Enzymatic Changes in Red Blood Cells of Diamond-Blackfan Anemia.

Diamond-Blackfan anemia is a congenital bone marrow failure syndrome characterized by red blood cell (RBC) aplasia with varied malformations in infants. Elevated activity of adenosine deaminase (ADA) has been considered as a useful biomarker of Diamond-Blackfan anemia, and ADA assay has been shown to be more sensitive than genetic diagnosis. Approximately, 80% of the examined patients showed elevated ADA activity, whereas genetic tests of ribosome subunit genes identified mutations in approximately 60% of the patients. We previously reported that reduced glutathione (GSH) levels in RBCs may serve as a biomarker of Diamond-Blackfan anemia. In this study, to confirm the universality of our data, we extended the analysis to seven RBC enzymes and GSH of 14 patients with Diamond-Blackfan anemia and performed a cross-analysis study using enzyme activity assay and recently reported proteome data. Statistical analysis revealed that both data exhibited high similarity, upregulation in the hexokinase and pentose-phosphate pathway, and downregulation in glycolytic enzymes such as phosphofructokinase and pyruvate kinase, in the RBCs obtained from the subjects with Diamond-Blackfan anemia. The only discrepancy between enzyme activity and proteome data was observed in glucose-6-phosphate dehydrogenase (G6PD), as increased G6PD activity showed no relation with the significant elevation in protein levels. These results suggest that our enzymatic activity data of Diamond-Blackfan anemia are universal and that the enzymatic activation of G6PD via a hitherto-unveiled mechanism is another metabolic feature of RBCs of Diamond-Blackfan anemia.

Development of a bsd-blasticidin selection system in Plasmodium berghei.

Plasmodium berghei is used as a rodent model for the study of malaria. However, multiple genetic manipulations are restricted by the paucity of selectable markers. The bsd-blasticidin selection system is widely used for eukaryotic cells; however, it could not previously be used for P. berghei due to toxicity to the rodent host. Here, we report the application of this selection system in P. berghei using an in vitro selection method. The desired bsd-integrated mutants are enriched by more than 90% within 2 weeks when using this system. Furthermore, the bsd marker can be used sequentially with established pyrimethamine- and puromycin-resistant markers. This system allows deeper understanding of malaria parasite biology through extensive genetic manipulation of P. berghei.

Treatment with tucumã oil (Astrocaryum vulgare) for diabetic mice prevents changes in seric enzymes of the purinergic system: Improvement of immune system.

The aim of this study was to evaluate the enzymatic activity of the purinergic system in sera samples from alloxan-induced diabetic mice treated with tucumã oil (Astrocaryum vulgare). For this, the mice were divided into four groups (n=6): control/water (the group CW); control/tucumã oil (the group CT); diabetic/water (the group DW), and diabetic/tucumã oil (the group DT) treated for 14days with 5.0mLkg-1 via oral gavage. On day 14 post-treatment, mice were submitted to euthanasia and blood samples were collected by cardiac puncture. Tucumã oil treatment significantly decreased (p<0.05) blood glucose levels in the group DT compared to the group DW. These results demonstrated an increase (p<0.05) in NTPDase (adenosine triphosphate (ATP substrate) or adenosine diphosphate (ADP substrate)), 5'-nucleotidase (AMP substrate) and adenosine deaminase (ADA; adenosine substrate) activities in serum from the group DW compared to the group CW. Tucumã oil treatment prevented these alterations in the group DT compared to the group DW, and restored these parameters near to the group CW. In summary, the treatment with tucumã oil was able to modulate the alterations caused by hyperglycemia probably by the presence of carotenoids compounds, maintaining normal levels of ATP, ADP, AMP and adenosine, molecules that could exhibit anti-inflammatory properties, depending on their concentration. Thus, the tucumã oil is a promising natural compound with protective action against diabetes and its side effects, such as changes in the purinergic system, improving the immune system.

Effects of iron supplementation on blood adenine deaminase activity and oxidative stress in Trypanosoma evansi infection of rats.

