Partial inactivation of Ankrd26 causes diabetes with enhanced insulin responsiveness of adipose tissue in mice.

AIMS/HYPOTHESIS: ANKRD26 is a newly described gene located at 10p12 in humans, a locus that has been identified with some forms of hereditary obesity. Previous studies have shown that partial inactivation of Ankrd26 in mice causes hyperphagia, obesity and gigantism. Hypothesising that Ankrd26 mutant (MT) mice could develop diabetes, we sought to establish whether the observed phenotype could be (1) solely related to the development of obesity or (2) caused by a direct action of ankyrin repeat domain 26 (ANKRD26) in peripheral tissues. METHODS: To test the hypothesis, we did a full metabolic characterisation of Ankrd26 MT mice that had free access to chow or were placed under two different energy-restricted dietary regimens. RESULTS: Highly obese Ankrd26 MT mice developed an unusual form of diabetes in which white adipose tissue is insulin-sensitive, while other tissues are insulin-resistant. When obese MT mice were placed on a food-restricted diet, their weight and glucose homeostasis returned to normal. In addition, when young MT mice were placed on a pair-feeding diet with normal mice, they maintained normal body weight, but showed better glucose tolerance than normal mice, an increased responsiveness of white adipose tissue to insulin and enhanced phosphorylation of the insulin receptor. CONCLUSIONS/INTERPRETATION: These findings show that the ANKRD26 protein has at least two functions in mice. One is to control the response of white adipose tissue to insulin; the other is to control appetite, which when Ankrd26 is mutated, leads to hyperphagia and diabetes in an obesity-dependent manner.

Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration.

The peritoneal mesothelium exhibits a high regenerative ability. Peritoneal regeneration is concomitant with the appearance, in the coelomic cavity, of a free-floating population of cells whose origin and functions are still under discussion. We have isolated and characterized this cell population and we have studied the process of mesothelial regeneration through flow cytometry and confocal microscopy in a murine model lethally irradiated and reconstituted with GFP-expressing bone marrow cells. In unoperated control mice, most free cells positive for mesothelin, a mesothelial marker, are green fluorescent protein (GFP). However, 24 hrs after peritoneal damage, free mesothelin(+)/GFP(+) cells appear in peritoneal lavages. Cultured lavage peritoneal cells show colocalization of GFP with mesothelial (mesothelin, cytokeratin) and fibroblastic markers. Immunohistochemical staining of the peritoneal wall also revealed colocalization of GFP with mesothelial markers and with procollagen-1 and smooth muscle α-actin. This was observed in the injured area as well as in the surrounding not-injured peritoneal surfaces. These cells, which we herein call peritoneal repairing cells (PRC), are very abundant 1 week after surgery covering both the damaged peritoneal wall and the surrounding uninjured area. However, they become very scarce 1 month later, when the mesothelium has completely healed. We suggest that PRC constitute a type of monocyte-derived cells, closely related with the tissue-repairing cells known as 'fibrocytes' and specifically involved in peritoneal reparation. Thus, our results constitute a synthesis of the different scenarios hitherto proposed about peritoneal regeneration, particularly recruitment of circulating progenitor cells and adhesion of free-floating coelomic cells.

Yogic exercises and health--a psycho-neuro immunological approach.

Relaxation potential of yogic exercises seems to play a vital role in establishing psycho-physical health in reversing the psycho-immunology of emotions under stress based on breath and body awareness. However, mechanism of yogic exercises for restoring health and fitness components operating through psycho-neuro-immunological pathways is unknown. Therefore, a hybrid model of human information processing-psycho-neuroendocrine (HIP-PNE) network has been proposed to reveal the importance of yogic information processing. This study focuses on two major pathways of information processing involving cortical and hypothalamo-pituitary-adrenal axis (HPA) interactions with a deep reach molecular action on cellular, neuro-humoral and immune system in reversing stress mediated diseases. Further, the proposed HIP-PNE model has ample of experimental potential for objective evaluation of yogic view of health and fitness.

