Serum Annexin V and Anti-Annexin V levels and their relationship with metabolic parameters in patients with type 2 diabetes

SUMMARY BACKGROUND We investigated the serum annexin V and anti-annexin V levels and their relationship with metabolic parameters in patients recently diagnosed type 2 diabetic. METHODS A total of 143 patients recently diagnosed type 2 diabetes and 133 control subjects were included in the study. Body mass index (BMI), hs-CRP, HOMA-IR, carotid intima-media thickness, and serum levels of annexin V and anti-annexin V were investigated. RESULTS HOMA-IR, serum hs-CRP, and carotid intima-media thickness were found to be statistically significant. The Pearson correlation analysis revealed a statistically significant positive relationship between the carotid intima-media thickness and the annexin V level (r=0.29, p=0.006*). A statistically significant positive relationship was also detected between the Annexin V level and level of serum hs-CRP (r=0.29 p=0.006*). CONCLUSION A positive relationship was observed between the carotid intima-media thickness and annexin V at the end of our investigation. In this regard, we also believe that serum levels of annexin V may be increased for cardiovascular protection in the elevation of carotid intima-media thickness.

RESUMO OBJETIVO Investigar os níveis séricos de anexina V e antianexina V e sua relação com os parâmetros metabólicos em pacientes diabéticos tipo 2 recém-diagnosticados. MÉTODOS Foram incluídos no estudo 143 pacientes e 133 controles com diabetes tipo 2 recém-diagnosticado. O índice de massa corporal (IMC), PCR-as, Homa-IR, espessura íntima média carotídea e níveis séricos de anexina V e antianexina V foram investigados. RESULTADOS O Homa-IR, a PCR-s do soro e a espessura média da carótida foram estatisticamente significantes. A análise de correlação de Pearson revelou uma relação positiva estatisticamente significante entre a espessura média da carótida e anexina V (r=0,29; p=0,006 *). Foi também detectada uma relação positiva estatisticamente significativa entre o nível de anexina V e o nível sérico de PCR-as (r=0,29, p=0,006*). CONCLUSÃO Também foi observada uma relação positiva entre a espessura média da carótida e anexina V no final da nossa investigação. A esse respeito, também pensamos que os níveis séricos de anexina V podem ser aumentados para proteção cardiovascular na elevação da espessura média da carótida.

Cloning and expression analysis of multiple proteins encoding P gene of Newcastle disease virus.

Viral gene oncotherapy is emerging as a biotherapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. Newcastle disease virus (NDV) has the property to cause selective oncolysis of tumor cells sparing normal cells. NDV has a single stranded negative sense RNA genome, which is 15,186 nucleotide long and consists of six genes, which codes for eight proteins. NDV like other paramyxoviruses has the ability to generate multiple proteins from the P gene. P protein is encoded by an unedited transcript of the P gene, whereas the V and W protein are the results of RNA editing event in which one and two G residues are inserted at a conserved editing site within the P gene mRNA resulting in V and W transcripts, respectively. Although NDV is known to cause oncolysis by triggering apoptosis, the role of different viral proteins in selective oncolysis is still unclear. P gene edited products are known for its anti-apoptotic property in homologous host. In the present study, NDV P gene and its RNA edited products were amplified, cloned, sequenced and in vitro expression was done in HeLa cells. Further constructs were assayed for their apoptosis inducing ability in HeLa cells. Preliminary study suggested that P, V and W proteins are not apoptotic to HeLa cells.

Heat stress induced apoptosis in BC-8 cells derived from AK-5 tumor involves downregulation of Bcl-2 and generation of reactive oxygen species.

BC-8, a rat histiocytoma undergoes apoptosis after heat shock, which is due to lack of an effective heat shock response. Heat shock induced generation of free radicals, which in turn are involved in the induction of apoptotic death in BC-8 cells. Treatment of BC-8 cells with N-acetylcysteine partially inhibited the heat induced apoptosis. Introduction of Bcl-2 gene in these cells did not protect them from apoptotic death, whereas transfection with hsp-70 gene did render these cells resistant to heat induced apoptosis transiently. Heat shock also downregulated the expression of Bcl-2 and p53 in these cells. These observations suggested that the heat shock induced apoptosis was mediated through reactive oxygen species and controlled upstream of Bcl-2 check point.

