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.

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

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.

Differential association of transfer RNAs with the genomes of murine, feline and primate retroviruses.

The tRNAs that are bound to the genomic RNAs of several murine, feline, and primate retroviruses have been identified. Transfer RNAs were divided into those loosely bound and those tightly bound by stepwise thermal dissociation of the 70 S RNA. They were then identified and semiquantitated by aminoacylation. Proline tRNA is the most tenaciously bound tRNA in several strains of murine leukemia virus, two strains of feline leukemia virus, and the primate viruses simian sarcoma, baboon endogenous, and gibbon ape lymphoma. In the feline xenotropic virus, RD-114, tRNAGly is enriched in the most tightly bound fraction. In Mason-Pfizer monkey virus, as in the murine mammary tumor virus, tRNALys is the tRNA most tenaciously bound to its genomic RNA. Besides the most tightly associated tRNA, one or more different tRNAs are found in relatively large amounts in association with the 70 S RNA. (For convenience, we refer to the largest RNA ccomplex (50-70 S) isolated from any of the retroviruses studies as '70 S' RNA.) These tRNAs can be distinguished from the most tightly bound tRNA by the fact that they can be dissociated at lower temperatures. However, they occur in the same relative abundance as the tightly bound tRNA.

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.

Amino acid substitution: its use in detection and analysis of genetic variants.

Techniques of chemical analysis, amino acid sequencing and autoradiography are being used to study the frequency of incorporation of normally noncoded amino acids into hemoglobins and seminal fluid proteins. We are studying, by the sequencing of radiolabeled proteins followed by the recovery of [3H]isoleucine phenylthiohydantoin by high-performance liquid chromatography, the frequency at which normally noncoded isoleucine is incorporated into hemoglobin because of base-substitution mutations versus translational errors. Irradiation increases the isoleucine content of human hemoglobin and the frequency of substitution of isoleucine for specific amino acids in rabbit hemoglobin. Studies to date indicate that these techniques have been developed sufficiently for initial analysis of the potential of drugs and environmental pollutants to induce base-substitution mutations in mammalian somatic cells.

Analysis of a mouse alpha-globin gene mutation induced by ethylnitrosourea.

A DBA/2 mouse treated with ethylnitrosourea sired an offspring whose hemoglobin showed an extra band following starch gel electrophoresis. The variant hemoglobin migrated to a more cathodal position in starch gel. Isoelectric focusing indicated that chain 5 of the mutant hemoglobin migrated to a more cathodal position than the normal chain 5 from DBA/2 mice and that the other alpha-globin, chain 1, was not affected. On focusing gels the phenotype of the mutant allele, Hbay9, was expressed without dominance to normal chain 5, and Hbay9/Hbay9 homozygotes were fully viable in the laboratory. The molecular basis for the germinal mutation was investigated by analyzing the amino acid sequence of chain 5y9, the mutant form of alpha-chain 5. A single amino acid substitution (His leads to Leu) at position 89 was found in chain 5y9. We propose that ethylnitrosourea induced an A leads to T transversion in the histidine codon at position 89 (CAC leads to CTC). This mutation has apparently not been observed previously in humans, mice or other mammals, and its novel occurrence may be indicative of other unusual mutational events that do not ordinarily occur in the absence of specific mutagen exposure.

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.

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.

Hemopoietic stem cell heterogeneity: use of cell cycle-specific drugs to look for age-associated alterations.

Hemopoietic tissue is vulnerable to perturbations, and data show that it is an appropriate tissue in which to look for age-associated alterations. This tissue has a high regenerative capacity, is composed of a heterogeneous population of stem cells that are capable of self renewal or differentiation, or both, and is sustained by a pool of resting cells. The heterogeneity of bone marrow has made characterization of the cellular elements difficult. Techniques commonly used to identify and quantify the various maturation levels of hemopoietic stem cells and the limitations of these techniques are discussed. Most techniques used to assay age-associated changes in bone marrow have not differentiated between specific cellular alterations or shifts in the distribution of the cellular elements. In particular, it has been difficult to determine the stability of the non-dividing stem cell because of the low incidence of this cell (6 per 1000) and the lack of a specific assay for this important cell type. The use of cell cycle-specific drugs has provided quantitative information on specific subpopulations of hemopoietic stem cells and seems to be the most promising approach towards determining qualitative and quantitative differences in the hemopoietic stem cells of young and old individuals.

Interaction of H-2 genotype and basal serum immunoglobulin A level influences longevity.

