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Biomedical subjects

S Kannan

Publications and source records attributed to S Kannan.

At least 91 records · Page 5Linked to original sources
PubMed1998

Purification and characterization of a recombinant human cripto-1 protein.

Cripto-1 (CR-1) is a novel protein that contains a modified EGF-like motif and that does not directly bind to any of the known erb B type-1 receptor tyrosine kinase receptors. To more clearly define the biological effects of CR-1 and to more adequately compare the structure-function relationships of CR-1 with other members of the EGF family of growth factors, we have expressed a modified, full-length recombinant human CR-1 protein (rhCR-1) in E. coli and have devised a procedure for the solubilization, refolding and purification of a biologically active form of this protein. We have generated the mature form of hCR-1 from computer assisted predictions of potential signal peptide cleavage sites. Expression of the modified rhCR-1 protein in E. coli was limited to the inclusion bodies. The rhCR-1 protein was found to be expressed at high levels in bacterial cells when fused to a histidine-tag sequence. Refolding of rhCR-1 was found to be difficult because of the large number of cysteine residues in the protein which results in protein aggregation. By chemically modifying the cysteine residues in the rhCR-1 protein with 3-trimethylammoniopropyl methanethiosulfonate, additional positive charges have been introduced into the protein by this disulfiding reagent. This modification facilitates solubilization of the protein when rhCR-1 is denatured. The solubilized, denatured protein was then purified by CM cation exchange and C4 reverse phase HPLC chromatography and refolded in a redox buffer. The refolded, modified rhCR-1 protein was found to be biologically active by its ability to inhibit beta-casein expression, to stimulate the tyrosine phosphorylation of Shc and the activation of MAPK and by its capacity to facilitate branching growth of mouse mammary epithelial cells in type I collagen gels.

Animals↗
PubMed1997

Chemical synthesis, structural modeling, and biological activity of the epidermal growth factor-like domain of human cripto.

Cripto, also known as human teratocarcinoma-derived growth factor 1 (TDGF-1), contains a 40 amino acid region with some similarity to the epidermal growth factor (EGF) domain. However, sequence homology is largely restricted to the classical cysteine/glycine motif with only limited similarities in other regions. Significant differences to human EGF include the absence of all seven residues between the two N-terminal half-cystines and a five-residue shorter loop between the third and fourth half-cystines. We examine the hypothesis that, in spite of these differences, cripto can adopt the characteristic EGF-like 1-3, 2-4, 5-6 disulfide bond pattern. A comparative structural model of the growth factor cripto was constructed on the basis of its similarity to EGF, transforming growth factor alpha (TGF-alpha), and the EGF-like domain of human clotting factor IX. The predicted disulfide bridges and disulfide-bridged loops were analyzed and appear viable in the modeled structure. Moreover, to ascertain the importance of disulfide arrangement for cripto bioactivity, two 47-residue peptides were synthesized and then refolded using either a simple oxidative or a controlled sequential refolding protocol. The cripto peptides were tested for their ability to stimulate MAP-kinase activity, for inhibition of beta-casein induction, and for Shc phosphorylation in MDA-MB 453 human mammary carcinoma cells and HC-11 mouse mammary epithelial cells. Data suggest that cripto does adopt the 1-3, 2-4, 5-6 disulfide pattern and thus forms the classical EGF-like fold in spite of the significant deletions within the folding domain. The predicted structure of cripto shows some of the characteristics of both the ErbB1- and ErbB3/ErbB4-binding growth factors.

Amino Acid Sequence↗
PubMed1997

Cripto enhances the tyrosine phosphorylation of Shc and activates mitogen-activated protein kinase (MAPK) in mammary epithelial cells.

