PRAP1 is a novel executor of p53-dependent mechanisms in cell survival after DNA damage.

p53 has a crucial role in governing cellular mechanisms in response to a broad range of genotoxic stresses. During DNA damage, p53 can either promote cell survival by activating senescence or cell-cycle arrest and DNA repair to maintain genomic integrity for cell survival or direct cells to undergo apoptosis to eliminate extensively damaged cells. The ability of p53 to execute these two opposing cell fates depends on distinct signaling pathways downstream of p53. In this study, we showed that under DNA damage conditions induced by chemotherapeutic drugs, gamma irradiation and hydrogen peroxide, p53 upregulates a novel protein, proline-rich acidic protein 1 (PRAP1). We identified functional p53-response elements within intron 1 of PRAP1 gene and showed that these regions interact directly with p53 using ChIP assays, indicating that PRAP1 is a novel p53 target gene. The induction of PRAP1 expression by p53 may promote resistance of cancer cells to chemotherapeutic drugs such as 5-fluorouracil (5-FU), as knockdown of PRAP1 increases apoptosis in cancer cells after 5-FU treatment. PRAP1 appears to protect cells from apoptosis by inducing cell-cycle arrest, suggesting that the induction of PRAP1 expression by p53 in response to DNA-damaging agents contributes to cancer cell survival. Our findings provide a greater insight into the mechanisms underlying the pro-survival role of p53 in response to cytotoxic treatments.

Annexin-1 interacts with NEMO and RIP1 to constitutively activate IKK complex and NF-κB: implication in breast cancer metastasis.

The molecular mechanisms underlying constitutive nuclear factor-κB (NF-κB) activation in solid tumors has not been elucidated. We show that Annexin-1 (ANXA1) is involved in this process, and suppression of ANXA1 in highly metastatic breast cancer cells impedes migration and metastasis capabilities in vitro and in vivo. ANXA1 expression correlates with NF-κB activity, suggesting that ANXA1 may be required for the constitutive activity of IκB kinase (IKK) and NF-κB in highly metatstatic breast cancer. Gel-filtration analysis demonstrated that ANXA1 co-elutes with the members of the IKK complex and NF-κB signaling pathway, and immunoprecipitation confirmed that ANXA1 can bind to and interact with IKKγ or NEMO, but not IKKα or IKKß. Importantly, silencing of ANXA1 prevents the interaction of NEMO and RIP1, which indicates that ANXA1 is required for the recruitment of RIP1 to the IKK complex, which may be important for the activation of NF-κB. Downstream targets of NF-κB include uPA and CXCR4, which can be modulated by ANXA1 silencing. CXCR4-mediated migration of breast cancer cell lines in response to CXCL12 was significantly modulated by ANXA1, indicating its importance in the tissue-specific migration of breast cancer cells. Chromatin immunoprecipitation experiments confirmed that in ANXA1 overexpressed cells, NF-κB was recruited to CXCR4 promoter without external stimulation, indicating that ANXA1 is critical for the constitutive activation of NF-κB in breast cancer to promote metastasis. Finally, we show that ANXA1 overexpression enhances metastasis and reduces survival in an intracardiac metastasis model, while ANXA1-deficient mice crossed with MMTV-PyMT mice display significantly less metastasis than their heterozygous littermates, indicating that ANXA1 is an important gene in breast cancer metastasis. Our data reveal that ANXA1 can constitutively activate NF-κB in breast cancer cells through the interaction with the IKK complex, and suggests that modulating ANXA1 levels has therapeutic potential to suppress breast cancer metastasis.

Desensitization for renal transplantation: depletion of donor-specific anti-HLA antibodies, preservation of memory antibodies, and clinical risks.

