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Biomedical subjects

J A Barnes

Publications and source records attributed to J A Barnes.

At least 19 recordsLinked to original sources
PubMed2002

Effects of heat shock protein 70 (Hsp70) on arsenite-induced genotoxicity.

Arsenic, a human carcinogen, is genotoxic, although its mechanism(s) of action for tumorigenesis is not well understood. Among the toxicity-related properties of this chemical are its clastogenic and aneugenic activities, as well as its capacity for inducing stress-response in the form of elevated heat shock protein (HSP) expression. In the present study, we evaluated the effects of Hsp70 expression on arsenite (As)-induced structural and numerical chromosome anomalies in human cells. Human MCF-7 Tet-off cells stably transfected with a pTRE/Hsp70-1 transgene construct were used to regulate Hsp70 levels prior to in vitro As exposures. Separate cultures of relatively high vs. low Hsp70-expressing cells were established. A cytokinesis block micronucleus assay with kinetochore immunostaining was used to detect micronuclei (MN) derived from chromosome breakage (K-MN) or loss (K+MN). These studies demonstrated significant increases in micronucleus frequencies in response to As following either a long exposure (5 or 10 microM for 46 hr), or short exposure (10 or 40 microM for 8 hr) protocol. Overall, the long protocol was more efficient in producing K+MN and cells with multiple MN. Overexpressing Hsp70 resulted in significant reductions in the percent of cells positive for MN for both the long and short As exposure protocols. Both K+ and K- types of As-induced MN were lower in cells with elevated Hsp70 as compared to cells without overexpression of Hsp70. We conclude that the dose and duration of As exposure influence the type as well as amount of chromosomal alteration produced and that inducible Hsp70 protects against both the clastogenic and aneugenic effects of this chemical.

Arsenites↗
PubMed2001

Expression of inducible Hsp70 enhances the proliferation of MCF-7 breast cancer cells and protects against the cytotoxic effects of hyperthermia.

Heat shock proteins (Hsps) are ubiquitous proteins that are induced following exposure to sublethal heat shock, are highly conserved during evolution, and protect cells from damage through their function as molecular chaperones. Some cancers demonstrate elevated levels of Hsp70, and their expression has been associated with cell proliferation, disease prognosis, and resistance to chemotherapy. In this study, we developed a tetracycline-regulated gene expression system to determine the specific effects of inducible Hsp70 on cell growth and protection against hyperthermia in MCF-7 breast cancer cells. MCF-7 cells expressing high levels of Hsp70 demonstrated a significantly faster doubling time (39 hours) compared with nonoverexpressing control cells (54 hours). The effect of elevated Hsp70 on cell proliferation was characterized further by 5-bromo-2'deoxyuridine labeling, which demonstrated a higher number of second and third division metaphases in cells at 42 and 69 hours, respectively. Estimates based on cell cycle analysis and mean doubling time indicated that Hsp70 may be exerting its growth-stimulating effect on MCF-7 cells primarily by shortening of the G0/G1 and S phases of the cell cycle. In addition to the effects on cell growth, we found that elevated levels of Hsp70 were sufficient to confer a significant level of protection against heat in MCF-7 cells. The results of this study support existing evidence linking Hsp70 expression with cell growth and cytoprotection in human cancer cells.

Breast Neoplasms↗
PubMed2001

Preductal aortic coarctation and patent ductus arteriosus in a Sumatran tiger (Panthera tigris sumatrae) cub.

Preductal aortic coarctation and patent ductus arteriosus are described in a neonatal Sumatran tiger, Panthera tigris sumatrae. Eight days postpartum, the cub appeared weak, and it was separated from the dam for hand rearing. On examination it was dehydrated and hypothermic. Despite treatment, the animal's condition worsened and the cub died 12 days postpartum. Gross postmortem and histologic examinations revealed a preductal aortic coarctation and patent ductus arteriosus with a patent foramen ovale and moderate dilatation of the right ventricle of the heart. Focal pneumonia and mild hepatitis were also present; however, diffuse pulmonary congestion and edema were considered to be the proximate cause of death.

Animals↗
PubMed2000

Selective degradation of annexins by chaperone-mediated autophagy.

Annexins are a family of proteins that bind phospholipids in a calcium-dependent manner. Analysis of the sequences of the different members of the annexin family revealed the presence of a pentapeptide biochemically related to KFERQ in some annexins but not in others. Such sequences have been proposed to be a targeting sequence for chaperone-mediated autophagy, a lysosomal pathway of protein degradation that is activated in confluent cells in response to removal of serum growth factors. We demonstrate that annexins II and VI, which contain KFERQ-like sequences, are degraded more rapidly in response to serum withdrawal, while annexins V and XI, without such sequences, are degraded at the same rate in the presence and absence of serum. Using isolated lysosomes, only the annexins containing KFERQ-like sequences are degraded by chaperone mediated-autophagy. Annexins V and XI could associate with lysosomes but did not enter the lysosomes and were not proteolytic substrates. Furthermore, four annexins containing KFERQ-like sequences, annexins I, II, IV, and VI, are enriched in lysosomes with high chaperone-mediated autophagy activity as expected for substrate proteins. These results provide striking evidence for the importance of KFERQ motifs in substrates of chaperone-mediated autophagy.