Mesothelin is not required for normal mouse development or reproduction.

Mesothelin is a glycosylphosphatidylinositol-linked glycoprotein highly expressed in mesothelial cells, mesotheliomas, and ovarian cancer, but the biological function(s) of the protein is not known. We have analyzed the expression of the mouse mesothelin gene in different developmental stages and in various adult tissues by Northern hybridization. The 2.5-kb mesothelin transcript was detected in the mRNA of E 7.0, E 15.0, and E 17.0 stages of mouse development. In adult tissues the mesothelin gene was expressed in lung, heart, spleen, liver, kidney, and testis. To directly assess the function of the mesothelin in vivo, we generated mutant mice in which the mesothelin gene was inactivated by replacing it with the neomycin resistance gene. In homozygous mutant mice neither mesothelin mRNA nor the protein product was detected. Null mutant mice were obtained in accordance with Mendelian laws, and both males and females produced offspring normally. No anatomical or histological abnormalities were detected in any tissues where mesothelin was reportedly expressed in wild-type mice. Our results demonstrate that mesothelin function is not essential for growth or reproduction in mice.

Cse1l is essential for early embryonic growth and development.

The CSE1L gene, the human homologue of the yeast chromosome segregation gene CSE1, is a nuclear transport factor that plays a role in proliferation as well as in apoptosis. CSE1 and CSE1L are essential genes in Saccharomyces cerevisiae and mammalian cells, as shown by conditional yeast mutants and mammalian cell culture experiments with antisense-mediated depletion of CSE1L. To analyze whether CSE1L is also essential in vivo and whether its absence can be compensated for by other genes or mechanisms, we have cloned the murine CSE1L gene (Cse1l) and analyzed its tissue- and development-specific expression: Cse1l was detected at embryonic day 7.0 (E7.0), E11.0, E15.0, and E17.0, and in adults, high expression was observed in proliferating tissues. Subsequently, we inactivated the Cse1l gene in embryonic stem cells to generate heterozygous and homozygous knockout mice. Mice heterozygous for Cse1l appear normal and are fertile. However, no homozygous pups were born after interbreeding of heterozygous mice. In 30 heterozygote interbreeding experiments, 50 Cse1l wild-type mice and 100 heterozygotes were born but no animal with both Cse1l alleles deleted was born. Embryo analyses showed that homozygous mutant embryos were already disorganized and degenerated by E5.5. This implicates with high significance (P < 0.0001, Pearson chi-square test) an embryonically lethal phenotype of homozygous murine CSE1 deficiency and suggests that Cse1l plays a critical role in early embryonic development.

Pharmacokinetics and antitumor activity of a bivalent disulfide-stabilized Fv immunotoxin with improved antigen binding to erbB2.

We have generated a stable bivalent Fv molecule [(dsFv)2] of the anti-erbB2 monoclonal antibody e23 in which the V(H) and V(L) domains of the Fv are linked to each other by a disulfide bond and the two Fvs are connected by a 15-amino acid linker (T. K. Bera et al., J. Mol. Biol., 281: 475-483, 1998). The e23 (dsFv)2 molecule is linked to a truncated form of Pseudomonas exotoxin (PE38) to generate a bivalent disulfide-stabilized immunotoxin e23 (dsFv)2-PE38. Compared to the monovalent immunotoxin, the (dsFv)2 immunotoxin showed greatly increased cytotoxicity to four cancer cell lines expressing low levels of erbB2 but not to four other cell lines with high erbB2 expression. e23 (dsFv)2-PE38 was administered i.v. to mice, and its half-life was determined. The t(1/2)alpha and t(1/2)beta were 20 and 325 min, respectively, whereas the corresponding values for the monovalent dsFv immunotoxin were shorter, 6 and 52 min. The antitumor activities of the monovalent and bivalent immunotoxin were compared using mice bearing A431 tumors. Despite the fact that e23 (dsFv)2-PE38 was 13-fold more active than e23 dsFv-PE38 on A431 cells in cell culture, its antitumor activity in mice was <2-fold that of the monovalent immunotoxin. These data show that a large increase in avidity does not always lead to an increase in cytotoxic activity. Furthermore, in one of the cases in which cytotoxic activity in vitro was greatly enhanced, there was only a small increase in antitumor activity.