Desensitization protocols reduce donor-specific anti-HLA antibodies (DSA) and enable renal transplantation in patients with a positive complement-dependent cytotoxic cross-match (CDC-CXM). The effect of this treatment on protective antibody and immunoglobulin levels is unknown. Thirteen patients with end-stage renal disease, DSA and positive CDC-CXM underwent desensitization. Sera collected pre- and post-transplantation were analysed for anti-tetanus and anti-pneumococcal antibodies, total immunoglobulin (Ig) levels and IgG subclasses and were compared to healthy controls and contemporaneous renal transplant recipients treated with standard immunosuppression alone. Ten patients developed negative CDC-CXM and enzyme-linked immunosorbent assay (ELISA) and underwent successful transplantation. Eight recipients achieved good graft function without antibody-mediated or late rejection, BK virus or cytomegalovirus infection. One patient had primary non-function due to recurrent oxalosis, and one patient with immediate graft function died from septicaemia. Seven recipients required post-operative transfusion and three developed septicaemia. DSA remained negative by ELISA at 12 months, but were detectable by Luminex(®) . Anti-tetanus and anti-pneumococcal antibodies, total Ig and IgG subclasses were below the normal range but comparable to levels in renal transplant recipients who had not undergone desensitization. Desensitization protocols effectively reduce DSA and allow successful transplantation. Post-operative bleeding and short-term infectious risk is increased. Protective antibody and serum immunoglobulin levels are relatively preserved.

One hundred years of physiology education in Singapore.

Physiology is the study of normal function in the body and how genes, proteins, organ systems interact to maintain health. It provides a foundation for the health sciences profession and life science research. Physiology education in Singapore began soon after the establishment of the Federated States Government Medical School in 1905. The importance of Physiology to medical education was recognised by the appointment of a separate lecturer in Physiology in 1906, followed by the appointment of Professor James Argyll Campbell as the first King Edward VII professor and endowed Chair in Physiology in 1912. The teaching of Physiology in the early days was focused on the basics of normal function with little correlation to clinical problems and application. However, by the 1970s, first-year medical students were given the opportunity to visit hospitals where they were tutored by clinicians to help them apply Basic Physiology to clinical problems. Curriculum changes in the subsequent years emphasised a reduction in content, integration among preclinical subjects, independent learning and clinical relevance. Physiology is taught not only to medical but also dental, pharmacy and life science students. The teaching of Physiology to science students is a collaborative effort between the Department of Physiology, Faculty of Medicine and the Department of Biological Sciences, Faculty of Science. A lot of the teaching of Physiology to life science students occurs not in classrooms but in the laboratories, where students work closely with research supervisors and mentors on research projects. There has been a very significant increase in the number of students doing research projects in Physiology in recent years, especially in the areas of Cell Physiology, Immunology and Neurobiology. The completion of the human genome sequence poses new challenges to understand function, especially how genes, proteins and organ systems interact to sustain function. Physiology education will be increasingly important in the undergraduate and graduate life science and medical curriculum. Further, the country's vision of being the biomedical R&D and healthcare hub for the region means that Physiology education must remain at the forefront to prepare the next generation of doctors, clinician-scientists, researchers and entrepreneurs.

The NUS MBBS-PhD programme: nurturing clinician-scientists for tomorrow.

The MBBS-PhD programme is a significant milestone in medical education in Singapore. In July 2000, the Faculty of Medicine, National University of Singapore launched this programme in collaboration with the Institute of Molecular and Cell Biology, with support from the Economic Development Board, and the Agency for Science, Technology and Research, Singapore. The objectives of the programme are to nurture and develop the talents of the brightest medical students by integrating clinical and basic biomedical research training, as well as to stimulate advanced basic and applied research in areas of growing importance to clinical medicine. The programme also aims to train clinician-scientists who will interface basic biology and clinical practice to solve biomedical problems and spearhead biomedical research initiatives in Singapore. Successful MBBS-PhD graduates can pursue career tracks in clinical research, basic biomedical research or in the biotechnology industry.

Inhibition of histone deacetylase 2 increases apoptosis and p21Cip1/WAF1 expression, independent of histone deacetylase 1.

