THE ROLE OF ADIPONECTIN AND TOLL-LIKE RECEPTOR 4 GENE POLYMORPHISMS ON NON-PROLIFERATIVE RETINOPATHY IN TYPE 2 DIABETES MELLITUS PATIENTS. A CASE-CONTROL STUDY IN ROMANIAN CAUCASIANS PATIENTS.

CONTEXT: Persistent inflammation and impaired neovascularization are important contributors to the development of diabetic retinopathy (DR). Gene polymorphisms of adiponectin (APN) were demonstrated to have an important role on the plasma level and activity of adiponectin. APN has anti-inflammatory, anti-diabetic and anti-atherogenic properties. Toll-Like Receptor 4 (TLR4) is a critical mediator of innate immunity. Polymorphisms in TLR-4 gene were shown to be associated with impaired inflammatory response in diabetes. OBJECTIVE: The aim of the study was to analyze the association of +276G>T variant of APN gene and Asp299Gly and Thr399Ile of TLR-4 gene variants in relationship with T2DM and DR in an Eastern European population group. DESIGN: The distribution of the mutant alleles in 198 T2DM patients with DR and 200 non-T2DM controls was examined. Genomic DNA from T2DM patients and healthy controls genotyped through the use of PCR-RFPL assay. RESULTS: Genotype and allele frequencies of the Asp299Gly and Thr399Ile polymorphisms differed between T2DM patients and non diabetic subjects (P<0.001). Moreover, the presence of the minor alleles of these polymorphisms were significantly identified as protective factors against T2DM, under a dominant model of Fisher's exact test (χ2=4.988, phi=0.745, OR=0.767, 95% CI=0.602-0.867, P<0.001; respectively χ2=5.254, phi=0.820, OR=0.487, 95% CI=0.211-0.648, P<0.001). Genotype analysis for the adiponectin 276G>T gene polymorphism yielded no significant association with T2DM, but revealed a borderline significance for the association with DR (χ2=5.632, phi=0.423, OR =1.101, 95% CI=0.887-1.203, P=0.009). CONCLUSIONS: We found an association between the TLR4 Asp299Gly and Thr399Ile polymorphisms and protection for DR. The APN genetic polymorphism is not associated with T2DM.

Multiple malignant tumors.

BACKGROUND: Due to the improvement in diagnosis and therapy for certain malignant tumors, we are now faced with patients who develop in time multiple malignancies. METHODS: We conducted a retrospective analysis of the patients diagnosed with at least two primary cancers that were admitted and treated in Cluj-Napoca Municipal Hospital. The study followed patients for a period of 7.5 years. RESULTS: We included in the present study 217 patients (4.33%) with two or more malignant primary tumors from 5003 cases diagnosed with a primary cancer. The most common sites for multiple malignant tumors were related to the breast, colorectum, urinary bladder, prostate and kidneys. CONCLUSIONS: We should always take into consideration the possibility of synchronous tumors and we have to keep in mind that a successful treatment of cancer might not prevent the onset of another primary mass.

GST gene variants in synchronous colorectal cancers and synchronous association of colorectal cancers with other cancers.

BACKGROUND: the present study evaluates genetic polymorphisms of three glutathione S-transferases (GSTM1, GSTT1and GSTP1) in patients with synchronous malignant colorectal tumors and the association of synchronous colorectal cancers with other cancers. MATERIAL AND METHODS: from 420 patients with a colorectal cancer admitted to our hospital between 2005-2012, we selected for genetic analysis 20 patients with multiple synchronous malignant colorectal tumors and 9 patients with asynchronous association of colorectal cancer with another cancer. We searched for GST genotypes, comparing the results with controls. RESULTS: the genetic analysis was possible only in 19 patients with colorectal synchronous cancers and 9 patients with asynchronous association of colorectal cancer with another cancer; we found a statistically significant difference for null GSTM1 genotype frequency between these patients and the control group; we found no differences regarding the frequency of null GSTT1 genotype and Ile105Val polymorphism of GSTP1 in patients with synchronous cancers compared with the control group. CONCLUSION: in our study we found the null GSTM1 genotype as a risk factor for multiple colorectal synchronous cancers and for an association of synchronous colorectal with other cancers

Rapid genotyping of mice with hemoglobinopathies and globin transgenes.