Amino Acid Motifs↗
PubMed2000

Spectroscopic characterization of the interaction between calmodulin-dependent protein kinase I and calmodulin.

Calmodulin-dependent protein kinase I (CaM kinase I) is a member of the expanding class of protein kinases that are regulated by calmodulin (CaM). Its putative CaM-binding region is believed to occur within a 22-residue sequence (amino acids 299-320). This sequence was chemically synthesized and utilized for CaM interaction studies. Gel band shift assays and densitometry experiments with intact CaM kinase I and the CaM-binding domain peptide (CaMKIp) reveal that they bind in an analogous manner, giving rise to 1:1 complexes. Fluorescence analysis using dansyl-CaM showed that conformational changes in CaM on binding CaM kinase I or CaMKIp were nearly identical, suggesting that the peptide mimicked the CaM-binding ability of the intact protein. In the presence of CaM, the peptide displays an enhancement of its unique Trp fluorescence as well as a marked blue shift of the emission maximum, reflecting a transfer to a more rigid, less polar environment. Quenching studies, using acrylamide, confirmed that the Trp in the peptide on binding CaM is no longer freely exposed to solvent as is the case for the free peptide. Studies with a series of Met mutants of CaM showed that the Trp-containing N-terminal end of CaMKIp was bound to the C-terminal lobe of CaM. Near-UV CD spectra also indicate that the Trp of the peptide and Phe residues of the protein are involved in the binding. These results show that the CaM-binding domain of CaM kinase I binds to CaM in a manner analogous to that of myosin light chain kinase.

Acrylamide↗
PubMed2000

The E1 helicase of human papillomavirus type 11 binds to the origin of replication with low sequence specificity.

Expression of the human papillomavirus type 11 E1 and E2 genes is necessary and sufficient to support viral DNA replication. The full-length E2 protein is a transcriptional modulator that also interacts with the E1 helicase to form an E1/E2 complex at the viral origin of replication. Previous studies indicated that efficient binding of this complex to the replication origin is site-specific and that the E2 homodimer was required for efficient E1 binding. Human papillomavirus type 11 E2 and E1 proteins have been purified and their cooperative binding to the HPV type 11 viral replication origin has been characterized. Low-affinity E1 binding to the HPV type 11 replication origin was demonstrated and found to be largely nonspecific. DNA binding by E1 does not require complex formation with E2 and appears to be independent of ATP binding or hydrolysis. E1 binding quantitatively increased with the addition of increasing amounts of E2 and mutations in the E2 binding site demonstrated that the E2BS site is required for E1 and E2 to specifically bind as a high-affinity complex at the replication origin. Analysis of the A/T-rich E1 binding site via mutation showed that it was nonessential for high-affinity E1/E2 complex formation. Thus, although the replication functions between the animal and the human papillomaviruses are well conserved, there are subtle differences in the DNA binding requirements for E1, which may portend mechanistic differences among the DNA replication systems of various papillomavirus types.

Base Sequence↗
PubMed2000

Replication-associated activities of purified human papillomavirus type 11 E1 helicase.

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.

Acid Anhydride Hydrolases↗
PubMed2000

Glucose-regulated protein 78 (GRP78) is elevated in embryonic mouse heart and induced following hypoglycemic stress.

This study investigates the distribution and heart levels of glucose regulated protein (GRP) 78 during normal development and in response to hypoglycemia in the mouse. Results demonstrate that GRP78 is strongly expressed with in the heart, neural tube, gut endoderm, somites, and surface ectoderm of mouse embryos during early organogenesis, and GRP78 staining remains prominent in the heart from gestational days 9.5 through 13.5. Cardiac myocytes are the primary site of GRP78 expression within the heart. GRP78 levels are highest in the heart during early organogenesis and levels decrease significantly by the fetal period. GRP78 expression is increased after 24 h of hypoglycemia in the early organogenesis-stage heart. Considering the tissue specific pattern of GRP expression and changes during development of the heart, GRPs may play significant roles in the normal differentiation and development of cardiac tissue. GRP induction may also be involved in hypoglycemia-induced cardiac dysmorphogenesis.

Animals↗
PubMed2000

Effective small-incision surgery for involutional lower eyelid entropion.