Histone deacetylases (HDACs) 1 and 2 share a high degree of homology and coexist within the same protein complexes. Despite their close association, each possesses unique functions. We show that the upregulation of HDAC2 in colorectal cancer occurred early at the polyp stage, was more robust and occurred more frequently than HDAC1. Similarly, while the expression of HDACs1 and 2 were increased in cervical dysplasia and invasive carcinoma, HDAC2 expression showed a clear demarcation of high-intensity staining at the transition region of dysplasia compared to HDAC1. Upon HDAC2 knockdown, cells displayed an increased number of cellular extensions reminiscent of cell differentiation. There was also an increase in apoptosis, associated with increased p21Cip1/WAF1 expression that was independent of p53. These results suggest that HDACs, especially HDAC2, are important enzymes involved in the early events of carcinogenesis, making them candidate markers for tumor progression and targets for cancer therapy.

Culture-proven bacterial keratitis in a Malaysian general hospital.

One hundred patients (101 eyes) with culture-proven bacterial keratitis were treated in the Department of Ophthalmology, Hospital Sultanah Aminah, Johor Bahru, over a 4-year period. The majority of patients was male (63%), Malay (60%), from the Johor Bahru district (62%) and aged between 41 to 50 years (20%). The ocular predisposing factors were ocular trauma (41 eyes), ocular surface disease (28 eyes) and contact lens wear (26 eyes). The corneal ulcers were mainly large (50.5%), central (59.4%) and colonized by Gram-negative bacteria (78.1%). The most frequently isolated microorganisms were Pseudomonas aeruginosa (67 eyes), Staphylococcus aureus (12 eyes), Acinetobacter baumanii (6 eyes), Klebsiella pneumoniae (5 eyes), Corynebacterium sp. (3 eyes:) and Streptococcus pneumonliae (3 eyes). Twelve eyes (11.8%) had polymicrobial infection. A good visual outcome occurred in 52.5% of eyes analysed. Prognostic factors for visual outcome include presenting Snellen visual acuity, time to presentation after onset of ocular symptoms, ocular predisposing factor, corneal ulcer location and corneal ulcer size.

Trabeculectomy outcomes in a Malaysian general hospital.

A retrospective study was conducted at the Hospital Sultanah Aminah Johor Bahru to determine the outcome of trabeculectomy surgeries over a period of 4 years. One hundred and two eyes were followed up to a maximum of 63 months (mean 34.2 months). The 2-year survival rates for plain trabeculectomies, 5-Fluorouracil augmented trabeculectomies and Mitomycin-C augmented trabeculectomies were 52.9%, 27.3% and 60.5% respectively. The commonest complications noted were cataract formation (25%) and hyphaema (11%). Mitomycin-C induced complications were rarely seen. At last follow-up, 54% of eyes had intraocular pressures below 21 mmHg without medication, while 34% of eyes had intraocular pressures below 21 mmHg with medication. Vitreous at the trabeculectomy site was a statistically significant predictor of operative failure.

Utilisation of ophthalmic services by foreign nationals in Johor: a review of 452 patients.

Between 1st January 1999 and 31st December 2000, 452 foreign nationals were treated at the Department of Ophthalmology, Hospital Sultanah Aminah, Johor Bahru. Eighty-five percent were male. The peak age range was from 21 to 30 years old. The patients were predominantly Indonesians (61%). A history of trauma was present in 63% of patients. Eight percent of eyes had severe visual impairment. Six patients (1.3%) were blind by WHO standards. Traumatic eye conditions, inflammatory/allergic eye conditions and degenerative eye conditions comprised 66%, 13% and 10% respectively of ocular pathology seen. The commonest ocular findings were corneal foreign body, corneal abrasion and subconjunctival haemorrhage.

Open-globe injuries: the experience at Hospital Sultanah Aminah, Johor Bahru.

Between 1st January 1999 and 31st December 2000, 152 patients (156 eyes) with open-globe injuries were treated in the Department of Ophthalmology, Hospital Sultanah Aminah, Johor Bahru. The majority were male (88.2%), Malay (63.2%), from the Johor Bahru district (51.3%) and aged between 21 and 30 years (23.7%). Most injuries were workplace-related (41.4%). Lens injury, retinal detachment, endophthalmitis, intraocular foreign bodies and phthisis occurred in 40.4%, 15.4%, 14.7%, 12.2% and 11.5% of eyes respectively. A favourable visual outcome occurred in 55.4% of eyes. Prognostic factors for visual outcome include presenting visual acuity, relative afferent pupillary defect, wound location, lens injury, retinal detachment and endophthalmitis.

