly proportional to inactivation of ATPase activity. Thus, inactivation of the Ca2+-ATPase by thiol reagents occurs because of denaturation not through oxidation of essential thiols. These results indicate that these thiol-specific heat shock inducers function by two mechanisms: (1) destabilization of proteins such that they thermally denature at 37 degrees C and (2) direct denaturation, apparently driven by thermal processes at room temperature, following more extensive reaction which results in extreme destabilization. We suggest that these are general mechanisms by which heat shock inducers damage proteins.

Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response.

While oxidative stress can induce a heat shock response, the primary signals that initiate activation have not been identified. To identify such signals, HepG2 and V 79 cells were exposed to menadione, a compound that redox-cycles to generate superoxide. The oxidative stress generated by menadione resulted in oxidation of protein thiols in a dose-dependent manner. This was followed by protein destabilization and denaturation, as determined by differential scanning calorimetry of whole cells. To directly evaluate the effect of non-native disulfides on protein conformation, Ca2(+)-ATPase, isolated from rabbit sarcoplasmic reticulum, was chemically modified to contain non-native intermolecular or glutathione (GHS)-mixed disulfides. Differential scanning calorimetry profiles and 1-anilinonaphthalene-8-sulfonic acid fluorescence indicated that formation of non-native disulfides produced protein destabilization, denaturation, and exposure of hydrophobic domains. Cellular proteins shown to contain oxidized thiols formed detergent-insoluble aggregates. Cells treated with menadione exhibited activation of HSF-1, accumulated Hsp 70 mRNA, and increased synthesis of Hsp 70. This work demonstrates that formation of physiologically relevant, non-native intermolecular and GSH-mixed disulfides causes proteins to destabilize, unfold such that hydrophobic domains are exposed, and initiate a signal for induction of the heat shock response.

Overexpression of autocrine TGF-beta 1 suppresses the growth of spindle epithelial cells in vitro and in vivo in the rat 4NQO model of oral carcinogenesis.

We examined the effect of the stable transfection of latent TGF-beta 1 cDNA, under the control of a cytomegalovirus promoter in the expression vector pcDNA3, into a 4NQO-induced clonal rat oral keratinocyte cell line that formed undifferentiated spindle cell tumours following subcutaneous transplantation to athymic mice. Test cells containing latent TGF-beta 1 cDNA produced a 2.3-fold increase in TGF-beta 1 protein compared to pcDNA3 controls as demonstrated by ELISA. Neutralisation experiments indicated that the majority of the protein was in the latent form. Untransfected and transfected (containing either TGF-beta 1 cDNA or pcDNA3) cell lines were keratin negative and vimentin positive. Cells transfected with TGF-beta 1 were inhibited more than pcDNA3 controls when cultured in an anchorage dependent or independent environment. Subcutaneous transplantation of cells overproducing TGF-beta 1 resulted in tumours of significantly smaller volume than vector-only controls. Further, orthotopic transplantation of cells containing TGF-beta 1 cDNA to the floor of the mouth in athymic mice markedly inhibited the development of pulmonary metastases compared to vector-only controls. Both test and control cell lines in athymic mice formed undifferentiated tumours with a complete absence of keratin elaboration. Subcutaneous xenografts were recultured and cells containing the TGF-beta 1 cDNA produced a similar amount of TGF-beta 1 peptide as the cells containing pcDNA3 only. The production of TGF-beta 1 by both of the xenograft-derived cell lines was significantly less than the parent, pre-transplanted cell lines and the untransfected cell line. All of the cell lines were inhibited by exogenous TGF-beta 1. Our results demonstrate that autocrine TGF-beta 1 functions as a tumour suppressor in vitro and in vivo in 4NQO-induced spindle tumour cells that are growth inhibited by the ligand. Furthermore, tumour formation in athymic mice is associated with selection for a cell phenotype with diminished autocrine TGF-beta 1 production.