Glycaemic evaluation of Psidium guajava in rats.

BACKGROUND & OBJECTIVE: Many plant polysaccharides exhibit hypoglycaemic effect. Though the fruit of Psidium guajava is known to contain free sugars, the fruit extract showed hypoglycaemic effect in alloxan treated mice and human subjects. The present study was aimed to determine the glycaemic potential of P. guajava fruit peel extract on blood glucose level (BGL) of normal and streptozotocininduced sub-diabetic rats during fasting blood glucose (FBG) and glucose tolerance test (GTT). METHODS: Female albino Wistar rats (n=42) were divided into seven equal groups, and were given different doses of fruit peel extract. Diabetes was induced by streptozotocin injection (ip) at a dose of 45 mg/kg body weight. Blood glucose levels were measured after collecting the blood from tail veins. RESULTS: The diabetic and sub-diabetic models showed hyperglycaemic effect from a single oral administration of variable doses of P. guajava fruit peel extract. The maximum rise of 26.51 per cent was observed in BGL from a dose of 400 mg/kg bw exactly after 8 h of administration in normal rats whereas the maximum rise of 90.7 per cent was observed with the same dose of 400 mg/kg bw after 2 h of glucose administration in sub-diabetic rats. INTERPRETATION & CONCLUSION: The hyperglycaemic effect of P. guajava fruit peel suggests that the diabetic patients should peel off the guava fruits before consuming. However, it can also be useful in controlling hypoglycaemia occasionally caused due to excess of insulin and other hypoglycaemic drugs.

A clinical, magnetic resonance imaging, and survival motor neuron gene deletion study of Hirayama disease.

BACKGROUND: Hirayama disease (HD) is a segmental nonprogressive spinal muscular atrophy found in male patients. OBJECTIVE: To report the results of a comprehensive evaluation of clinical, magnetic resonance imaging (MRI), electromyography (EMG), and survival motor neuron (SMN) gene analysis of HD. DESIGN: Clinical, MRI, and SMN gene deletion study. SETTING: Tertiary care teaching hospital. PATIENTS: Patients with HD diagnosed according to defined criteria were included in the study. INTERVENTIONS: Patients underwent a neurologic evaluation and pedigree charting. Concentric needle EMG was performed on a number of muscles. Motor nerve conduction study of the median, ulnar, and peroneal nerves and sensory conduction study of the median, ulnar, and sural nerves were also performed. Spinal MRI of the cervical region was performed with the 2-T scanner operating at 1.5 T. Gene deletion study of SMN1 and SMN2 was performed in all patients. MAIN OUTCOME MEASURES: History of trauma, occupation, exercise, associated medical disease, and cold paresis and muscle wasting, power, reflex changes, and tone. RESULTS: Fifteen male patients with HD from 14 families participated in the study (mean age at the onset of disease, 18 years; range, 15-23 years). Muscle weakness and wasting were noted in the right upper limb in 12 and the left upper limb in 3, which became bilateral in 8 patients. Cold paresis was present in 6 patients and polyminimyoclonus in all patients. The EMG revealed fibrillations in 10, fasciculations in 15, and neurogenic motor unit potentials in C7, C8, and T1 myotomes in all patients. The EMG abnormalities were unilateral in 5, bilateral in 10, and subclinical in 2 patients. Spinal MRI revealed cord atrophy in 3 of 11 patients. Although family history was present in 1 brother only, the results of both SMN1 and SMN2 gene deletion studies were negative in all patients. CONCLUSIONS: The SMN gene deletion is not found in HD. Exclusive occurrence in male patients and the presence of this disease in 2 brothers suggest a possible role of the X chromosome, which needs further evaluation.

Spontaneous programmed cell death in infiltrating duct carcinoma: association with p53, BCL-2, hormone receptors and tumor proliferation.

