Stability and degradation of fibroblast growth factor 23 (FGF23): the effect of time and temperature and assay type.

UNLABELLED: There is growing need for a reliable assay for measuring fibroblast growth factor 23 (FGF23), a regulator of phosphorus and vitamin D. In this work, we analyze and compare the performance of three available assays, including the effect of temperature and time. This knowledge will allow for better understanding of FGF23 in the future. INTRODUCTION: Intact and C-terminal FGF23 (iFGF23 and cFGF23) concentrations are important in the diagnosis of hypo- and hyperphosphatemic diseases. The effects of temperature, storage, and specimen handling on FGF23 levels are not well known. We investigated the effects of various factors on plasma and serum measurement of FGF23 using three different assays. METHODS: Serum and plasma FGF23 were measured using three commercially available ELISA assays-two measuring iFGF23 and one measuring cFGF23. Samples from subjects with known FGF23 disorders were stored at 4, 22, and 37 °C and analyzed at different intervals up to 48 hours (h). A subset of samples underwent repeated freeze-thaw cycles, and samples frozen at -80 °C for up to 60 months were reanalyzed. The effect of adding a furin convertase inhibitor on FGF23 degradation was investigated using samples stored at 37 °C for 48 h. Intact FGF23 levels were measured from plasma samples of four different groups to test the correlation of the two assays. RESULTS: Plasma FGF23 levels were stable when stored at 4 and 22 °C for 48 h. Both plasma and serum FGF23 levels demonstrated relative stability after five freeze-thaw cycles. Long-term storage at -80 °C for 40 months induced some variability in FGF23 levels. The addition of a furin inhibitor did not affect FGF23 degradation. Intact FGF23 levels showed good correlation only at the upper limit of the assay range when comparing the two assays. CONCLUSIONS: Sample type, handling, and choice of assay are factors that affect FGF23 levels and should be considered when measuring this hormone.

Transcriptional regulation of microRNA-100, -146a, and -150 genes by p53 and NFκB p65/RelA in mouse striatal STHdh(Q7)/ Hdh(Q7) cells and human cervical carcinoma HeLa cells.

MicroRNA (miRNA) genes generally share many features common to those of protein coding genes. Various transcription factors (TFs) and co-regulators are also known to regulate miRNA genes. Here we identify novel p53 and NFκB p65/RelA responsive miRNAs and demonstrate that these 2 TFs bind to the regulatory sequences of miR-100, -146a and -150 in both mouse striatal and human cervical carcinoma cells and regulate their expression. p53 represses the miRNAs while NFκB p65/RelA induces them. Further, we provide evidence that exogenous p53 inhibits NFκB p65/RelA activity by reducing its nuclear content and competing with it for CBP binding. This suggests for the existence of a functional cross-talk between the 2 TFs in regulating miRNA expression. Moreover, promoter occupancy assay reveals that exogenous p53 excludes NFκB p65/RelA from its binding site in the upstream sequence of miR-100 gene thereby causing its repression. Thus, our work identifies novel p53 and NFκB p65/RelA responsive miRNAs in human and mouse and uncovers possible mechanisms of co-regulation of miR-100. It is to be mentioned here that cross-talks between p53 and NFκB p65/RelA have been observed to define the outcome of several biological processes and that the pro-apoptotic effect of p53 and the pro-survival functions of NFκB can be largely mediated via the biological roles of the miRNAs these TFs regulate. Our observation with cell lines thus provides an important platform upon which further work is to be done to establish the biological significance of such co-regulation of miRNAs by p53 and NFκB p65/RelA.

A pentaplex PCR assay for detection and characterization of Vibrio vulnificus and Vibrio parahaemolyticus isolates.