The hematology of the laboratory mouse has been well characterized. Normal genetic differences at the alpha- and beta-globin gene loci serve as useful markers for a wide variety of types of experimental studies. There are a number of naturally occurring or induced mutations that disrupt globin expression and produce thalassemic phenotypes. In addition, much has been learned of the workings of the globin locus control region from studies of transgenic mice, including those with mutations induced by targeted site-specific modifications. After a new mutation or transgene has been created, it must be maintained in living mice, and the genotypes of the offspring must be ascertained. While it is possible to determine genotypes by DNA analyses, such assays are time consuming and relatively expensive. An osmotic challenge test--originally developed for the genotyping of large-deletion alpha-thalassemia mutations in mice--has proven useful in detecting both severe and milder alpha- and beta-thalassemias, as well as some transgenic genotypes in mice carrying human globin genes. Reliable genotyping can, in some cases, be completed within a few minutes with minimal expense. Quantification of red cell fragility for a variety of thalassemic and transgenic mice is described here, along with a simplified test suitable for rapid, routine genotyping. The osmotic challenge test is perfectly reliable for distinguishing genotypes that cause significantly decreased release of hemoglobin from the red cells, but it is also useful for some of the conditions in which overall erythrocyte osmotic fragility is essentially normal.

Mutations in the murine fitness 1 gene result in defective hematopoiesis.

Identification and characterization of mutations that disrupt normal hematopoiesis are essential for understanding the genetic pathways that control the development and regulation of the mammalian hematopoietic system. Previously, the fitness 1 gene was identified by five, independent mutations in N-ethyl-N-nitrosourea (ENU) saturation mutagenesis experiments within the albino (c) region of mouse chromosome 7 (MMU7). We report here that fit1 mutants are anemic, display numerous peripheral blood defects, and are deficient in early hematopoietic progenitor cell populations. The number of both erythroid and myeloid progenitors, as well as B cells, are reduced. These results implicate fit1 involvement in normal hematopoiesis and suggest that further characterization of the fit1 gene, and the five presumed point mutations of the gene, will lead to an improved understanding of normal hematopoiesis in the mouse.

A transgenic mouse model of hemoglobin S Antilles disease.

Hemoglobin (Hb) S Antilles is a naturally occurring form of sickling human Hb but causes a more severe phenotype than Hb S. Two homozygous viable Hb S Antilles transgene insertions from Tg58Ru and Tg98Ru mice were bred into MHOAH mice that express high oxygen affinity (P50 approximately 24.5 mm Hg) rather than normal (P50 approximately 40 mm Hg) mouse Hbs. The rationale was that the high oxygen affinity MHOAH Hb, the lower oxygen affinity of Hb S Antilles than Hb S (P50 approximately 40 v 26.5 mm Hg), and the lower solubility of deoxygenated Hb S Antilles than Hb S (approximately 11 v 18 g/dL) would favor deoxygenation and polymerization of human Hb S Antilles in MHOAH mouse red blood cells (RBCs). The Tg58 x Tg98 mice produced have a high and balanced expression (approximately 50% each) of h alpha and h beta(S Antilles) globins, 25% to 35% of their RBCs are misshapen in vivo, and in vitro deoxygenation of their blood induces 30% to 50% of the RBCs to form classical looking, elongated sickle cells with pointed ends. Tg58 x Tg98 mice exhibit reticulocytosis, an elevated white blood cell count and lung and kidney pathology commonly found in sickle cell patients, which should make these mice useful for experimental studies on possible therapeutic intervention of sickle cell disease.

Functional analysis of the c-myb proto-oncogene.