Neuroprotective Effect of Cycloheximide on Hypoxic-Ischemic Brain Injury in Neonatal Rats

This study was done to determine the neuroprotective effect of cycloheximide on neonatal hypoxic-ischemic brain injury. Seven day-old newborn rat pups were subjected to 90 min of 8% oxygen following a unilateral carotid artery ligation. The extent of cerebral infarction was evaluated at 1 and 4 week of recovery. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluoresceinated annexin V and propidium iodide. Brain infarction area was significantly increased at 4 week compared to 1 week after hypoxia-ischemia in the control group. With cycloheximide treatment, the number of TUNEL positive cells in the ipsilateral cerebral cortex at 48 hr and peri-infarct area at 1 and 4 week of recovery was significantly reduced, both apoptotic and necrotic cells by flow cytometry 48 hr after the injury were significantly reduced, and the extent of cerebral infarction at 1 and 4 week of recovery was also significantly attenuated compared to the hypoxia-ischemia control group. In summary, our data suggest that apoptosis plays an important role in the development of delayed infarction, and inhibition of apoptosis with cycloheximide significantly reduces the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia.

Activation of the intrinsic mitochondrial apoptotic pathway in swine influenza virus-mediated cell death

The mitochondrial pathway of swine influenza virus (SIV)-induced apoptosis was investigated using porcine kidney (PK-15) cells, swine testicle (ST) cells, and HeLa cervical carcinoma cells which are known not to support viral replication. As judged by cell morphology, annexin V staining, and DNA fragmentation, PK-15 and ST cells infected with three different subtypes of SIV (H1N1, H3N2, and H1N2) were obviously killed by apoptosis, not necrosis. SIV infection in PK-15 and HeLa cells was shown to decrease the cellular levels of Bcl-2 protein compared to that of mock-infected control cells at 24 h post-infection, whereas expression levels of Bax protein increased in the PK-15 cells, but did not increase in HeLa cells by SIV infection. Cytochrome c upregulation was also observed in cytosolic fractions of the PK-15 and HeLa cells infected with SIV. Apoptosome (a multi-protein complex consisting of cytochrome c, Apaf-1, caspase-9, and ATP) formation was confirmed by immunoprecipitation using cytochrome c antibody. Furthermore, SIV infection increased the cellular levels of TAJ, an activator of the JNK-stressing pathway, and the c-Jun protein in the PK-15 and HeLa cells. Taken together, these results suggest that the mitochondrial pathway should be implicated in the apoptosis of PK-15 cells induced by SIV infection.

Mutation of highly conserved arginine residues disrupts the Structure and Function of Annexin V

Background. Annexins are a family of structurally related proteins that bind to phospholipid membranes in a Ca²+-dependent manner. Annexins are characterized by highly conserved canonical domains of approximately 70 amino acids. Anexin V contains four such domains. Each of these domains has a highly conserved arginine (R). Methods. To evaluate the role of the conserved arginines in the molecular structure of annexin V, negatively charged amino acids were substituted for arginines at positions R43, R115, R199, and R274 using site-directed mutagenesis. Results. Mutants R199D and R274E were rapidly degraded when expressed in bacteria, and were not further characterized. R43E exhibited an electrophoretic mobility similar to the wild-type protein, while R115E migrated significantly in a slower fashion, suggesting a less compact conformation, R43E and R115E exhibited much grater susceptibility to proteolytic digestion than the wild type. While Ca²+-dependence for phospholipid binding was similar in both mutants (half-maximal 50-80 µ; Ca²+), R43E and R115E exhibited a phospholipid affinities of the annexins, a phospholipid-dependent clotting reaction, the activated partial thromboplastin time (aPTT), was significantly prolonged by the wild-type protein and mutants R115E and R115A. The aPTT was unaffected by R43E. Conclusions. Our data suggest that mutation of these highly conserved arginine residus in each of the four canonical domains of annexin have differential effects on the phospholipid binding tertiary structure, and proteolytic susceptibility of annexin V. The site I mutation , R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolytic susceptibility. The site II mutation, R115E, produced a small change in phospholipid binding but a significant modification of electrophoretic mobility. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of ammexin V by establishing hydrogen bonds and ionic bridges