Differential expression of Hox A5 in human colon cancer cell differentiation: a quantitative study using real-time RT-PCR.

Fifteen different homeobox genes were identified from normal colon mucosa, untreated COLO 205 and herbimycin A treated COLO 205 cells in a degenerate primer RT-PCR screen. Several of the homeobox genes, including Cdx-1, Cdx-2, Pdx-1 and Hox A5, showed a trend toward differential expression in normal colon mucosa, and undifferentiated COLO 205 cells. Hox A5 was recently shown to suppress growth and induce p53-dependent apoptosis. To determine if Hox A5 was differentially expressed in differentiation of colon epithelial cells, we quantified Hox A5 expression by real-time quantitative RT-PCR. Expression of Hox A5 was 5.3- and 4.8-fold higher in normal colon mucosa compared to COLO 205 and HT-29 cells, respectively, suggesting that Hox A5 expression was higher in differentiated compared to undifferentiated colon epithelial cells. To avoid the complexity of tissue specimens and the influence of individual variation in Hox A5 expression, the effect of differentiation on Hox A5 expression was studied in COLO 205 cells treated with herbimycin A. The quantitative study showed that Hox A5 expression was increased when COLO 205 cells were induced to differentiate. The expression of Hox A5 was about 2-fold higher in the cells treated for 48 h compared to the untreated poorly-differentiated cells. The present study shows that Hox A5 may be involved in intestinal cell differentiation, in addition to its role in apoptosis.

Characterization and expression of the mouse pregnant specific uterus protein gene and its rat homologue in the intestine and uterus.

The pregnant specific uterus protein gene (Psup) is a novel mouse gene expressed in pregnant uterus. This paper describes the identification and expression of the rat homologue of Psup. The gene is highly expressed in the duodenum. Expression decreases in a proximal-distal gradient in the small intestine and was not detected in the cecum and colon. The pattern of expression in the mouse was similar. Expression of Psup in the mouse was localized to the epithelial cells in the intestine and pregnant uterus by in situ hybridization. The data show tissue-specific expression of Psup.

Ribosomal protein L7a gene is up-regulated but not fused to the tyrosine kinase receptor as chimeric trk oncogene in human colorectal carcinoma.

Ribosomal protein L7a (rp L7a) was identified in a subtractive hybridization screen as a gene up-regulated in human colorectal cancer. Expression of rp L7a was greater than 2-fold higher in tumors compared to adjacent normal mucosa in 72% of the patients studied (n=36). rp L7a was also up-regulated in concomitant polyps. The number of patients with rp L7a T/N ratio of >2 was significantly higher in the female (16/18) than in the male (10/18). rp L7a expression was also significantly higher in females with lymph node involvement compared to males. These results indicate that rp L7a expression is related to tumor growth in colorectal cancer especially in females, where it may also be related to tumor spread. There was no correlation of rp L7a expression with tumor cell differentiation. We also show that rp L7a does not exist as a fusion oncogene (trk-2h) in colorectal cancer.

Syncollin is differentially expressed in rat proximal small intestine and regulated by feeding behavior.

Gradients of gene expression are maintained along the proximal-distal axis of the mammalian small intestine despite a continuously regenerating epithelium. To study the molecular mechanisms responsible for this phenomenon, we utilized a subtractive hybridization strategy to isolate genes differentially expressed in the duodenum but not ileum. We isolated and sequenced 15 clones. The clones were fragments of genes encoding lipases, proteases, and an esterase. A novel clone was characterized and subsequently shown to encode syncollin, a secretory granule protein that binds to syntaxin in a calcium-sensitive manner. RT-PCR and S1 nuclease protection assay were used to clarify the 5'-end of syncollin. Syncollin was expressed in the rat pancreas, spleen, duodenum, and colon. In situ hybridization localized syncollin expression in the pancreas to acinar cells and in the duodenum to villus epithelial cells.