Cripto-1 (CR-1), a recently discovered protein of the epidermal growth factor (EGF) family, was found to interact with a high affinity, saturable binding site(s) on HC-11 mouse mammary epithelial cells and on several different human breast cancer cell lines. This receptor exhibits specificity for CR-1, since other EGF-related peptides including EGF, transforming growth factor alpha, heparin-binding EGF-like growth factor, amphiregulin, epiregulin, betacellulin, or heregulin beta1 that bind to either the EGF receptor or to other type 1 receptor tyrosine kinases such as erb B-3 or erb B-4 fail to compete for binding. Conversely, CR-1 was found not to directly bind to or to activate the tyrosine kinases associated with the EGFR, erb B-2, erb B-3, or erb B-4 either alone or in various pairwise combinations which have been ectopically expressed in Ba/F3 mouse pro-B lymphocyte cells. However, exogenous CR-1 could induce an increase in the tyrosine phosphorylation of 185- and 120-kDa proteins and a rapid (within 3-5 min) increase in the tyrosine phosphorylation of the SH2-containing adaptor proteins p66, p52, and p46 Shc in mouse mammary HC-11 epithelial cells and in human MDA-MB-453 and SKBr-3 breast cancer cells. CR-1 was also found to promote an increase in the association of the adaptor Grb2-guanine nucleotide exchange factor-mouse son of sevenless (mSOS) signaling complex with tyrosine-phosphorylated Shc in HC-11 cells. Finally, CR-1 was able to increase p42(erk-2) mitogen-activated protein kinase (MAPK) activity in HC-11 cells within 5-10 min of treatment. These data demonstrate that CR-1 can function through a receptor which activates intracellular components in the ras/raf/MEK/MAPK pathway.

Animals↗
PubMed1997

Telomerase activity in premalignant and malignant lesions of human oral mucosa.

Recent studies have demonstrated a strong association between carcinogenesis and re-activation of telomerase in various human tumors. In the present study, we have analyzed the telomerase activity in 105 oral mucosal samples, including normal mucosa and premalignant and malignant lesions, by using the telomeric repeat amplification protocol assay. The telomerase activity was detected in normal oral squamous epithelium and in 75% of the oral leukoplakias and oral carcinomas. Although the telomerase activity was observed in normal and hyperplastic squamous epithelium, it showed some relationship with certain clinico-pathological factors in malignant lesions. Telomerase activity was found to have a relationship with the grade of tumor differentiation. Of 34 well-differentiated squamous cell carcinomas, only 10 (30%) exhibited high telomerase activity, whereas in moderately or poorly differentiated squamous cell carcinomas, all seven (100%) tumors displayed high activity. In addition, the level of telomerase activity had an inverse correlation with the treatment response in the early-stage tumors, and the activity differed significantly between the tumors in the following intraoral sites: nonkeratinizing mucosa (buccal mucosa, alveolus, and floor of mouth) and tongue. This preliminary result shows that telomerase activity is present in normal oral squamous epithelium, as it is in normal hematopoietic cells and in carcinomas, and that telomerase activity has a relationship with degree of tumor differentiation and treatment response. Thus, assessing the telomerase activity may be a useful prognostic marker in oral squamous cell carcinomas.

Biomarkers, Tumor↗
PubMed1997

Serum levels of copper, zinc, iron and ceruplasmin in oral leukoplakia and squamous cell carcinoma.

Trace elements have been extensively studied in recent years to assess whether they have any modifying effects in the etiology of cancer. In the present study serum levels of copper, zinc, ceruloplasmin, total iron and total protein were estimated in 92 patients with oral leukoplakia and squamous cell carcinoma and 45 age and sex matched controls. Copper was significantly increased in oral leukoplakia and cancer in both sexes. The level of zinc decreased significantly only in male patients with leukoplakia and cancer. Copper zinc ratio was found significantly elevated in oral leukoplakia and cancer. A marked increase in serum ceruloplasmin was seen in oral leukoplakia and cancer in both sexes. A significant decrease of serum total iron and proteins were observed only in carcinomas. These findings suggest that reduction in the serum total iron and total protein may be due to malnutrition caused, on its turn, by tumour burden. Serum ceruloplasmin level and copper-zinc ratio demonstrated significant differences in the patients of both sexes from those of controls, while, comparing leukoplakia with cancer, a significant difference is only observed in male patients. Thus, the present study shows that these factors have diagnostic value only in differentiating malignancies from normal and a little value as biomarkers of disease progression. However, the exact mechanism responsible for the alteration of these factors in oral lesions is still not clear and a detailed study on large sample size is therefore needed.

Adult↗
PubMed1997

Nonsteroidal anti-inflammatory drug induced duodenal web.