Recent evidence has emphasized the importance of programmed cell death or apoptosis in the maintenance of tissue homeostasis and pathogenesis of tumors. This study, analyzed in breast cancer, investigates the significance of apoptosis in relation to the expression of p53 and bcl-2 proteins, tissue proliferation defined by Ki-67 expression, hormone receptors and tumor grade. The extent of apoptosis was defined by morphological criteria and the TUNEL (Tdt-mediated dUTP biotin nick end labelling) assay. Immunocytochemistry was performed for p53, bcl-2, estrogen receptor, progesterone receptor and Ki-67 expression. Mutant p53 protein was detected using a mutant specific ELISA. Immunoreactivity of p53 significantly correlated with the presence of mutant p53 protein detected by ELISA (r = 0.654, p = 0.00001). An inverse correlation was observed between bcl-2 expression and the extent of apoptosis (r = -0.33369, p = 0.01912). The extent of apoptosis directly correlated with p53 protein accumulation (r = 0.485, p = 0.00041), Ki-67 immunoreactivity (r = 0.435, p = 0.001), histopathological grade (r = 0.492, p = 0.0003), tumor size (r = 0.326, p = 0.023) and lymph node status (r = 0.287, p = 0.047). A direct correlation was also observed between p53 expression and Ki-67 immunoreactivity (r = 0.623, p = 0.0002). There was no statistically significant association between estrogen and progesterone receptor status and apoptosis. In addition, the TNM stage of the disease correlated with immunoreactivity of p53 (r = 0.572, p = 0.00012) and Ki-67 (r = 0.3744, p = 0.00818). Bcl-2, by inhibiting apoptosis, may cause a shift in tissue kinetics towards the preservation of genetically aberrant cells, thereby facilitating tumor progression. These results imply that rapidly proliferating tumors appear to have a high "cell turnover state" in which there may be an increased chance of apoptosis amongst the proliferating cells. The ability of apoptosis to also occur in the presence of mutant p53 protein suggests the existence of at least two p53-dependent apoptotic pathways, one requiring activation of specific target genes and the other independent of it.

Apoptosis in epithelial ovarian tumors.

It is now recognized that apoptosis plays an important role in the pathogenesis of tumors. This study evaluated the extent of apoptosis in different grades of ovarian tumors and correlated it with the expression of apoptosis regulatory genes, p53 and bcl-2 and with the total proliferative compartment of the tumor defined by the expression of the proliferating cell nuclear antigen (PCNA). Apoptosis was evaluated by the TUNEL (Tdt-mediated dUTP biotin nick end labelling) assay. Expressions of p53, bcl-2 and PCNA were analyzed by immunohistochemistry. A negative correlation was observed between the expression of bcl-2 and the extent of apoptosis (r = -0.3336, p = 0.019). P53 accumulation directly correlated with the extent of apoptosis (r = 0.485, p = 0.00041). The labelling index of PCNA also showed correlation with expression of p53 (r = 0.49, p = 0.00000). Apoptosis was significantly higher in poorly differentiated tumors when compared to the well- and moderately-differentiated tumors (r = 0.49152, p = 0.00034). Such poorly-differentiated tumors also showed high p53 overexpression and loss of bcl-2 expression. The present study thus provides evidence that dysregulation of apoptosis and its regulatory genes is associated with increasing malignant potential and may thus contribute to the pathogenesis of ovarian tumors.

Clinicopathological significance of tissue homeostasis in Indian breast cancer.

BACKGROUND: Tissue homeostasis and the maintenance of cell populations depend on a delicate balance between the rates of cell proliferation and cell death. Disruption of this balance is an important factor in development and progression of tumors. In the present study we evaluated the growth index in a large group of breast cancer patients and correlated it with various clinical and histopathological features of the tumor. METHODS: Estimation of apoptosis was determined by TUNEL assay while immunocytochemistry for proliferating nuclear cell antigen (PCNA) was used as a measure of proliferation. Necrosis was identified morphologically by haematoxylin and eosin staining. RESULTS: A positive correlation was observed between the percentage of PCNA positive cells and the frequency of mitosis (r=0.6117, p<0.0001). A highly statistical significant correlation was observed between type of tissue analyzed and growth index (r=0.46869, p<0.0001). No significant association was observed between hormone receptor status and growth index. CONCLUSIONS: The growth index was found to be higher in carcinoma cells that metastasised into lymph nodes compared with primary lesions with no nodal metastasis. Growth index was particularly prominent in high-grade tumors in which increased proliferative activity was evident. Apoptotic cells were detected more frequently in tumor cells with higher rather than lower proliferative activity. This suggests that not only proliferative activity but also capacity for apoptosis is altered in breast tumors.