Heparin/heparan sulfate interacting protein gene expression is up-regulated in human colorectal carcinoma and correlated with differentiation status and metastasis.

We applied a subtractive hybridization strategy to obtain genes that are differentially expressed in colorectal carcinoma. Heparin/heparan sulfate interacting protein (HIP) was shown to be up-regulated in colorectal carcinoma. A study of 53 patients with documented colorectal carcinoma showed that 70% of the tumors had HIP tumor-to-normal ratios (expression in tumor tissue compared to expression in normal mucosa) of >2. In six patients with concomitant polyps, HIP expression in the polyps was similar to the carcinoma, showing that up-regulation of HIP may be an early event in tumorigenesis. A significant inverse correlation between HIP levels and the presence of distant metastasis (Duke's stage D) was noted. Similarly, HIP expression was also related to differentiation status in human colorectal carcinoma cell lines. HIP expression was lower in the poorly differentiated COLO 205 cell line compared to the well-differentiated HT-29 cell line. The correlation was further strengthened by studies in COLO 205 cells that were induced to differentiate with herbimycin A treatment. HIP expression was significantly higher when the cells were induced to differentiate. Withdrawal of herbimycin A resulted in a reversal of morphological changes associated with differentiation and an associated decrease in HIP expression. These studies indicate that HIP is an important molecule for cell-cell and cell-extracellular matrix adhesion. The up-regulation of HIP may be an early event in tumorigenesis, and its increased expression may facilitate growth and local invasion. A lower expression of HIP in tumors results in decreased cell adhesion, favoring metastasis. HIP is a candidate marker of abnormal cell growth in the colon and a prognostic marker for colorectal carcinoma.

Identification and characterization of a novel rat triosephosphate isomerase gene in remnant ileum after massive small bowel resection.

This paper describes the identification and characterization of a novel cDNA encoding a putative protein of 254 amino acids that is highly homologous to triosephosphate isomerase. The cDNA was isolated by subtractive hybridization and was differentially expressed in the remnant rat ileum after massive small bowel resection. The novel triosephosphate isomerase was named rsTPI (resection-induced TPI) and the putative protein encoded RSTPI. The nucleotide and amino acid sequences of rsTPI and RSTPI were about 60% and 62% homologous to Giardia lamblia TPI and TPI, respectively. Active catalytic sites (Lys 13, His 95, and Glu 167) and the peptide motifs, AYEPVWSIGT and GGASLKPEF found in other triosephosphate isomerases were conserved in RSTPI. rsTPI expression was detected in normal ileum and pancreas by reverse transcription-polymerase chain reaction. Expression of rsTPI in remnant rat ileum was detectable by northern blot analysis one week after massive small bowel resection. Expression increased significantly by 2.8-fold between one and two weeks after surgery. High levels were maintained for at least one month after surgery. The up-regulation of triosephosphate isomerase expression in the remnant small intestine after massive resection indicates that it may play an important role in the adaptive process.

Stimulation of anterior pituitary galanin and prolactin gene expression in suckling rats.

Recent evidence suggests that galanin may regulate prolactin (PRL) secretion during lactation. In this article, we describe the regulation of anterior pituitary galanin and PRL gene expression during pregnancy and after parturition in the rat. Expression of galanin and PRL in the anterior pituitary were significantly higher at d 20 of pregnancy compared to diestrus. One day after parturition, galanin mRNA levels increased a further 4.5-fold. This post partum increase in gene expression was not observed for PRL. The increase in galanin gene expression was maintained above the diestrous level for at least 10 d after parturition. PRL mRNA expression, on the other hand, was largely unchanged after parturition. Although the increase in galanin gene expression 1 d after parturition was independent of suckling, subsequently, galanin gene expression was significantly higher in nursing mothers. Anterior pituitary galanin gene expression was 12-fold higher in nursing mothers compared with those that were not, 3 d after parturition. Similarly, PRL gene expression was significantly lower in mothers who were not suckling their pups 3 d after parturition. Initiation of suckling alone was insufficient to stimulate galanin and PRL expression. Despite suckling for 2 d, removal of the suckling stimulus subsequently resulted in a rapid decrease in galanin gene expression. Hence, the stimulatory effect of suckling on galanin expression requires a sustained suckling stimulus. In conclusion, the data support the hypothesis that anterior pituitary galanin plays an important role during lactation, likely acting to amplify lactotroph stimulation through paracrine and autocrine mechanisms.