Vibrio parahaemolyticus and Vibrio vulnificus are the leading causes of seafood-related illnesses and also can cause wound infections. These bacteria often co-exist in marine and estuarine environments. However, there have been no reported protocols that can detect and characterize (i.e. pathogenic or nonpathogenic) them in a single PCR. In this study, we developed a pPCR assay with a combination of two species-specific and three pathogenic-specific PCR primers to simultaneously detect virulent (viuB in V. vulnificus and tdh/trh in V. parahaemolyticus) and nonvirulent (vvhA in V. vulnificus and tlh in V. parahaemolyticus) markers of the two species in bacterial isolates. The assay was validated by three methods. First, the pPCR was used to characterize 300 bacterial isolates consisting of seven reference strains and 293 environmental strains isolated from the Gulf of Mexico water. Results were compared with characterizations based on single-gene PCR amplifications and previously published multiplex PCR protocols. Second, 51 isolates characterized with the pPCR were analysed by 16S rRNA sequencing to confirm any false-negative/positive reaction. Finally, the effectiveness of the assay for heterogeneous bacterial samples was validated. The pPCR correctly characterized isolates from the Gulf with an efficiency of 96·6-98·7%.

Transcription regulation of caspase-1 by R393 of HIPPI and its molecular partner HIP-1.

Earlier we have shown that exogenous expression of HIPPI, a molecular partner of Huntingtin interacting protein HIP-1, induces apoptosis and increases expression of caspases-1, -8 and -10 in HeLa and Neuro2A cells. The C-terminal pseudo death effector domain of HIPPI (pDED-HIPPI) specifically interacts with the putative promoter sequences of these genes. In the present manuscript, we predict from structural modeling of pDED-HIPPI that R393 of HIPPI is important for such interaction. R393E mutation in pDED-HIPPI decreases the interaction with the putative promoter of caspase-1 in cells. Expression of caspase-1 is decreased in cells expressing mutant pDED-HIPPI in comparison to that observed in cells expressing wild type pDED-HIPPI. Using HIP-1 knocked down cells as well as over expressing HIP-1 with mutation at its nuclear localization signal and other deletion mutations, we demonstrate that translocation of HIPPI to the nucleus is mediated by HIP-1 for the increased expression of caspase-1. HIPPI-HIP-1 heterodimer is detected in cytoplasm as well as in the nucleus and is associated with transcription complex in cells. Taking together, we are able to show the importance of R393 of HIPPI and the role of HIPPI-HIP-1 heterodimer in the transcription regulation of caspase-1.

Assessment of availability, ecological feature, and habitat preference of the medicinal herb Houttuynia cordata Thunb in the Brahmaputra Valley of Assam, India.

Houttuynia cordata Thunb. is a rhizome-bearing aromatic medicinal herb and is restricted to specialized moist habitats. The plant is collected from natural habitats for local consumption and trade. The status of the species and its variations in physiological performance in different habitats were studied in selected sites of geographically different areas of Brahmaputra valley in eastern India. The surveys were conducted in two different growth stages of the plant during 2005-2007. The sites where the species was encountered were marked and a distribution map was prepared. The frequency and density of the plant was higher in the moist habitats with higher organic carbon (0.85+/-0.05%). Generally, the density, biomass production and growth had significant (P<0.05) positive relationship with the soil physicochemical properties (linear curve fit). Soil moisture was the most dependent factor for the plant growth and the optimum growth was recorded at 78+/-5.6% (r2=0.9; P

Characterization of drug resistance and genetic diversity of Plasmodium falciparum parasites from Tripura, Northeast India.

Monitoring of anti-malarial drug resistance is vital in Northeast India as this region shares its international border with Southeast Asia. Genetic diversity of Plasmodium parasites regulates transmission dynamics, disease severity and vaccine efficacy. P. falciparum chloroquine resistance transporter (Pfcrt), multidrug resistance-1 (Pfmdr-1) and kelch 13 propeller (PfK-13) genes which govern antimalarial drug resistance and three genetic diversity markers, merozoite surface protein 1 and 2 (Pfmsp-1, Pfmsp-2) and glutamate rich protein (Pfglurp) were evaluated from Tripura, Northeast India using molecular tools. In the Pfcrt gene, 87% isolates showed triple mutations at codons M74I, N75E and K76T. 12.5% isolates in Pfmdr-1 gene showed mutation at N86Y. No polymorphism in PfK-13 propeller was found. Polyclonal infections were observed in 53.85% isolates and more commonly in adults (p = 0.0494). In the Pfmsp-1 locus, the K1 allelic family was predominant (71.2%) followed by the 3D7/IC family (69.2%) in the Pfmsp-2 locus. RII region of Pfglurp exhibited nine alleles with expected heterozygosity of 0.85. The multiplicity of infection for Pfmsp-1, Pfmsp-2 and Pfglurp were 1.56, 1.31 and 1.06 respectively. Overall, the study demonstrated a high level of chloroquine resistance and extensive parasite diversity in the region, necessitating regular surveillance in this population group.