Targeted mutagenesis studies were initiated to determine the normal biological function of the c-myb proto-oncogene. While heterozygous mice are phenotypically indistinguishable from their wild-type littermates, homozygous mutant fetuses die at approximately 15.5 days of gestation apparently due to anemia, which results from an inability to switch from embryonic yolk sac to fetal liver erythropoiesis. Studies are currently being done to determine the extent of hematopoietic abnormalities in the homozygous mutant fetuses. In vitro assays for hematopoietic colony-forming cells have been used to determine the frequency of both erythroid and myeloid progenitors in the fetal livers of wild-type, heterozygous, and homozygous mutant c-myb fetuses. The reduced number of erythroid progenitors was not unexpected considering the mutant fetus's pale color and reduced hematocrit. The dramatically reduced number of colonies derived from myeloid progenitors in the mutant fetuses in comparison to the number detected in phenotypically normal littermates suggests that expression of the c-myb proto-oncogene is critical for the proliferation and/or differentiation of early hematopoietic progenitors and possibly hematopoietic stem cells. Other possible explanations would include a hematopoietic progenitor migration problem from the yolk sac to the fetal liver or a defect in the microenvironment of the liver. Whether the lymphoid lineage is also adversely affected by the lack of c-myb expression remains to be determined. RT-PCR and Northern blot analyses were used in an attempt to identify downstream genes which may be directly or indirectly regulated by the Myb gene product. While the levels of expression of several genes involved in erythropoiesis (GATA-1, NF-E2, SCL, and EpoR) were reduced in the livers of homozygous mutant fetuses in comparison to phenotypically normal littermates and one gene, Kit ligand (KL), was expressed at higher levels in the mutant livers, these results must be viewed with caution. The livers of the mutant fetuses have been shown to be hypocellular in comparison to those of phenotypically normal littermates (35). It is possible that the Myb gene product is directly or indirectly modulating the expression of these genes. Conversely, the alteration in expression may be due to the reduced number or absence of specific hematopoietic lineages in the livers of the mutant fetuses. Differential display has also been used to identify putative novel genes that are involved in hematopoiesis. Preliminary studies suggest that this may be a powerful methodology to compare the expression pattern of genes in the fetal liver of wild-type, heterozygous, and homozygous mutant littermates at 14.5 days of gestation. To date nearly 60% of the partial cDNAs subcloned analyzed have been shown to be differentially expressed. More importantly, 75% of the differentially expressed cDNAs that have been sequenced appear to encode novel genes. Whether any of these novel genes are involved in the c-myb transcriptional cascade remains to be determined. Overall, analysis of the c-myb mutant fetuses have provided valuable insight into the biological function of this interesting proto-oncogene. The continued analysis of this resource will undoubtedly provide additional information concerning the role of the c-myb gene in hematopoiesis.

Pestivirus translation initiation occurs by internal ribosome entry.

The role of the 385 nucleotide 5' noncoding region (NCR) in the translation of the pestivirus genome was investigated. In vitro translation of an RNA transcript containing the 5' NCR of the bovine viral diarrhea virus (BVDV) genome followed by the coding sequence of the first gene product (p20) of the BVDV large open reading frame resulted in the synthesis of a 20-kDa polypeptide. Results from hybrid-arrest translation studies identified a region involving a predicted RNA stem-loop structure spanning nucleotides 154-261 within the 5' NCR that was important for p20 synthesis. An additional inhibitory oligonucleotide was complementary to the sequence at the base of this stem-loop and encompassed the initiating AUG at nucleotide 386. Antisense oligonucleotides both upstream and downstream of those that were inhibitory had no effect on p20 translation. RNA from a dicistronic expression vector in which the BVDV 5' NCR was inserted between two reporter genes, CAT and LUC, showed strong expression of the second (LUC) cistron upon in vitro translation. This expression was dramatically reduced in an analogous construct in which nucleotides 173-236 of the 5' NCR were deleted. Similar results were obtained when RNA from these same vectors was evaluated for expression after transfection into BHK cells. These results suggest that the BVDV 5' NCR contains an internal ribosome entry site for translation initiation. This translational mechanism is similar to that shown for hepatitis C virus, further demonstrating the close relationship between viruses of these two genera within the family Flaviviridae.

Oxygen association-dissociation and stability analysis on mouse hemoglobins with mutant alpha- and beta-globins.