Regulation of leptin expression and secretion by corticosteroids and insulin. Implications for body weight.

Leptin is an important hormone that has potent effects on appetite and body weight. The regulation of leptin gene expression and secretion by corticosteroids and insulin was studied in the rat. Adrenalectomy resulted in a significant reduction in leptin gene expression and secretion. The reduction was corrected by hormonal replacement with corticosterone pellets, showing that normal levels of circulating corticosteroids are required to maintain leptin expression and secretion in the body. Chronic treatment with dexamethasone (DEX) over 3 wk did not significantly increase leptin gene expression and secretion, contrary to earlier reports using shorter treatment paradigms. The profound weight loss associated with chronic DEX treatment may have abrogated the direct stimulatory effect of DEX on leptin gene expression and secretion, indicating a possible crosstalk between corticosteroids and leptin in the regulation of body weight. Shorter-term treatment of animals with DEX (3.7 micrograms/g body wt; 24 h) increased leptin gene expression and secretion about 2-fold and 1.4-fold, respectively. The increase was independent of circulating insulin concentrations. In streptozotocin-treated rats, short-term DEX treatment increased leptin gene expression and secretion about 3.5-fold and 2-fold, respectively. The data indicate that circulating leptin concentrations and adipose tissue leptin expression are dependent on corticosteroids and insulin. Although acute DEX treatment resulted in a stimulatory effect on leptin secretion and expression, chronic DEX treatment did not. The stimulatory effect of DEX on leptin is independent of circulating insulin concentrations.

Increase in plasma leptin and Lep mRNA concentrations by food intake is dependent on insulin.

Obese (Lep) gene expression and leptin secretion are regulated by changes in food intake. However, the mechanism by which leptin concentrations are altered by fasting and feeding is unclear. Since these changes occur in parallel with changes in plasma insulin, it is possible that the changes observed are mediated by insulin. To test this hypothesis, we studied the role of insulin in the regulation of Lep gene expression in epididymal fat and leptin secretion during feeding. As shown previously, fasted animals showed significant reductions in Lep mRNA, plasma leptin, and plasma insulin concentrations. Conversely, feeding increased plasma insulin, Lep mRNA, and plasma leptin. In streptozotocin (STZ)-treated animals, plasma insulin concentrations were low. This was associated with low Lep mRNA and plasma leptin concentrations. Changes in food intake, whether fasting or feeding, did not significantly alter plasma insulin levels in STZ-treated animals. Under these circumstances, Lep mRNA and plasma leptin concentrations also remained low. Our results demonstrate that the decrease in Lep mRNA and plasma leptin during fasting and the increase with feeding are dependent on changes in the plasma insulin concentration.

Regulation of anterior pituitary galanin gene expression by thyroid hormone.

Thyroid hormone is required for basal and estrogen-induced expression of anterior pituitary galanin. Steady-state anterior pituitary galanin mRNA levels decreased 6-fold in hypothyroid rats after 3 weeks of treatment. Similarly, hypothyroidism resulted in a 2.6-fold decrease in estrogen induction of galanin gene expression. The effect of thyroid hormone on anterior pituitary galanin gene expression appears to be exerted, at least in part, at the pituitary itself. Transient expression assays in GH3 cells suggest the involvement of transcriptional mechanisms in the regulation of galanin gene expression by thyroid hormone. A region between -41 and -132 bp upstream of the transcriptional start site confers thyroid hormone responsiveness to the galanin gene. Gel-mobility shift assays show specific binding of 'SPI-like' proteins in GH3 nuclear extracts to this region of the galanin gene. This binding was greatly enhanced by thyroid hormone.