The aim of this study was to assess the effects of iron supplementation on oxidative stress and on the activity of the adenosine deaminase (ADA) in rats experimentally infected by Trypanosoma evansi. For this purpose, 20 rats were divided into four experimental groups with five animals each as follows: groups A and B were composed by healthy animals, while animals from groups C and D were infected by T. evansi. Additionally, groups B and D received two subcutaneous doses of iron (60 mg kg(-1)) within an interval of 5 days. Blood samples were drawn on day 8 post infection in order to assess hematological and biochemical variables. Among the main results are: (1) animals from group C showed reduced erythrogram (with tendency to anemia); however the same results were not observed for group D; this might be a direct effect of free iron on trypanosomes which helped to reduce the parasitemia and the damage to erythrocytes caused by the infection; (2) iron supplementation was able to reduce NOx levels by inhibiting iNOS, and thus, providing an antioxidant action and, indirectly, reducing the ALT levels in groups Band D; (3) increase FRAP levels in group D; (4) reduce ADA activity in serum and erythrocytes in group C; however, this supplementation (5) increased the protein oxidation in groups B and D, as well as group C (positive control). Therefore, iron showed antioxidant and oxidant effects on animals that received supplementation; and it maintained the activity of E-ADA stable in infected/supplemented animals.

Blood-based biomarkers can differentiate ulcerative colitis from Crohn's disease and noninflammatory diarrhea.

BACKGROUND: Blood gene expression profiling has been used in several studies to identify patients with a number of conditions and diseases. A blood test with the ability to differentiate Crohn's disease (CD) from ulcerative colitis (UC) and noninflammatory diarrhea would be useful in the clinical management of these diseases. METHODS: Affymetrix U133Plus 2.0 GeneChip oligonucleotide arrays were used to generate whole blood gene expression profiles for 21 patients with UC, 24 patients with CD, and 10 control patients with diarrhea, but without colonic pathology. RESULTS: A supervised learning method (logistic regression) was used to identify specific panels of probe sets which were able to discriminate between UC and CD and from controls. The UC panel consisted of the four genes, CD300A, KPNA4, IL1R2, and ELAVL1; the CD panel comprised the four genes CAP1, BID, NIT2, and NPL. These panels clearly differentiated between CD and UC. CONCLUSIONS: Gene expression profiles from blood can differentiate patients with CD from those with UC and from noninflammatory diarrheal disorders.

[Clinico-pathogenetic significance of enzymes regulating nucleic metabolism in erythrocyte lysates and sera of patients with osteoarthrosis].

Lysates of erythrocytes and sera of 52 patients with osteoarthrosis (OA) and 30 healthy subjects were used used to determine adenosine deaminidase (ADA, AMP deaminase (AMPDA) and adenine deaminidase (AD). Their activities were unrelated to the age and sex of the patients. At admission, patients with OA showed enhanced activity of ADA in sera and reduced activity of AMPDA and AD in lysates compared with normal values. These changes depended on clinical features of OA and were especially pronounced in patients with poly-OA and synovitis. These data suggest participation of enzymes of the adenyl branch of purine metabolism in pathogenesis of OA. Treatment of hospitalized patients allowed to achieve positive dynamics of the above activities coupled to their improved clinical conditions even though enzymatic activity of erythrocyte lysates remained different from that in healthy subjects. It is concluded that enzymatic assays used in the study may be used as additional diagnostic methods for the assessment of synovitis and optimizatic of control over efficiency of OA therapy.

Enzyme rate assay for 5-fluorocytosine.

Modification of an enzyme assay for 5-fluorocytosine, from an end point to a rate analysis, has resulted in a method that can provide results within 10 minutes. The modification also rendered the assay less susceptible to interference from bilirubin and triglycerides. This obviated the need to correct 5-fluorocytosine results for icteric or moderately lipemic samples. The performance history of the enzyme rate method has shown that it is reliable, quick, cost-effective, and suitable for use in a clinical laboratory.

[Activity of guanase in serum and liver function tests in HBe antigen-positive chronic hepatitis].

Forty-one patients with chronic hepatitis were divided into an HBe antigen-positive group (n = 13) and an HBe antigen-negative group (n = 28) to clarify the relationship between the presence of HBe antigen and liver function. In the HBe antigen-positive group, the activities of serum alanine aminotransferase (p less than 0.01), aspartate aminotransferase (p less than 0.05) and guanase (p less than 0.01) were significantly higher than those in the HBe antigen-negative group. The correlation coefficient between the HBe antigen titer and guanase activity was 0.528, which was higher than the corresponding values for alanine aminotransferase and aspartate aminotransferase. The determination of guanase activity in serum may be useful for evaluating the clinical severity of HBe antigen-positive chronic hepatitis.

Clinical significance of serum guanase activity in various liver diseases.

Serum guanase activity was measured using a sensitive colorimetric method in patients with liver diseases. Guanase activity was correlated with GPT, GOT in acute viral hepatitis and chronic hepatitis, however, in liver cirrhosis and hepatocellular carcinoma it was correlated with total bilirubin as well as aminotransferases. In addition, the GPT-to-guanase ratio differed chronic hepatitis from liver cirrhosis. These findings suggest that determination of guanase and aminotransferases in useful in differentiation of liver diseases as well as assessing liver damage.