Oxygen association-dissociation and hemoglobin stability analysis were performed on mouse hemoglobins with amino acid substitutions in an alpha-globin (alpha 89, His to Leu) and a beta-globin (beta 59, Lys to Ile). The variant alpha-globin, designated chain 5m in the Hbag2 haplotype, had an high oxygen affinity and was stable. The variant beta-globin, (beta s2) of the Hbbs2 haplotype, also had an elevated oxygen affinity and in addition was moderately unstable in 19% isopropanol. Hemoglobins from the expected nine (Hbag2/Hbag2;Hbbs/Hbbs x Hbaa/Hbaa;Hbbs2/Hbbs2) F2 genotypes can be grouped into five classes of P50 values characterized by strict additivity and dependency on mutant globin gene dosage; physiologically, both globin variants gave indistinguishable effects on oxygen affinity. The hemoglobin of normal mice (Hbaa/Hbaa;Hbbs/Hbbs) had a P50 = 40 mm Hg and the hemoglobin of Hbag2/Hbag2;Hbbs2/Hbbs2 F2 mice had a P50 = 25 mm Hg (human P50 = 26 mm Hg). Peripheral blood from Hbag2/Hbag2;Hbbs/Hbbs, Hbaa/Hbaa;Hbbs2/Hbbs2 and Hbag2/Hbag2;Hbbs2/Hbbs2 mice exhibited normal hematological values except for a slightly higher hematocrit for Hbag2/Hbag2;Hbbs/Hbbs and Hbag2/Hbag2;Hbbs2/Hbbs2 mice, slightly elevated red cell counts for mice of the three mutant genotypes, and significantly lower values for the mean corpuscular volume and mean corpuscular hemoglobin for Hbag2/Hbag2;Hbbs2/Hbbs2 mice.

Changes in alpha-globin gene expression in mice of two alpha-globin haplotypes during development.

Adult alpha-globin in mice is synthesized in large amounts during development, first in the primitive, nucleated erythrocytes of yolk sac origin and later in the definitive, nonnucleated erythrocytes that differentiate in the fetal liver, spleen, and bone marrow. Isoelectric focusing analysis of hemoglobins of mice with the Hbag2 and Hbac haplotypes shows that the ratios of alpha chain 1 to chain 5m and alpha chain 1 to chain 4 in adult hemoglobins from Hbag2 and Hbac mice, respectively, change between day 11.5 and day 16.5 of gestation in nucleated red cells, while no change occurs in nonnucleated red cells. The percentage ratios of the two different alpha-globin chains are different in Hbag2 and Hbac mice for EII, EIII, and adult hemoglobin. In nucleated red cells of yolk sac origin, differences and changes in alpha-globin ratios are a composite of changing globin gene transcription and posttranslational competitive affinities among globins to form embryonic and adult hemoglobin tetramers.

A comparison of stem cell populations and hemoglobin switching in normal versus beta-thalassemic mice.

The hematopoietic stem cell concentrations in tissues of homozygous beta-thalassemic and non-thalassemic fetuses and neonates were compared by using the spleen colony-forming units (CFU-S) assay. The relative quantities of embryonic and adult hemoglobins were also determined for fetuses. Beta-thalassemic fetuses had a reduced incidence of CFU-S in the liver throughout gestation, but after birth the beta-thalassemic neonates maintained a greater number of CFU-S in the liver for an extended period. The incidence of CFU-S in the bone marrow was not different for the two groups. The beta-thalassemic mice exhibited a significant expansion of CFU-S in the spleen beyond 11 days after birth. The switch from the synthesis of primarily embryonic to primarily adult hemoglobins in circulating erythrocytes in beta-thalassemic fetuses appeared later than the switch in normal fetuses. These observations establish that the developmental timing and expansion of hematopoiesis are perturbed in beta-thalassemic mice.

An overview of occupational health promotion.

1. Occupational health promotion has been institutionalized into many companies in the United States and new programs are being started up across the country. 2. Several stimuli affect this growth in programs, including cost-containment, employee recruitment, and governmental support. 3. It is essential that companies involved in occupational health promotion understand their reasons for espousing this trend. 4. Health promotion activities should be planned thoroughly and plans should especially address cost analyses and ethical issues early in the planning phase.

Resistance to infection in murine beta-thalassemia.