Duodenal webs represent an unusual cause of intestinal obstruction in adults. These anomalies are generally considered to be congenital in origin and usually present in infancy. However, they occasionally become symptomatic in adulthood. In these cases, because of the delay in symptoms, the etiology of duodenal webs in adults is uncertain. Gastrointestinal webs in adults have also been reported in the small intestine and colon. It is generally accepted that these lesions are an acquired defect related to long term nonsteroidal anti-inflammatory drug (NSAID) use. We report a patient with a history of long term NSAID use who presented with symptoms of gastric outlet obstruction due to the presence of a duodenal web.

Adult↗
PubMed1997

Cripto-1 inhibits beta-casein expression in mammary epithelial cells through a p21ras-and phosphatidylinositol 3'-kinase-dependent pathway.

Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that does not directly activate any of the known erbB type 1 tyrosine kinase receptors. Also, CR-1 stimulates the growth of HC-11 mouse mammary epithelial cells. We found that prior treatment of HC-11 cells with exogenous CR-1 induced a competency response to the lactogenic hormones dexamethasone, insulin, and prolactin (DIP) with respect to the induction of the milk protein beta-casein. In contrast, simultaneous treatment of mouse HC-11 cells with CR-1 in the presence of DIP inhibited beta-casein expression. The inhibitory effects of CR-1 on beta-casein expression in response to DIP were not unique to this mouse mammary epithelial cell line, because beta-casein and whey acidic protein expression in primary mouse mammary explant cultures established from midpregnant mice were also differentially inhibited by several epidermal growth factor-related peptides including CR-1. The mitogenic and differentiation effects of CR-1 are mediated by the binding of CR-1 to a cell surface receptor that is known to activate the ras/raf/mitogen-activated protein kinase (MAPK)/MAPK kinase pathway. The inhibitory response of CR-1 in HC-11 cells on beta-casein expression after treatment with DIP can be attenuated by B581, a peptidomimetic farnesyltransferase inhibitor that blocks p21ras farnesylation and activation, and by the phosphatidylinositol 3'-kinase (PI3k) inhibitor LY 294002 but not by PD 98059, a MAPK kinase inhibitor that blocks MAPK activation. These data suggest that the ability of CR-1 to block lactogenic hormone-induced expression of beta-casein is mediated through a p21ras-dependent, PI3k-mediated pathway. This is further substantiated by the observation that CR-1 is able to stimulate the tyrosine phosphorylation of the p85 PI3k regulatory subunit and to increase the activity of PI3k in HC-11 cells.

Animals↗
PubMed1996

Enhanced expression of heregulin in c-erb B-2 and c-Ha-ras transformed mouse and human mammary epithelial cells.

Heregulin beta 1 was found to stimulate the anchorage-dependent, serum-free growth of nontransformed human MCF-10A mammary epithelial cells. Unlike epidermal growth factor, transforming growth factor alpha, or amphiregulin, heregulin beta 1 was also able to induce the anchorage-independent growth of MCF-10A cells. In contrast, the anchorage-independent, serum-free growth of c-Ha-ras or c-erb B-2 transformed MCF-10A cells was unaffected by heregulin beta 1, whereas heregulin beta 1 was able to stimulate the anchorage-independent growth of these cells. c-Ha-ras or c-erb B-2 (c-neu) transformed MCF-10A or mouse NOG-8 mammary epithelial cells express elevated levels of 2.5, 5.0, 6.5, 6.8, and 8.5 kb heregulin mRNA transcripts and/or synthesize cell-associated 25, 29, 50, and 115 kDa isoforms of heregulin. Since the MCF-10A cells and transformants also express c-erb B-3, these data suggest that endogenous heregulin might function as an autocrine growth factor for Ha-ras or erb B-2 transformed mammary epithelial cells.

Animals↗
PubMed1996

Differential expression of jackfruit-lectin-specific glycoconjugates in metastatic adenocarcinoma and reactive mesothelial cells-a diagnostic aid in effusion cytology.