XAGE-1, a new gene that is frequently expressed in Ewing's sarcoma.

Our previous expressed sequence tag database analysis indicates that XAGE-1 is frequently found in Ewing's sarcoma and alveolar rhabdomyosarcoma (U. Brinkmann et al., Cancer Res., 59: 1445-1448, 1999). Using Northern blots and RNA dot blots, we have now found that XAGE-1 is highly expressed in normal testis, in seven of eight Ewing's cell lines, in four of nine Ewing's sarcoma patient samples, and in one of one alveolar rhabdomyosarcoma patient sample. The gene is located on the X chromosome. The full-length cDNA contains 611 bp and predicts a protein of Mr 16,300 with a potential transmembrane domain at the NH2 terminus. XAGE-1 shares homology with GAGE/PAGE proteins in the COOH-terminal end. These findings could be valuable for cancer diagnosis and cancer immunotherapy.

A bivalent disulfide-stabilized Fv with improved antigen binding to erbB2.

We have used protein engineering to generate a stable bivalent Fv molecule of the anti-erbB2 monoclonal antibody e23. The VH and VL domains of the Fv are linked to each other by a disulfide bond and the two Fvs are connected by a flexible 15 amino acid residue (Gly4-Ser)3 linker. The e23 (dsFv)2 molecule is fused to a truncated form of Pseudomonas exotoxin to generate a bivalent disulfide-stabilized, (dsFv)2, immunotoxin. The immunotoxin was expressed in Escherichia coli, refolded in vitro and purified to about 95% purity. Binding studies demonstrated that the (dsFv)2 molecule has a much higher affinity for erbB2 than a monovalent dsFv molecule and a similar binding affinity as the parental antibody e23. The (dsFv)2 immunotoxin was 5 to 20-fold more cytotoxic to two e23 antigen-positive cell lines than the monovalent dsFv immunotoxin. The bivalent dsFv molecule is very stable, retaining 94% of its activity after a 24 hours incubation in human serum at 37 degreesC. Two other molecules with shorter linkers five and ten amino acid residues in length were produced and showed similar activities as the molecule containing a 15 amino acid residue linker. The bivalency, stability and the relative ease of purification makes these e23 (dsFv)2 molecules valuable reagents for cancer immunotherapy and diagnosis.

Bivalent disulfide-stabilized fragment variable immunotoxin directed against mesotheliomas and ovarian cancer.

We have used protein engineering to generate a stable bivalent fragment variable (Fv) molecule from the antimesothelin antibody SS, in which the VH and VL domains of the Fv are linked to each other by a disulfide bond, and the two Fvs are connected by a flexible 15-amino acid (Gly4-Ser)3 linker. The SS (dsFv)2 molecule is fused to a M(r) 38,000 truncated form of Pseudomonas exotoxin to generate a bivalent, disulfide stabilized, (dsFv)2 immunotoxin. The immunotoxin was expressed in Escherichia coli, refolded in vitro, and purified to approximately 95% purity with a high yield of > 10%. Binding studies demonstrated that the (dsFv)2 molecule has 40 times higher apparent affinity for recombinant mesothelin than a monovalent dsFv molecule. The (dsFv)2 immunotoxin was 4-10-fold more cytotoxic to three mesothelin antigen-positive cell lines than the monovalent dsFv immunotoxin. However, when tested in mice bearing tumor cells expressing mesothelin, the antitumor activity of the bivalent immunotoxin is very similar to the activity of the lower affinity monovalent immunotoxin. Our data indicate that increasing affinity of an antibody fragment does not necessarily lead to higher antitumor activity of an immunotoxin in vivo.