OBJECTIVE: The aim of this study was to develop an effective and minimally invasive operation to correct lower eyelid entropion that would address both the horizontal and vertical laxity. DESIGN: A prospective, noncomparative, interventional case series. PARTICIPANTS: Thirty-five consecutive patients with involutional entropion, aged 62 to 92 years (mean, 77.1 years), had surgery on 45 lower eyelids. Of the 45 procedures, 33 (73%) had a primary procedure and 12 (27%) were reoperations. INTERVENTION: A lateral tarsal strip with diagonal tightening of the orbital septum and lower lid retractors to the lateral orbital rim was performed via a 1-cm lateral canthal incision. MAIN OUTCOME MEASURES: Complications and surgical outcome were monitored clinically for between 12 and 24 months after surgery. RESULTS: The results were analyzed from 42 eyelids (33 patients) with a mean follow-up of 17.1 months (range 12-24 months). Two patients died and one dropped out of the study 3 months after the second eyelid operation. In 36 cases (86%), the entropion was cured. Transient lateral orbital rim tenderness was noted in six cases (14%), and one patient had a wound infection. Anatomic recurrences were detected in six eyelids of six patients, and five of these (83%) were asymptomatic. CONCLUSIONS: This surgical approach has been found effective in 86% of eyelids. Adequate clinical followup has proven essential for accurate evaluation of entropion surgery.

Aged↗
PubMed2000

Fatty acid composition and possible health effects of coconut constituents.

The link between excessive consumption of dietary saturated fats and coronary heart disease (CHD) is now well established. Because of its high content of saturated fatty acids, the consumption of foods containing coconut oil may therefore be a risk factor for CHD. While the fatty acid composition of coconut oil is well established, relatively little is known about the other constituents of coconut: the milk, water, cream and meat fractions. In this study, we show that while the water fraction is low in lipid content, the milk contains about 24% of the fat content of oil and the cream and meat fractions about 34%. The other coconut constituents contain significant amounts of medium-chain triglycerides that are formed from fatty acids of chain length 8:0 to 14:0. It is these fatty acids, primarily 14:0, that are thought to be atherogenic. On the other hand, medium-chain triglycerides may be advantageous under some circumstances in that they are absorbed intact and do not undergo degradation and re-esterification processes. As a result, medium-chain triglycerides provide a ready source of energy and may be useful in baby foods or in diet therapy. Nevertheless, the possible negative effects of the saturated fatty acids and the absence of the essential fatty acid linolenic acid from all coconut constituents suggest that the coconut milk, oil and cream should not be used on a regular basis in adults.

Cocos↗
PubMed1999

Oscillation and light induction of timeless mRNA in the mammalian circadian clock.

Circadian rhythms in Drosophila melanogaster depend on a molecular feedback loop generated by oscillating products of the period (per) and timeless (tim) genes. In mammals, three per homologs are cyclically expressed in the suprachiasmatic nucleus (SCN), site of the circadian clock, and two of these, mPer1 and mPer2, are induced in response to light. Although this light response distinguishes the mammalian clock from its Drosophila counterpart, overall regulation, including homologous transcriptional activators, appears to be similar. Thus, the basic mechanisms used to generate circadian timing have been conserved. However, contrary to expectations, the recently isolated mammalian tim homolog was reported not to cycle. In this study, we examined mRNA levels of the same tim homolog using a different probe. We observed a significant (approximately threefold) diurnal variation in mTim expression within mouse SCN using two independent methods. Peak levels were evident at the day-to-night transition in light-entrained animals, and the oscillation persisted on the second day in constant conditions. Furthermore, light pulses known to induce phase delays caused significant elevation in mTim mRNA. In contrast, phase-advancing light pulses did not affect mTim levels. The mTim expression profile and the response to nocturnal light are similar to mPer2 and are delayed compared with mPer1. We conclude that temporal ordering of mTim and mPer2 parallels that of their fly homologs. We predict that mTIM may be the preferred functional partner for mPER2 and that expression of mTim and mPer2 may, in fact, be driven by mPER1.

Animals↗
PubMed1999

Expression of glucose-regulated proteins (GRP78 and GRP94) in hearts and fore-limb buds of mouse embryos exposed to hypoglycemia in vitro.