An N-terminal fragment of insulin-like growth factor binding protein-3 (IGFBP-3) induces apoptosis in human prostate cancer cells in an IGF-independent manner.

OBJECTIVE: IGF-binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by direct, IGF-independent mechanisms that are poorly understood. IGFBP-3 undergoes limited proteolysis by plasmin and other proteases to generate small N-terminal fragments (e.g., amino acids 1-97) that have lost their affinity for IGF-I and IGF-II yet still can inhibit mitogenesis. The present study examines whether the N-terminal 1-97-IGFBP-3 fragment can induce apoptosis in human prostate cancer cells in an IGF-independent manner. DESIGN: N-terminal 1-97-IGFBP-3 with or without a signal prepeptide was fused to yellow fluorescent protein (YFP) and expressed in PC-3 human prostate cancer cells. In some cases, the N-terminal IGF-binding site was mutated. Subcellular localization was determined by confocal microscopy. Loss of cell viability was determined by Annexin V-APC staining in the presence and absence of a general caspase inhibitor, z-VAD-fmk. RESULTS: All of the fusion proteins, including those synthesized with a signal peptide, were predominantly intracellular, suggesting that they had been internalized following secretion. YFP-1-97-IGFBP-3 is present at comparable concentrations in the nucleus and cytoplasm, indicating that it does not contain a nuclear localization signal. Cells transfected with YFP-1-97-IGFBP-3 lost viability. Cell death was blocked by incubation with a caspase inhibitor suggesting that it resulted from apoptosis. Similar results were obtained with YFP-1-97-IGFBP-3 mutants that do not bind IGFs. CONCLUSIONS: The N-terminal 1-97-IGFBP-3 fragment induces apoptosis in human prostate cancer cells in an IGF-independent manner. Generation of the fragment might contribute to the proapoptotic activity of IGFBP-3 in vivo.

A study of proliferative activity, angiogenesis and nuclear grading in renal cell carcinoma.

To evaluate the role of proliferative marker, proliferating cell nuclear antigen (PCNA) and microvessel density (MVD) as prognostic markers in renal cell carcinoma (RCC) and to see their relationship with the clinical stage and nuclear grades, we studied 30 cases of RCC for nuclear grading (Fuhrman's nuclear grade), MVD (using anti CD-34 antibody), and PCNA labeling index (using anti-PCNA antibody) over a period of 2.5 years. Staging was assessed by peroperative and radiologic findings. The area of highest MVD within the tumor was selected for microvessel count (MVC) per high-power field (0.1885 mm 2 area). PCNA labeling index was determined by counting percentage of positively stained tumor cell nuclei. PCNA labeling index above 60% was taken as high PCNA index and up to 60% was considered low. There was significant positive correlation between PCNA labeling index with both nuclear grade and clinical stage using Spearman's correlation coefficient. No association was noted between MVC with PCNA, nuclear grade, and clinical stages. Evaluation of proliferative status of RCC is a useful adjunct as a prognostic parameter as it is seen to correlate well with both clinical stage and nuclear grade. In our study, MVD was not seen to correlate with either of these.

Zoledronic acid increases the prevalence of medication-related osteonecrosis of the jaw in a dose dependent manner in rice rats (Oryzomys palustris) with localized periodontitis.