ERGL, a novel gene related to ERGIC-53 that is highly expressed in normal and neoplastic prostate and several other tissues.

We have identified a new gene, that is highly expressed in normal and neoplastic prostate, and is also expressed in cardiac atrium, salivary gland, spleen and selective cells in the CNS. Database analyses of ESTs indicated prostate specificity but experimental results showed the expression in other tissues. The full length transcript is 1800 bp with an open reading frame of 526 aa. The amino-terminal 230 residues of the expressed protein has high homology to a family of lectins, especially to the sugar binding domain of ERGIC-53. We therefore designate the new gene ERGL (ERGIC-53-like). There is a transmembrane domain at amino acid positions 468-482 suggesting that the product of ERGL is a type-I membrane protein. In prostate there are two fully processed transcripts one of which is a splice variant with a deletion in the region of the transmembrane domain of the protein.

Expression of MAT1/PEA-15 mRNA isoforms during physiological and neoplastic changes in the mouse mammary gland.

MAT1 is a novel transforming gene which was cloned from a mouse mammary tumor induced by N-methyl-N-nitrosourea in vitro in the presence of lithium as a mitogen. Later, it was found to be identical to the 3' untranslated region (UTR) of the 2.5 kb isoform of PEA-15 (phosphoprotein enriched in astrocytes-15 kDa). We re-cloned MAT1/PEA-15 cDNAs and showed 2.5, 2.0 and 1.8 kb isoforms and confirmed MAT1 localization as reported. The 2.0 and 1.8 kb isoforms were produced by alternative splicing and alternative polyadenylation at the 3' UTR, respectively. To analyze the role of MAT1/PEA-15, we examined the expression of MAT1/PEA-15 mRNA in normal mammary tissues and in mammary tumors. The mammary gland during pregnancy, lactation and weaning showed weak but stable expression. Compared with normal mammary gland, mammary tumors showed stronger expression. Aberrant expression of MAT1/PEA-15 isoforms was found in mouse mammary epithelial cell lines, FSK7 and TM6, which lost the 2.5/2.0 and 2.5 kb isoforms, respectively. In contrast to other oncogenes like c-myc, MAT1/PEA-15 mRNA was extremely stable after actinomycin D and cycloheximide treatments suggesting that other protein expression is prerequisite for degradation of MAT1/PEA-15 mRNA. It evoked the possibility of the 3' UTR of MAT1/PEA-15 (designated as MAT1-T) as a riboregulator in mammary tumorigenesis and necessity for further analysis of human breast cancers as well as mouse mammary tumors.

Body composition, cardiovascular endurance and anaerobic power of yogic practitioner.

Forty male high school students, age 12-15 yrs, participated for a study of yoga in relation to body composition, cardiovascular endurance and anaerobic power. Ths Ss were placed into two subsets viz., yoga group and control group. Body composition, cardiovascular endurance anaerobic power were measured using standard method. The duration of experiment was one year. The result of ANCOVA revealed that a significant improvement in ideal body weight, body density, cardiovascular endurance and anaerobic power was observed as a result of yoga training. This study could not show significant change in body fat (midaxillary), skeletal diameters and most of the body circumferences. It was evident that some of the fat-folds (tricep, subscapular, suprailiac, umbilical, thigh and calf) and body circumferences (waist, umbilical and hip) were reduced significantly.

Effect of selected yogic practices on the management of hypertension.

On the basis of medical officers diagnosis, thirty three (N = 33) hypertensives, aged 35-65 years, from Govt. General Hospital, Pondicherry, were examined with four variables viz, systolic and diastolic blood pressure, pulse rate and body weight. The subjects were randomly assigned into three groups. The exp. group-I underwent selected yoga practices, exp. group-II received medical treatment by the physician of the said hospital and the control group did not participate in any of the treatment stimuli. Yoga imparted in the morning and in the evening with 1 hr/session. day-1 for a total period of 11-weeks. Medical treatment comprised drug intake every day for the whole experimental period. The result of pre-post test with ANCOVA revealed that both the treatment stimuli (i.e., yoga and drug) were effective in controlling the variables of hypertension.