Hypoglycemia, the classic inducer of glucose-related protein (GRP) synthesis, is dysmorphogenic in rodent embryos and detrimentally affects the heart. This study compares GRP induction in a target vs non-target tissue by evaluating GRP expression in hearts and fore-limb buds of mouse embryos following exposure to hypoglycemia in vitro. Gestational day 9.5 embryos were exposed to 2, 6, and 24 h of either mild (80 mg/dl glucose) or severe (40 mg/dl glucose) hypoglycemia using the method of whole-embryo culture. GRP78 increased in a dose- and time-dependent fashion in embryonic hearts exposed to either 40 mg/dl or 80 mg/dl glucose, whereas GRP94 levels increased in hearts only after 24 h of hypoglycemia. In contrast to the heart, GRP induction in fore-limb buds occurred only with GRP78 following the most severe level and duration of hypoglycemia. RT-PCR analysis demonstrated an elevation in GRP78 and GRP94 message levels in embryonic hearts following severe hypoglycemia. However, mRNA levels did not increase in response to mild hypoglycemia. Overall, these data demonstrate the preferential induction of GRPs in the heart as compared to fore-limb buds in mouse embryos exposed to hypoglycemia. Increases in GRP protein levels may be a more reliable biomarker of stress than message levels. However, both tissues and methods should be examined for enhanced biomarker sensitivity.

Animals↗
PubMed1998

Assuring quality in extramural studies: the perceptions of practitioners.

This paper presents the results of a questionnaire completed by 331 UK veterinary practitioners which focused on the quality assurance of extramural studies. The questionnaire considered the attributes of practitioners as extramural teachers and the suitability of host practices for extramural studies, the students' evaluation of their quality, and the role of the veterinary schools. The questionnaire identified two broad roles for the practitioner, first, as teacher and, secondly, as professional role model, and there was strong support for students being allowed to evaluate their role as teacher. The findings are discussed in relation to aspects of good practice associated with quality assurance, and in relation to the need to develop stronger links between the schools and the veterinary profession.

Animal Diseases↗
PubMed1998

Molecular targets for human papillomaviruses: prospects for antiviral therapy.

A substantial medical need exists for the development of antiviral medicines for the treatment of diseases associated with infection by human papillomaviruses (HPVs). HPVs are associated with various benign and malignant lesions including benign genital condyloma, common skin warts, laryngeal papillomas and anogenital cancer. Since treatment options are limited and typically not very satisfactory, the development of safe and effective antiviral drugs for HPV could have substantial clinical impact. In the last few years, exciting advances have been made in our understanding of papillomavirus replication and the effects that the virus has on growth of the host cell. Although still somewhat rudimentary, techniques have been developed for limited virion production in vitro offering the promise of more rapid advances in the dissection and understanding of the virus life cycle. Of the 8-10 HPV gene products that are made during infection, only one encodes enzymatic activities, the E1 helicase. Successful antiviral therapies have traditionally targeted viral enzymes such as polymerases, kinases and proteases. In contrast, macromolecular interactions which mediate the functions of E6, E7 and E2 are thought to be more difficult targets for small molecule therapy.

Antiviral Agents↗
PubMed1997

Towards a strategy for learning: the response of practices to the Liverpool pilot scheme for extramural study.

This paper presents the responses of veterinary surgeons to a pilot scheme initiated by the SILVER project. The scheme was designed to increase communication between a veterinary school, host practices and students undertaking extramural study ("seeing practice') and to provide a more structured use of the students' time. The work is set in the context of recent interest in work-based learning as part of undergraduate education, the approach being based on experience gained from a number of professions, including veterinary science. Students were required to identify and communicate their personal learning objectives to host practices in advance of their arrival, and practices were asked to collaborate in a new system for recording students' progress. The paper presents an analysis of the evidence obtained from questionnaires sent to pilot practices. It shows that all the elements of the scheme had been received favourably and had had a positive impact on student learning. The findings are discussed in relation to the development and implications of an educational strategy for extramural study.

Education, Veterinary↗
PubMed1997

Immunolocalization and heart levels of GRP94 in the mouse during post-implantation development.

Glucose-regulated proteins (GRPs), which belong to the highly conserved family of stress proteins, are resident to the endoplasmic reticulum and function as molecular chaperones. Heat shock proteins have been shown to be developmentally regulated, but little work has been done to investigate the expression of GRPs during embryogenesis. Therefore, this study examined the distribution of GRP94 within mouse embryos during the period of organogenesis and characterized levels of GRP94 within the developing heart during organogenesis and late fetal stages. Our results demonstrate that the GRP94 protein is constitutively expressed within mouse embryos during early stages of organogenesis and is localized particularly within the developing heart, neuroepithelium, and surface ectoderm tissues. Positive staining for GRP94 remains within developing heart tissues throughout organogenesis and is found primarily within the atrial and ventricular myocardial cells. Western blot analysis of GRP94 expression demonstrates a significantly higher level of GRP94 in embryonic hearts during early stages of organogenesis than in later stages of organogenesis or the fetal period. These results demonstrate that the stress protein GRP94 is constitutively expressed within specific tissues during post-implantation mouse development and suggest that GRPs may play an important role in the process of myocardial cell differentiation and heart development.

Animals↗