Clinical evidence suggests that individuals with chronic iron overload may be at increased risk of bacterial infection. We studied this question by using a unique model in which mice homozygous for a deletion in the gene encoding for the beta-major globin develop moderate anemia, splenomegaly, and tissue iron overload, a syndrome similar to beta-thalassemia in humans. Mice heterozygous for the gene deletion were phenotypically normal. Homozygous mice were significantly more susceptible to infection with Listeria monocytogenes than were heterozygous mice (P less than 0.01). This increased susceptibility was associated with a greater number of organisms in the liver and spleen than was found in heterozygous mice (P less than 0.05). However, histologic studies demonstrated similar inflammatory responses within these organs in homozygous and heterozygous mice. The increased susceptibility of homozygous mice to infection with L. monocytogenes was not seen when homozygotes were immunized with a low dose of L. monocytogenes. Although the results were not as striking as with L. monocytogenes, homozygous mice were also found to be more susceptible to infection with Salmonella typhimurium than were heterozygous mice (P less than 0.05). Splenic mononuclear cells from homozygous mice demonstrated less responsiveness in vitro to the mitogens concanavalin A and phytohemagglutinin than did those from heterozygotes (P less than 0.05). These data suggest that there is a generalized defect in innate immunity in homozygous mice which makes them more susceptible to infection by L. monocytogenes and S. typhimurium. The site of this immunological defect is not known but is most likely in the mononuclear phagocyte and may be due to tissue iron overload.

Genetics of ocular NAD+-dependent alcohol dehydrogenase and aldehyde dehydrogenase in the mouse: evidence for genetic identity with stomach isozymes and localization of Ahd-4 on chromosome 11 near trembler.

Electrophoretic and activity variation of the stomach and ocular isozyme of aldehyde dehydrogenase (designated AHD-4) was observed between C57BL/6J and SWR/J inbred strains of mice. The phenotypes were inherited in a normal mendelian fashion, with two alleles at a single locus (Ahd-4) showing codominant expression. The alleles assorted independently of those at Adh-3 [encoding the stomach and ocular isozyme of alcohol dehydrogenase (ADH-C2)] on chromosome 3. Three chromosome 11 markers, hemoglobin alpha-chain (Hba), trembler (Tr), and rex (Re), were used in backcross analyses which established that Ahd-4 is closely linked to trembler. The distribution patterns for stomach and ocular AHD-4 phenotypes were examined among SWXL recombinant inbred mice, and those for stomach and ocular ADH-C2 among BXD recombinant inbred strains. The data provided evidence for the genetic identity of stomach and ocular ADH-C2 and of stomach and ocular AHD-4.

Mouse globin gene nomenclature.

A new system of nomenclature for haplotypes, genes, alleles, and mutant alleles in the mouse alpha- and beta-globin gene complexes was formulated at a meeting of workers in the field and is presented here.

A unique alpha chain in hemoglobin of "Skive" Danish Mus musculus.

The primary structures of the alpha chains in hemoglobins from three stocks of mice with the Hbaw2, Hbaw3, and Hbaw4 haplotypes were determined to establish whether the tentative alpha-chain assignments based on the results of isoelectric focusing patterns were correct. These Hba haplotypes were identified in laboratory descendants of feral mice captured in different parts of the world. Hemoglobin from "Centreville", Maryland, Mus musculus domesticus (Hbaw2) contains equal amounts of alpha chains 1 and 3. Hemoglobin from "Czech" Mus musculus musculus (Hbaw4) contains equal amounts of alpha chains 3 and 4. Amino acid analysis of the alpha-globins of "Skive" Danish Mus musculus musculus (Hbaw3) establishes that its hemoglobin is comprised of about one-third alpha chain 2 as expected plus a greater amount of a unique alpha chain that has not been described previously. This unique alpha chain has glycine at position 25, isoleucine at position 62, and serine at position 68; it is called chain 7. It may represent an intermediate in the evolution of genes that code for chain 2 (which has glycine, valine, and serine at positions 25, 62, and 68, respectively) and chain 4 (which has valine, isoleucine, and serine at positions 25, 62, and 62, respectively).