Recovery from stress in two different postures and in Shavasana--a yogic relaxation posture.

The recovery from induced physiological stress in Shavasana (a yogic relaxation posture) and two other postures (resting in chair and resting supine posture) was compared. Twenty one males and 6 females (age 21-30 yrs) were allowed to take rest in one of the above postures immediately after completing the scheduled treadmill running. The recovery was assessed in terms of Heart Rate (HR) and Blood pressure (BP). HR and BP were measured before and every two minutes after the treadmill running till they returned to the initial level. The results revealed that the effects of stress was reversed in significantly (P < 0.01) shorter time in Shavasana, compared to the resting posture in chair and a supine posture.

CAPC negatively regulates NF-κB activation and suppresses tumor growth and metastasis.

CAPC, also known as LRRC26, is expressed in normal prostate and salivary gland. We developed a mAb to CAPC and used it to characterize the protein and study its function. CAPC protein was detected in normal prostate and salivary gland, in several human breast cancer cell lines and in the prostate cancer cell line LNCaP. Knockdown of CAPC by siRNA in LNCaP cells enhanced anchorage-independent growth in soft agar. Conversely, overexpression of CAPC in MDA-231 breast cancer cells and A431 epidermoid cancer cells inhibited growth in soft agar and tumorigenesis in nude mice, and suppressed the metastasis of MDA-231 cells to the lung. Overexpression of CAPC downregulated NF-κB activity and its target genes, including GM-CSF (CSF2), CXCL1, IL8 and LTB1. It also suppressed genes encoding the serine protease mesotrypsin (PRSS3) and cystatin SN (CST1). CAPC expressing tumors showed a decrease in the number of proliferating cells and a large increase in ECM. The role of CAPC in the suppression of tumor growth and metastasis may be through its alteration of the tumor microenvironment.

Effect of yogic exercise on super oxide dismutase levels in diabetics.

CONTEXT: Reactive oxygen species are known to aggravate disease progression. To counteract their harmful effects, the body produces various antioxidant enzymes, viz, superoxide dismutase, glutathione reductase etc. Literature reviews revealed that exercises help to enhance antioxidant enzyme systems; hence, yogic exercises may be useful to combat various diseases. AIMS: This study aims to record the efficacy of yoga on superoxide dismutase, glycosylated hemoglobin (Hb) and fasting blood glucose levels in diabetics. SETTINGS AND DESIGN: Forty diabetics aged 40-55 years were assigned to experimental (30) and control (10) groups. The experimental subjects underwent a Yoga program comprising of various Asanas (isometric type exercises) and Pranayamas (breathing exercises) along with regular anti-diabetic therapy whereas the control group received anti-diabetic therapy only. MATERIALS AND METHODS: Heparinized blood samples were used to determine erythrocyte superoxide dismutase (SOD) activity and glycosylated Hb levels and fasting blood specimens collected in fluoride Vacutainers were used for assessing blood glucose. STATISTICAL ANALYSIS USED: Data were analyzed by using 2 × 2 × 3 Factorial ANOVA followed by Scheffe's posthoc test. RESULTS: The results revealed that Yogic exercise enhanced the levels of Superoxide dismutase and reduced glycosylated Hb and glucose levels in the experimental group as compared to the control group. CONCLUSION: The findings conclude that Yogic exercises have enhanced the antioxidant defence mechanism in diabetics by reducing oxidative stress.

Comparison of recombinant immunotoxins against LeY antigen expressing tumor cells: influence of affinity, size, and stability.