OBJECTIVE: Investigate role of dose/duration of zoledronic acid (ZOL), a powerful anti-resorptive (pAR), on prevalence of medication-related osteonecrosis of the jaw (MRONJ) in rice rats (Oryzomys palustris), a species with natural susceptibility to food impaction-induced localized periodontitis (FILP). We hypothesize that ZOL induces MRONJ lesions in rice rats with FILP, and that the prevalence of MRONJ rises with increasing dose and duration of ZOL treatment. METHODS: We performed a toxicology experiment with clinically-relevant doses of ZOL in female rats (N=230) fed standard (STD) rodent chow. At age 4weeks (baseline), 12 rats were necropsied. The rest were randomized into five groups that began to receive 0, 8, 20, 50 or 125µg/kg ZOL IV/q 4weeks. After 12, 18, 24 and 30weeks, subgroups (N=9-16) from each of the dose groups were necropsied. High-resolution macroscopic photos of all jaw quadrants were given a gross quadrant grade (GQG) (0-4 or MRONJ) that classified FILP lesion severity and determined presence of gross MRONJ. Quadrants with GQG≥1 were examined histopathologically. Logistic regression analysis (ZOL dose/duration) of MRONJ prevalence was completed. RESULTS: We found: 1) 75% of 0µg/kg ZOL rats developed FILP lesions; 2) baseline rats and rats treated with 0µg/kg ZOL had no MRONJ; 3) 29 gross MRONJ cases were identified; 4) all gross MRONJ cases were confirmed histopathologically by the observation of exposed necrotic bone, and 53 new cases were discovered (total=82); 5) ZOL dose (P<0.001), but not duration (P=0.326), was a significant predictor of MRONJ prevalence; 6) 13% prevalence of gross MRONJ among all rats, with 22% prevalence among rats exposed to ZOL oncologic doses (20-125µg/kg); 7) 38% prevalence of histopathologic MRONJ among all rats, with 73% prevalence among rats exposed to ZOL oncologic doses. CONCLUSIONS: This is the first experiment to show a dose response relationship between clinically relevant doses of ZOL and MRONJ prevalence.

Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans.

Leukocytes are coated with a layer of heterogeneous carbohydrates (glycans) that modulate immune function, in part by governing specific interactions with glycan-binding proteins (lectins). Although nearly all membrane proteins bear glycans, the identity and function of most of these sugars on leukocytes remain unexplored. Here, we characterize the N-glycan repertoire (N-glycome) of human tonsillar B cells. We observe that naive and memory B cells express an N-glycan repertoire conferring strong binding to the immunoregulatory lectin galectin-9 (Gal-9). Germinal center B cells, by contrast, show sharply diminished binding to Gal-9 due to upregulation of I-branched N-glycans, catalyzed by the ß1,6-N-acetylglucosaminyltransferase GCNT2. Functionally, we find that Gal-9 is autologously produced by naive B cells, binds CD45, suppresses calcium signaling via a Lyn-CD22-SHP-1 dependent mechanism, and blunts B cell activation. Thus, our findings suggest Gal-9 intrinsically regulates B cell activation and may differentially modulate BCR signaling at steady state and within germinal centers.

Interactions of HIPPI, a molecular partner of Huntingtin interacting protein HIP1, with the specific motif present at the putative promoter sequence of the caspase-1, caspase-8 and caspase-10 genes.

To investigate the mechanism of increased expression of caspase-1 caused by exogenous Hippi, observed earlier in HeLa and Neuro2A cells, in this work we identified a specific motif AAAGACATG (- 101 to - 93) at the caspase-1 gene upstream sequence where HIPPI could bind. Various mutations in this specific sequence compromised the interaction, showing the specificity of the interactions. In the luciferase reporter assay, when the reporter gene was driven by caspase-1 gene upstream sequences (- 151 to - 92) with the mutation G to T at position - 98, luciferase activity was decreased significantly in green fluorescent protein-Hippi-expressing HeLa cells in comparison to that obtained with the wild-type caspase-1 gene 60 bp upstream sequence, indicating the biological significance of such binding. It was observed that the C-terminal 'pseudo' death effector domain of HIPPI interacted with the 60 bp (- 151 to - 92) upstream sequence of the caspase-1 gene containing the motif. We further observed that expression of caspase-8 and caspase-10 was increased in green fluorescent protein-Hippi-expressing HeLa cells. In addition, HIPPI interacted in vitro with putative promoter sequences of these genes, containing a similar motif. In summary, we identified a novel function of HIPPI; it binds to specific upstream sequences of the caspase-1, caspase-8 and caspase-10 genes and alters the expression of the genes. This result showed the motif-specific interaction of HIPPI with DNA, and indicates that it could act as transcription regulator.