A clinical and genetic study of spinal muscular atrophy.

AIMS: This study evaluates clinical, electromyography (EMG) and genetic analysis of consecutive patients with spinal muscular atrophy (SMA) in a tertiary care adult neurology practice in India. METHODS: Consecutive patients with SMA attending the neurology out patient department during 2001-2003 were included. They were subjected to a detailed clinical examination, nerve conduction and EMG and muscle biopsy. Clinically patients were classified into generalised and segmental SMA. SMN gene deletion study was carried out in all the patients. RESULTS: There were 15 patients with type III and type IV SMA and 15 with segmental SMA (Hirayama disease). The age ranged between 5 and 23 years in type III SMA, 33-50 years in type IV SMA and 16-30 years in Hirayama disease (HD). The latter was found exclusively in males. Family history was observed in 1 patient each in all the groups. In SMA III mother and brother were affected, in SMA IV two siblings and in HD one brother had similar disease. One type III SMA family was associated with deafness and one type IV family had strong association with maturity onset diabetes in young. The EMG was characterised by lack of fibrillations in all type III and IV SMA patients except 1 whereas in HD, 11 out of 15 had fibrillations suggesting ongoing denervation. The EMG was suggestive of reinnervation in generalised SMA in both upper and lower limb muscles where as these abnormalities were restricted to C7-T1 mytomes in HD. Muscle biopsy in 10 patients with generalised SMA revealed group atrophy in all, and loss of fascicular architecture in 3, clumping of nuclei in 7 and hypertrophic fibers in 4. SMN1 gene deletion was present in 3 patients with type III but none in type IV and HD. CONCLUSION: SMN gene deletion was positive in 33% type III SMA whereas it was negative in type IV and HD. Presence of HD only in males may be consistent with X-linked disorder.

Clinical and molecular diagnosis of spinal muscular atrophy.

The spinal muscular atrophies are a group of disorders characterized by flaccid limb weakness. It is necessary to differentiate these from other causes and identify the SMA variants. In classical SMA, majority of the patients shows homozygous deletion of the telomeric SMN gene (SMN1) on chromosome 5q. The availability of DNA analysis has allowed proper genetic counseling and prenatal diagnosis in the affected families. Application of newer techniques has enabled more accurate carrier detection. Our objective is to stress the variability in the clinical features and recent advances in the molecular diagnosis for SMA.

Re-evaluation of reading frame-shift hypothesis in Duchenne and Becker muscular dystrophy.

The reading frame hypothesis has been proposed to explain the molecular basis of two allelic forms of muscular dystrophies, Duchenne/Becker muscular dystrophy (D/BMD). To evaluate the hypothesis in Indian D/BMD patients, we analyzed deletion of dystrophin exons in 147 DMD and 19 BMD patients. Our studies showed deviation of more than 30% from the reading frame hypothesis in DMD patients (47/147). The present results implicate a need to reevaluate the reading frame hypothesis.

Fighting cancer in the information age: the role of Internet.

Cancer is a major health problem worldwide which is likely to assume alarming proportions in the next two decades. Communication and information have increasingly been considered important in helping people to cope with cancer. The arrival of Internet offers the opportunity to fundamentally reinvent medicine and health care delivery. Medical professionals can now use the Internet for continuing medical education, access latest medical information, for fast confirmation of diagnosis, exchange opinion on treatment strategies and in palliative care. Internet can provide cost-effective and timely ways to deliver a complex mix of interesting and high-quality information and expertise to cancer patients. Patients can also independently search the Internet to know about their illness and treatment options. However, of concern is the quality of information that is available in the 'Net'. Some Internet sites may contain erroneous information on cancer and can pose serious problems. There are also many good sites, which provide quality information on cancer for medical professionals, researchers and patients. This article focuses on how the Internet will aid us in fight against cancer.