Distinguishing reactive mesothelial cells from adenocarcinoma cells in serous effusions on the basis of morphological criteria alone is often difficult. Interest has therefore been focused on identifying reliable methods to supplement the conventional cytological techniques. Plant lectins have been reported as diagnostic markers for malignant cells. We studied 51 aspirated samples of benign and malignant effusions using horseradish-peroxidase-conjugated jackfruit lectin. No significant difference was observed between the cells of pleural and peritoneal fluids. The reactively proliferated mesothelial cells of benign effusions showed a predominance of mild staining while moderate and intense staining was predominant in malignant effusions. Intense and irregular lectin binding was observed in macrophages irrespective of the cause of effusion. The lectin staining method therefore appears to have some clinical significance as an additional diagnostic aid for use in effusion cytology.

Adenocarcinoma↗
PubMed1996

Lectin histochemistry of oral premalignant and malignant lesions: correlation of JFL and PNA binding pattern with tumour progression.

The expression of glycoconjugates specific to Jack fruit lectin (JFL) and peanut agglutinin (PNA) in various clinicopathological stages of tumour progression in the oral mucosa were studied. These included various clinical forms of dysplastic and non-dysplastic oral leucoplakias, carcinomas, normal keratinising (gingiva) and non-keratinising (buccal mucosa) epithelia. It was seen that the binding patterns of PNA and JFL in the epithelial cells of various types of oral lesions were more or less similar. Normal non-keratinising epithelium showed mild membrane staining only in the spinal layers, while normal keratinising epithelium showed a moderate membrane staining and mild cytoplasmic staining in all layers. Moderate membrane and mild cytoplasmic staining were observed in leucoplakias, irrespective of various clinical or histological types. In carcinomas, the intensity of lectin binding was high, particularly in the membrane of differentiated cells. Correlation analysis of the binding pattern of PNA and JFL showed significant correlation in the membrane and cytoplasm of all layers with histological stages of tumour progression. The present study thus showed that PNA and JFL may be used as cytochemical probes in differentiating malignancy from benign lesions of the oral mucosa.

Carcinoma, Squamous Cell↗
PubMed1996

Ultrastructural analysis of the adjacent epithelium of oral squamous cell carcinoma.

Fifteen biopsies of the immediate adjacent epithelium of oral squamous cell carcinoma were examined under light and electron microscopy. Light microscopic examination of one micron thick sections revealed that the majority of lesions (67%) had hyperplastic or mildly dysplastic epithelium while the remaining (33%) had moderate to severe dysplasia. Ultrastructural observations showed that all these lesions had subcellular alterations similar to those seen in frank malignant oral tissue, particularly in the lower half of the epithelium. Important ultrastructural changes observed included bizarre nuclei of basal and lower spinal cells, enlarged and multiple nucleoli, presence of interchromatin and perichromatin granules, loss of desmosomes and marked spongiosis as well as disturbed cellular maturation sequences in the keratinocytes evidenced by abnormal and irregular distribution of maturation markers such as keratohyalin granules and tonofilaments. The present study thus shows the value of electron microscopy in the detection of malignant changes in the adjacent epithelium of oral squamous cell carcinoma.

Carcinoma, Squamous Cell↗
PubMed1996

Expression of p53 in leukoplakia and squamous cell carcinoma of the oral mucosa: correlation with expression of Ki67.

Aim-To study p53 expression in relation to proliferative status in normal and nondysplastic, dysplastic and malignant lesions of the oral mucosa.Method-The standard avidin-biotin complex (ABC) immunohistochemical staining method was used to study the expression of p53 and Ki67 on frozen sections of oral leukoplakias and carcinomas.Results-Of the leukoplakia and carcinoma samples, 70% expressed p53 in over 5% of cells. In normal mucosa less than 5% of cells expressed p53. The proliferation index, as assessed by expression of Ki67, was highest in the malignant lesions (43%) and lowest in normal mucosa (11%). Statistical analysis revealed that expression of both p53 and Ki67 was correlated significantly with the histopathological stage of the tumour. However, expression of p53 was not correlated with that of Ki67. In leukoplakia lesions with proliferative features p53 immunostaining was less intense than in non-proliferative lesions; this difference was statistically significant.Conclusions-These results emphasise the potential of Ki67 and p53 as biomarkers of carcinogenesis in oral cancer and may also serve as intermediate points for cancer prevention programmes, such as the oral chemopreventive trials. Factors other than p53 may have a more important role in the deregulation of proliferation in pre-malignant oral lesions.