Loss of heterozygosity and base substitution at the APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines.

We determined the nature of mutations occurring at the autosomal APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines. The analysis of mutations that result in APRT deficiency in a mismatch-repair-deficient strain of DLD-1 heterozygous for this locus enabled us to measure the rate of loss of the wild-type gene through deletion, recombination, or gene conversion as well as the rate of point mutation. The overall rate of mutation at the APRT locus in DLD-1 was elevated 100-fold compared with the mismatch-repair-proficient colorectal carcinoma cell line SW620. Loss of heterozygosity (LOH) at APRT accounted for only 4 to 9% of mutant strains derived from DLD-1, indicating a rate for these types of events of 4 x 10(-7) to 9 x 10(-7). In SW620 the rate of LOH at APRT was about 10-fold higher. LOH was not found at polymorphic markers within the same chromosome subband as APRT, indicating that only a limited portion of the chromosome was affected by these alterations. Chromosome painting of SWS620 mutants revealed that the loss of APRT occurred together with a substantial portion of the long arm of chromosome 16. Differences in the nature of base substitutions at APRT (e.g., the proportion of mutations resulting from transitions or transversions) in these tumor cell lines were also detected. There was also an important similarity---the presence of a mutant APRT gene with multiple base substitutions that may be the result of some sort of error-prone DNA synthesis.

Effect of nucleotide excision repair in human cells on intrachromosomal homologous recombination induced by UV and 1-nitrosopyrene.

To study the role of nucleotide excision repair in the induction of intrachromosomal homologous recombination in mammalian cells, we introduced a plasmid containing a substrate for recombination into three human cell lines that differ in their repair capacity and compared the frequency of recombination induced by UV radiation and by 1-nitrosopyrene. One strain had a normal capacity for nucleotide excision repair, the second exhibited an intermediate rate of repair, and the third, derived from a patient with xeroderma pigmentosum, had no ability to repair UV- or 1-nitrosopyrene-induced DNA damage. The endogenous thymidine kinase genes in these cell strains had been inactivated, and the cells contained an integrated copy of a plasmid carrying duplicated copies of the herpes simplex virus type 1 thymidine kinase (Htk) gene, each inactivated by an 8-base-pair XhoI site inserted at a unique site. A functional tk gene can only be generated by a productive recombination event between the two Htk genes. In all three stains, UV and 1-nitrosopyrene induced dose-dependent increases in the frequency of recombinants. However, the doses required to cause a specific increase in recombination in the repair-deficient strains were 10 to 30 times lower than the dose required for the cell strain with a normal capacity for repair. These results strongly suggest that unexcised DNA lesions, rather than excision repair per se, stimulate intrachromosomal homologous recombination. Southern blot analysis of DNA from representative recombinants indicated that in all cases one of the two Htk genes had become wild type (XhoI resistant). The majority (90%) retained the Htk duplication, consistent with nonreciprocal transfer of genetic information (gene conversion).

Retinoid-induced chromatin structure alterations in the retinoic acid receptor beta2 promoter.

Transcription of the retinoic acid receptor beta2 (RARbeta2) gene is induced by retinoic acid (RA) in mouse P19 embryonal carcinoma (EC) cells. Here we studied RA-induced chromatin structure alterations in the endogenous RARbeta2 promoter and in an integrated, multicopy RARbeta2 promoter in EC cells. RA markedly increased restriction site accessibility within the promoter, including a site near the RA responsive element (RARE) to which the nuclear receptor retinoid X receptor (RXR)-RAR heterodimer binds. These changes coincided with RA-induced alterations in the DNase I hypersensitivity pattern in and around the promoter. These changes became undetectable upon removal of RA, which coincided with the extinction of transcription. Analyses with receptor-selective ligands and an antagonist showed that increase in restriction site accessibility correlates with transcriptional activation, which parallels the RA-induced in vivo footprint of the promoter. Despite these changes, the micrococcal nuclease digestion profile of this promoter was not altered by RA. These results indicate that concurrent with the binding of the RXR-RAR heterodimer to the RARE, the local chromatin structure undergoes dynamic, reversible changes in and around the promoter without globally affecting the nucleosomal organization.