The congenic pair of mice, C57BL/10 (B10) and C57BL/10.F (B10.F), differ at the H-2 locus and have mean ages at death of 706 and 456 days, respectively. B10.F also has reduced basal serum IgA levels compared with B10, 63 and 256 mg/dl, respectively. Controlled matings between the two strains of mice were used to identify genetic factors that govern longevity. F2 and backcross progeny from reciprocal F1 hybrids were classified for H-2 genotype and serum IgA levels and allowed to live out their lifespan. F2 and backcross progeny homozygous for the H-2 allele of B10.F had a mean age at death (602 days) significantly reduced from that of progeny homozygous for the H-2 allele of B10 (689 days). However, the greatest reduction of lifespan occurred among progeny of the (B10.F X B10)F1 mothers, 693 compared with 540 days. The strain of the maternal parent also has been shown to affect the segregation of IgA phenotypes. An increased incidence of low IgA phenotype associated with H-2 genotype was observed among progeny of (B10.F X B10)F1 mothers. Survival curves demonstrated a relationship between low serum IgA levels and shortened lifespan and no maternal effect was observed. The basis of the shortened lifespan among progeny of F1 hybrids in which the maternal parent was B10.F was the increased incidence of offspring with low IgA phenotypes. The apparent association of H-2 and shortened lifespan also was because the low IgA phenotype was more frequent among progeny that carried the H-2 allele of the B10.F strain. The B10.F mice spontaneously shed an endogenous ecotropic retrovirus which may be responsible for the maternal effect on immunoglobulin levels and lifespan.

Hematology of a murine beta-thalassemia: a longitudinal study.

Mice homozygous for a spontaneous mutation, in which the beta-major globin gene is deleted, have clinical symptoms of beta-thalassemia. These mice have a hypocellular, hypochromic, microcytic anemia that becomes more severe with increasing age. The defective red cell morphology, decreased osmotic fragility of erythrocytes and shortened red cell life span found in beta-thalassemic mice are similar to those observed in human beta-thalassemia. Synthesis of beta-globin is depressed but not as much as might be expected because the expression of the beta-minor globin gene is enhanced to encode two to three times more globin than in normal mice. Splenomegaly, an enlarged pool of stem cells for erythropoiesis, and iron overloading occur in older mice. The fact that these mice remain moderately healthy makes them a very suitable animal model in which to develop and test alternative techniques of gene therapy that could be successfully applied to the treatment of human thalassemia. Homozygous beta-thalassemic mice have large deposits of iron in their tissues, which might make these mice also useful for in vivo tests of the effectiveness and possible long-term side effects of newly developed iron chelators.

Critical sample sizes for determining the statistical significance of mutation frequencies.

Based on the assumption that the numbers of mutations observed in an untreated and treated sample of individuals are binomial random variables, a method is presented to compute the probability of observing a specific number of mutations as a function of the sample sizes and the number of mutations in the untreated control sample. Knowledge of the true mutation frequencies is not required. The formalism is then used to compute critical sample sizes for testing hypotheses concerning mutation frequencies in the two populations.

Radiation-induced alpha-thalassemia in mice.

The clinical hematologic change in 2 groups of progeny from mice carrying radiation-induced strain SEC alpha-chain deficiencies was found to be similar to the hematologic alterations in persons with alpha-thalassemia. The heterozygous deletion or inactivation of the alpha-chain gene in mice caused an anemia similar to alpha-thalassemina minor in persons. The alpha-chain deficiency in mice created an erythrocytosis, reticulocytosis, and microcytic, hypochromic anemia comparable with the changes in human alpha-thalassemia minor resulting from deletion of the alpha-chain gene. These mouse mutants are the only known animal models of human thalassemia. A comparison of hematologic values obtained from progeny possessing an alpha-chain gene deficiency and from progeny possessing a beta-chain duplication suggested that the deficiency of alpha-chain synthesis, rather than a simple imbalance between the amounts of alpha- and beta-chains produced, was primarily responsible for the altered hematologic characteristics in these alpha-thalassemic mice.

Preschool children's understanding of children with orthopedic disabilities and their expectations.

The present study investigated whether preschool children (N = 121) had a realistic understanding of the limitations imposed by various orthopedic disabilities. The results have indicated that the preschoolers were aware that orthopedic disabilities may impose some physical limitations. Ss tended to perceive able-bodied children more capable in performing various tasks than disabled children. This evaluative judgment does not necessarily mean that the preschoolers were discriminating against the disabled children, but it may be a reflection of a sense of realism that the able-bodied children have less trouble in doing these tasks than their disabled counterpart. It was suggested that, with increased understanding of the realistic limitations imposed by orthopedic disabilities, children may be come less biased toward their disabled peers. This change in attitude would have significant impact on the self-concepts of disabled children.

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.