Journal Article↗
PubMed1995

Anaesthetic management of a child with Bartter's syndrome.

We report the anaesthetic management of an eight-year-old asthmatic boy with Bartter's syndrome who had bilateral orchidopexy with caudal epidural analgesia. Bartter's syndrome is a rare congenital disorder characterized by hypokalaemic hypochloraemic metabolic alkalosis, hyperaldosteronism, hyperreninaemia and hyperplasia of the juxtaglomerular apparatus of the kidneys. Characteristically, although these patients are normotensive they may be hypovolaemic. They may have unstable baroreceptor responses and show marked resistance to vasopressors. Hence, fluid, acid-base and electrolyte imbalances along with haemodynamic instability pose particular problems in their anaesthetic management. Previous case reports have described the management of these patients with general anaesthesia, our patient had his orchidopexy with caudal epidural analgesia using plain bupivacaine 0.5%. The patient was haemodynamically stable throughout surgery and was comfortable with caudal analgesia as the sole anaesthetic. Hypovalaemia, acid-base status and electrolyte imbalance were treated before instituting caudal epidural analgesia. We present this case report which describes the anaesthetic considerations in the light of the pathophysiology of Bartter's syndrome.

Anesthesia↗
PubMed1995

Hen's teeth and whale's feet: generalized characters and their compatibility.

We propose a new model of computation for deriving phylogenetic trees based upon a generalization of qualitative characters. The model we propose is based upon recent experimental research in molecular biology. We show that the general case of determining perfect compatibility of generalized ordered characters is an NP-complete problem, but can be solved in polynomial time for a special case.

Algorithms↗
PubMed1995

Implications of gynaecological abnormalities in pre-selection criteria for cervical screening: preliminary evaluation of 3602 subjects in south India.

Early detection and eradication of cervical cancer and its precursor lesions through organized mass cytological screening programmes have recently gained considerable attention in developing countries. Strategies for both cost saving and effective implementation are however required for mass cervical screening in developing countries. In an early cancer detection programme conducted in South India, we analysed cytological abnormalities in 3602 women and correlated the results with other factors, including age, gynaecological complaints, number of years of married life and parity to see if pre-selection for cytologic screening was possible. Only lower grades of dysplasia were found in asymptomatic women below the age of 40 years. In asymptomatic women, malignancy and higher grades of dysplasia were confined to women with a clinically abnormal cervix only. Univariate analysis also revealed that subjects with a parity of more than 3 and a married life of more than 20 years had a significantly higher number of cytological abnormalities. However, on a multivariate analysis the increased number of marital years was not found to be an independent variable. These results suggest that asymptomatic women below the age of 40 years with a married life of less than 20 years and parity below 3, may be excluded from screening campaigns, and that pre-selection for cytologic screening is possible by introducing a programme of clinical and speculum examination of the cervix.

Adult↗
PubMed1994

Expression of galactoside-specific endogenous lectins and their ligands in human oral squamous cell carcinoma.

Human endogenous lectins have a wide spectrum of biological functions. The present study analyses the expression of beta-galactoside specific and N-acetyl-D-galactosamine specific endogenous lectins in oral squamous cell carcinomas using biotinylated neoglycoproteins. The expression pattern of beta-galactosyl-containing glycoconjugates or ligands of beta-galactoside specific lectins in these tissues was also studied using an endogenous biotinylated lectin, the human 14-kDa lectin. For comparison a galactoside specific plant lectin from mistletoe, Viscum album was also employed. The results demonstrate that oral squamous cell carcinomas mainly express accessible binding sites for lactosylated neoglycoprotein (90%) while few carcinomas expressed mild amount of N-acetyl-D-galactosamine specific binding sites (40%). There was no difference in the binding patterns of these probes between well and less differentiated carcinomas. Expression of these neoglycoprotein binding sites were mostly concentrated in immature basaloid cells, indicating a possible association with cell proliferation. The binding pattern of D-galactosyl specific lectins (human 14-kDa and mistletoe lectins) showed conspicuous differences. This feature emphasizes the caution that needs to be exercised in interpreting the biological significance of results attained using plant lectins on human tissue.

Acetylgalactosamine↗