Monoclonal antibody B3 (MAb B3) reacts with many epithelial cancers. It recognizes a carbohydrate antigen (Ley) which is expressed in a variety of solid tumors including breast and colon. We have used the Fab portion of MAb B3 and a portion of the constant domain of human IgG1 to make recombinant immunotoxins of different compositions. The toxin component employed is a truncated form of Pseudomonas exotoxin (PE38). The light chain or Fd of the antibody was cloned from hybridoma RNA and fused to PE38. Immunotoxin (IT) was then expressed in Escherichia coli as a fusion protein and refolded with either the Fd or the light chain. We have also made B3(Fab) immunotoxins of different sizes ranging 85-140 kDa, by introducing different portions of the constant domain of human IgG1 at the junction of Fd and PE38 fusion site. We compared the properties of the resulting immunotoxins with existing anti-Ley immunotoxins side by side. All recombinant Fab-immunotoxins made in this study were cytotoxic to antigen-positive cancer cell lines. However, in contrast to the B3(scFv) immunotoxin, the B3(Fab) immunotoxins are very stable, retaining 90% of their activity after 24 h of incubation in human serum albumin at 37 degreesC. A pharmacokinetics study with these immunotoxin molecules showed a longer survival in the circulation of mice compared to the smaller Fv immunotoxins. The smaller size of the Fab immunotoxins compared to B3Lys-PE38 and the increased T1/2 value compared to B3(scFv)-PE38 and B3(dsFv)-PE38 make these recombinant immunotoxins alternative therapeutic agents to treat Ley antigen positive cancers.

Cloning and characterization of mutL and mutS genes of Vibrio cholerae: nucleotide sequence of the mutL gene.

The mutL and mutS genes of Vibrio cholerae have been identified using interspecific complementation of Escherichia coli mutL and mutS mutants with plasmids containing the gene bank of V. cholerae. The recombinant plasmid pJT470, containing a 4.7 kb fragment of V. cholerae DNA codes for a protein of molecular weight 92,000. The product of this gene reduces the spontaneous mutation frequency of the E. coli mutS mutant. The plasmid, designated pJT250, containing a 2.5 kb DNA fragment of V. cholerae and coding for a protein of molecular weight 62,000, complements the mutL gene function of E. coli mutL mutants. These gene products are involved in the repair of mismatches in DNA. The complete nucleotide sequence of mutL gene of V. cholerae has been determined.

MRP8, a new member of ABC transporter superfamily, identified by EST database mining and gene prediction program, is highly expressed in breast cancer.

BACKGROUND: With the completion of the human draft genome sequence, efforts are now devoted to identifying new genes. We have developed a computer-based strategy that utilizes the EST database to identify new genes that could be targets for the immunotherapy of cancer or could be involved in the multistep process of cancer. MATERIALS AND METHODS: Utilizing our computer-based screening strategy, we identified a cluster of expressed sequence tags (ESTs) that are highly expressed in breast cancer. Northern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses demonstrated the tissue specificity of the computer-generated cluster and comparison with the human genome sequence assisted in isolating a full-length cDNA clone. RESULTS: We identified a new gene that is highly expressed in breast cancer. This gene is expressed at moderate levels in normal breast and testis and at very low levels in liver, brain, and placenta. The gene has two major transcripts of 4.5 kb and 4.1 kb. The 4.5-kb transcript is very abundant in breast cancer, and has an open reading frame of 1382 amino acids. The predicted protein sequence of the 4.5-kb transcript reveals that it has high homology with MRP5, a member of multidrug resistant-associated protein family (MRP). There are seven reported members in the MRP family; we designate this gene as MRP8 (ABCC11). The 4.5-kb MRP8 transcript consists of 31 exons and is located in a genomic region of over 80.4 kb on chromosome 16q12.1. The smaller 4.1-kb transcript of MRP8 is found in testis and may initiate within intron 6 of the gene. CONCLUSION: The selective expression of MRP8 (ABCC11), a new member of ATP-binding cassette transporter superfamily could be a molecular target for the treatment of breast cancer.