SPP1 genotype is a determinant of disease severity in Duchenne muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is the most common single-gene lethal disorder. Substantial patient-patient variability in disease onset and progression and response to glucocorticoids is seen, suggesting genetic or environmental modifiers. METHODS: Two DMD cohorts were used as test and validation groups to define genetic modifiers: a Padova longitudinal cohort (n = 106) and the Cooperative International Neuromuscular Research Group (CINRG) cross-sectional natural history cohort (n = 156). Single nucleotide polymorphisms to be genotyped were selected from mRNA profiling in patients with severe vs mild DMD, and genome-wide association studies in metabolism and polymorphisms influencing muscle phenotypes in normal volunteers were studied. RESULTS: Effects on both disease progression and response to glucocorticoids were observed with polymorphism rs28357094 in the gene promoter of SPP1 (osteopontin). The G allele (dominant model; 35% of subjects) was associated with more rapid progression (Padova cohort log rank p = 0.003), and 12%-19% less grip strength (CINRG cohort p = 0.0003). CONCLUSIONS: Osteopontin genotype is a genetic modifier of disease severity in Duchenne dystrophy. Inclusion of genotype data as a covariate or in inclusion criteria in DMD clinical trials would reduce intersubject variance, and increase sensitivity of the trials, particularly in older subjects.

Cellular manifestations of human papillomavirus infection in the oral mucosa.

BACKGROUND AND OBJECTIVES: Human papillomavirus infection has been suggested to play a role in the development of epithelial carcinomas, particularly those of the uterine cervix. Less information is available on the role of the virus in oral lesions. It has been proposed that the viral oncoproteins specifically complex with the products of cellular tumor suppressor gene, namely E6 with p53 and E7 with retinoblastoma gene product. Inactivation or mutation in p53 gene is also known to result in loss of control over the cell cycle and increases in tumor proliferation rates. The present study examines the role of HPV infection in relation to p53 and the extent of the tumor proliferative compartment reflected by cyclin D1 and Ki-67 expression during various phases of tumor progression in the oral epithelium. METHOD: Nonisotopic in situ hybridization (NISH) was performed to detect HPV 6/11 and 16/18. Expression of p53, cyclin D1, Ki-67, and the HPV 16/18 E6 protein were detected by immunocytochemistry. RESULTS: There was significant correlation between the extent of histological abnormality and HPV infection. A correlation (r = 0.250, P = 0.0089) was evident between the presence of HPV 16 and occurrence of invasive cancer. Expression of the tumor suppressor p53 protein also showed significant positive correlation with histology (r = 0.475, P = 0.00004). The tumor proliferative fraction also increased with the extent of histological abnormality (r = 0.387, P = 0.0003 for cyclin D1 and r = 0.463, P = 0.0001 for Ki 67). Accumulation of p53 and increase in tumor proliferation also correlated to the presence of HPV infection (r = 0.511, P = 0.00003 for p53; r = 0.478, P = 0.00002 for cyclin D1 and r = 0.521, P = 0.00004 for Ki-67). CONCLUSIONS: The present study thus demonstrates the importance of HPV infection in oral tissue. Expression of the high-risk HPV 16/18 E6 protein also appears to be a critical event along with aberrant p53 expression. These results are of significance to the molecular epidemiology of oral cancer and may also be used to supplement and elaborate the diagnosis of oral lesions.

Expression of the antiapoptotic protein bcl-2 is not dependent on the tumor suppressor p53 protein in Indian breast carcinoma.

Tissue homeostasis and the maintenance of cell populations depend on a delicate balance between the rates of cell proliferation and cell death. Programmed cell death or apoptosis is believed to play a major role in physiological processes which, when defective, could contribute to the pathogenesis and progression of tumors. A role for altered programmed cell death in cancer stems from the description of alterations on tumor-associated genes involved in the regulation of apoptosis such as p53 and bcl-2. The p53 gene promotes apoptosis in cells with genetic damage, while bcl-2 is an antiapoptotic gene. It is therefore possible that the balance between p53 and bcl-2 may have significant implications for the pathobiology of breast cancer. This study was therefore undertaken to evaluate the expression of these two proteins with opposite functions and their relation to the total growth fraction of the tumor as measured by PCNA immunoreactivity. A significant correlation was observed between expression of p53 and PCNA. In contrast, bcl-2 expression did not correlate with the expression of p53. There was also no correlation observed between expression of bcl-2 and PCNA. A significant correlation was observed between expression of p53 and the grade of the tumor and stage of the disease. Our results thus support the hypothesis that accumulation of p53 is associated with a high tumor proliferation rate, an association that might be expected in view of the role of wild-type p53 as a negative regulator of cell proliferation. Another important observation was the lack of relationship between bcl-2 expression and PCNA immunoreactivity, supporting the hypothesis that bcl-2 is not a major regulator of proliferation.