Retinoid X receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand and enhances retinoid-dependent gene expression.

Retinoic acid receptor (RAR) and retinoid X receptor (RXR) form heterodimers and regulate retinoid-mediated gene expression. We studied binding of RXR- and RAR-selective ligands to the RXR-RAR heterodimer and subsequent transcription. In limited proteolysis analyses, both RXR and RAR in the heterodimer bound their respective ligands and underwent a conformational change in the presence of a retinoic acid-responsive element. In reporter analyses, the RAR ligand (but not the RXR ligand), when added singly, activated transcription, but coaddition of the two ligands led to synergistic activation of transcription. This activation required the AF-2 domain of both RXR and RAR. Genomic footprinting analysis was performed with P19 embryonal carcinoma cells, in which transcription of the RARbeta gene is induced upon retinoid addition. Paralleling the reporter activation data, only the RAR ligand induced in vivo occupancy of the RARbeta2 promoter when added singly. However, at suboptimal concentrations of RAR ligand, coaddition of the RXR ligand increased the stability of promoter occupancy. Thus, liganded RXR and RAR both participate in transcription. Finally, when these ligands were tested for teratogenic effects on zebra fish and Xenopus embryos, we found that coadministration of the RXR and RAR ligands caused more severe abnormalities in these embryos than either ligand alone, providing biological support for the synergistic action of the two ligands.

Inhibition of telomerase activity and induction of apoptosis by curcumin in K-562 cells.

Telomerase, a reverse transcriptase that maintains telomere length, is highly activated in tumor cells and practically absent in somatic cells and hence considered a potential marker for tumorigenesis. A connection between telomerase activity and resistance to apoptosis has been established. Telomerase, therefore, has been proposed to represent a novel and potentially selective target for cancer therapy. Several synthetic compounds have been developed in recent years with a view to inhibit telomerase activity with telomere shortening below a critical length resulting in apoptosis. Such compounds are always highly toxic. Many plant-derived products act through the induction of apoptosis as a mechanism to suppress carcinogenesis. Curcumin, a phenolic compound isolated from the rhizome of the plant Curcuma longa Linn., has been reported to possess anti-tumor, apoptotic and anti-angiogenic properties. Apoptosis has emerged as the major mechanism by which anti-tumor agents eliminate pre-neoplastic cells or cells progressed to malignancy. The present study was undertaken to examine the mechanism of curcumin-induced apoptosis in human leukemia cell line K-562 with particular emphasis on the role of curcumin on telomerase activity. Induction of apoptosis by curcumin is initiated by the release of cytochrome c from mitochondria into the cytosol, and evidenced by the increase in DNA content in the sub-G1 region as obtained from FACS analysis. Apoptosis is mediated by the activation of caspases 3 and 8, up-regulation of the apoptotic gene bax with concomitant down-regulation of the anti-apoptotic gene bcl-2. Using TRAP assay it has been observed that curcumin inhibits telomerase activity in a dose and time-dependent manner, the inhibition being due to suppression of translocation of telomerase reverse transcriptase (TERT), a catalytic subunit, from cytosol to nucleus. Most significantly, the inhibition of telomerase activity by curcumin correlates with several parameters of apoptosis. The results suggest that telomerase status plays an important role in the induction of apoptosis in K-562 cells by curcumin.

A rare case of diffuse neonatal hemangimatosis.