Tumor proliferative fraction in infiltrating duct carcinoma.

Deregulated cell proliferation is a key factor in malignancy and therefore may be of prognostic significance. Both Proliferating Cell Nuclear Antigen (PCNA) and silver binding Argyrophilic Nucleolar Organizer Regions (AgNORs) have been established as a valuable reflection of the tissue proliferative compartment and hence could be of value in studying the biologic behavior of malignant cells. In the present study, we evaluated the proliferative activity of 120 infiltrating duct carcinomas by analysing for PCNA and AgNOR expression in relation to pathologic variables. The AgNOR scores were significantly greater in the malignant tissue than in benign lesions. Expression of PCNA was also found to be increased in malignant breast tumors, when compared to controls. A strong positive correlation between PCNA immunoreactivity and AgNORs was evident. There was also a positive correlation between histologic grading and AgNOR score in breast cancer (r = 0.92, p = 0.000). Tumor with elevated AgNOR counts were often poorly differentiated. It is therefore apparent that evaluation of AgNORs and PCNA may help in the elaboration of histopathologic grading of infiltrating duct carcinoma and may be of prognostic significance.

Prostate cancer cell growth inhibition by tamoxifen is associated with inhibition of protein kinase C and induction of p21(waf1/cip1).

BACKGROUND: Inhibition of protein kinase C (PKC) and modulation of transforming growth factor-beta (TGF-beta) are both associated with tamoxifen treatment, and both appear to be important in the regulation of prostate cancer cell growth. Investigations were performed which sought to measure the efficacy, and to elucidate the mechanism of growth inhibition by tamoxifen, in hormone-refractory prostate cancer. METHODS: Growth assays were performed on PC3, PC3-M, and DU145 prostate cancer cells. TGF-beta was measured by ELISA; p21(waf1/cip1) and retinoblastoma (Rb) protein levels were measured by Western blot; PKC activity was measured by kinase assay; and effects upon cell cycle were measured by flow cytometric analysis. RESULTS: IC50s for growth inhibition ranged from 5.5-10 microM, and were not affected by estrogen. Tamoxifen-mediated growth inhibition was not associated with induction of TGF-beta. However, tamoxifen treatment was associated with inhibition of PKC, which was followed by induction of p21(waf1/cip1), Rb dephosphorylation, and G1/S phase cell cycle arrest. Similar effects were observed with the known PKC inhibitor, Ro31-8220. CONCLUSIONS: These data suggest that micromolar concentrations of tamoxifen inhibit prostate cancer cell growth by inhibition of PKC, resulting in induction of the p21(waf1/cip1) protein.

p53 tumor suppressor protein and tissue proliferative fraction in infiltrating duct carcinoma.

BACKGROUND AND OBJECTIVES: Breast cancer continues to frustrate oncologists worldwide. In India, it is the second most common neoplasm among women and is increasing in incidence. Several molecular markers have been associated with a poor prognosis in patients with breast cancer, and the presence of these markers is often thought to provide information on the biological behavior of the malignant breast tumor. Much attention has recently been focused on the tumor suppressor gene p53. Mutation or alteration in this gene leads to the loss of negative growth regulation and hence to rapid cell proliferation. The present study was designed to evaluate the connection between expression of the p53 protein and its relation to the tissue proliferative compartment as measured by expression of the proliferating cell nuclear antigen (PCNA). METHODS: Expression of p53 and PCNA were detected by immunocytochemistry in paraffin-embedded sections of infiltrating duct carcinoma and control breast tissue (normal tissue and adenoma). RESULTS: A significant correlation was observed between expression of p53 and PCNA. A significant correlation was also observed between expression of p53 and grade of tumor and stage of disease. CONCLUSIONS: Our results support the hypothesis that accumulation of p53 is associated with a high tumor proliferation rate an association that might be expected in view of the role of wild p53 as a negative regulator of cell proliferation.