Diffuse neonatal hemangiomatosis (DNH) is a rare neonatal condition in which cutaneous and visceral hemangiomas coexist. If left untreated, DNH is usually fatal at an early age. We report a case of a 6-month-old male infant who was brought to our institution with hepatosplenomegaly and a history of anemia and thrombocytopenia since 1 month of age. Cytogenetic analysis and liver biopsy were normal and bone marrow aspirate was nondiagnostic. Congenital red blood cell abnormality was ruled out. Ultrasound confirmed an increase in size of the spleen from 5 to 15 cm, and magnetic resonance imaging demonstrated intense splenic enhancement consistent with a hemangioma or vascular malformation. Despite severe thrombocytopenia, an exploratory laparotomy was done and the patient underwent a splenectomy and omentectomy. The final pathology confirmed hemangiomatosis of the spleen and omentum. In the neonate with unexplained anemia and thrombocytopenia, DNH should be considered as part of the differential diagnosis. In our case, the patient not only exhibited no obvious cutaneous involvement, but also had rare splenic involvement. Although there are risks involved when operating on a thrombocytopenic patient, the benefits of operating on a patient with DNH far outweigh the risks, and operative intervention should proceed without delay.

Response of colon cancer cell lines to the introduction of APC, a colon-specific tumor suppressor gene.

The APC gene, mutations in which are responsible for the inherited colon cancer syndrome adenomatous polyposis coli (APC), is described as a tumor suppressor gene. A full-length, wild-type APC gene was introduced by transfection into three human colon carcinoma cell lines, each characterized for mutations at loci involved in colon tumor formation. The response of each cell line to the introduction of APC differed with the genotype of the cell line. Some of the cell clones derived from these transfections displayed altered morphologies; some showed suppression of tumorigenicity based on growth in soft agar and tumor formation in nude mice. One cell line, SW480, could not be stably transfected with the APC gene. These results provide the first direct evidence that the APC gene can alter the transformation properties of colon carcinoma cells.

Molecular anatomy of CTG expansion in myotonin protein kinase gene among myotonic dystrophy patients from eastern India.

We have studied the CTG repeat sizes in the DMPK gene and six biallelic markers which are in complete linkage disequlibrium with Caucasian DM patients, to identify any common founder haplotype in 30 clinically diagnosed unrelated DM patients from eastern India. Our results revealed that in 27 patients (90%), CTG expansion took place on a DraIII(-) - HhaI(-) - Alu(+) - HinfI(+) - Fnu4H I(-) - TaqI(+) haplotype (haplotype I), similar to what have been published for Caucasoid and other DM patients. However, in three patients (10%), the expansion of CTG repeat was on DraIII(+) - HhaI(+) - Alu(+) - HinfI(-) - Fnu4H I(+) - TaqI(-) background (haplotype II), indicating a new haplotype. The distribution of haplotypes in 52 normal individuals of eastern India revealed that percentage of haplotypes I and II were 23.1% and 7.7% respectively in normal chromosomes. Haplotype II is absent among Caucasian DM patients as well as normal individuals indicating that this particular haplotype may be characteristic of the Indian population. Hum Mutat 16:372, 2000.

Analysis of CAG and CCG repeats in Huntingtin gene among HD patients and normal populations of India.

We have analysed the distribution of CAG and adjacent polymorphic CCG repeats in the Huntingtin gene in 28 clinically diagnosed unrelated Huntington's disease (HD) patients and in normal individuals belonging to different ethnic groups of India. The range of expanded CAG repeats in HD patients varied from 41 to 56 repeats, whereas in normal individuals this number varied between 11 and 31 repeats. We identified six CCG alleles from a total of 380 normal chromosomes that were pooled across different ethnic populations of India. There were two predominant alleles: (CCG)7 (72.6%) and (CCG)10 (20%). We report here for the first time one four-repeat CCG allele which has not been found in any population so far. We found 30 haplotypes (two loci CAG-CCG) for 380 normal chromosomes. In the present study, no statistically significant preponderance of expanded HD alleles was found on either (CCG)7 or (CCG)10 backgrounds. Our studies suggest that the overall prevalence of HD in Indian populations may not be as high as in Western populations. Further studies are necessary to identify the origin of HD mutation in these populations.