Simple ultraviolet spectrophotometric method for the determination of serum guanase activity.

We describe a simple, kinetic method for the determination of serum guanase activity that involves enzymatic coupling to xanthine oxidase and measurement of the rate of uric acid formation by spectrophotometric monitoring of the absorption at 300 nm. At this wavelength, the absorption of uric acid is about 80% of its maximal absorption at 293 nm, but the difference in molar extinction coefficient between guanine and uric acid is similar (9,000 at 293 nm vs 8,400 at 300 nm). There are three advantages to the use of the higher wavelength: first, the absorption of serum proteins is only one third of the absorption at 293 nm resulting in a significant reduction in noise level. Second, the lower absorption of serum proteins allows increasing sensitivity of the assay by increasing the amount of serum in the reaction mixture. Third, the higher wavelength allows the use of automated centrifugal analyzers that are generally not designed for measurements below 300 nm. The between-day coefficient of variation was 5.8% (n = 27) at an activity of 17 U/L and 8.2% at an activity of 2 U/L. The reference range for 50 sera from males and females was 0.4 to 1.8 U/L (n = 50; mean +/- 2SD). The method is linear to 40 U/L.

Prevention of posttransfusional non-A, non-B hepatitis using the screening test for guanase activity of donor blood.

A total of 107 recipients, who did not show any evidence of hepatic disorders in pretransfusional liver function tests and gave a negative reaction for HBsAg, were observed from 3 weeks to 3 months after blood transfusion of 711 units of blood. Of 107 recipients, 18 (17%) developed posttransfusional non-A, non-B hepatitis (PTH). It was detected in 2 of 71 recipients (3%) with blood guanase activities below 3.5 U/L and in 16 of 36 recipients (44%) with blood guanase activities above 3.6 U/L. It is considered that development of PTH could be reduced by avoiding use of donor blood with high guanase activity. We adopted an automated method for measuring guanase in donor blood in the Tokushima Red Cross blood center and examined the incidence of PTH when donor blood with high guanase activity was excluded. Of 112 recipients, 8 (7%) developed PTH. The incidence of PTH was 17% before adoption of the guanase screening test and 7% after its adoption. This work shows that for prevention of PTH it is very important to screen donor blood for guanase activity and discard blood with high guanase activity.

Clinical value of the guanase screening test in donor blood for prevention of posttransfusional non-A, non-B hepatitis.

We adopted an automated method for measuring guanase in donor blood and examined the incidence of posttransfusional non-A, non-B hepatitis when donor blood with high guanase activities was excluded. Sixty-seven (2.4%) of 2,826 units were excluded from use in transfusion because they had guanase activities above 1.71 units per liter. Of 112 recipients, 8 (7%) developed posttransfusional non-A, non-B hepatitis. The incidence of posttransfusional non-A, non-B hepatitis was 17% before adoption of the guanase screening test and 7% after its adoption. Thus, the incidence of posttransfusional non-A, non-B hepatitis was significantly decreased after adoption of this screening test. This study shows that, for prevention of posttransfusional non-A, non-B hepatitis, it is important to screen donor blood for guanase activity and discard blood with high activities.

Localization of GTP cyclohydrolase I in human peripheral blood smears using a specific monoclonal antibody and an immune-alkaline phosphatase labeling technique.

GTP cyclohydrolase I, the enzyme catalyzing the first step in the cofactor biosynthesis for the aromatic amino acid hydroxylases, has been localized in situ. By the use of a monoclonal antibody specific to human GTP cyclohydrolase I, the enzyme has been visualized immuno-enzymatically by alkaline phosphatase monoclonal anti-alkaline phosphatase labeling. In routine blood smears lymphocytes, monocytes/macrophages, and granulocytes show strong intraplasmatic staining. Premature erythrocytes show clear staining of the reticulated cytoplasmatic structure, while mature erythrocytes are completely negative. Neither is there any staining for GTP cyclohydrolase I in the blast cells of a case of T-cell acute lymphoblastic leukemia. These results closely confirm the prior finding that mature erythrocytes as well as most malignant mononuclear cells lack GTP cyclohydrolase I activity, and they indicate that in these cells the enzyme protein may be absent.

Relationship between guanase activity in donor blood and the incidence of posttransfusional non-A, non-B hepatitis, and a possible method for preventing posttransfusional hepatitis.

A total of 107 recipients, who did not show any evidence of hepatic disorders in pretransfusional liver function tests and gave a negative reaction for HBsAg, were observed from 3 weeks to 3 months after blood transfusion of 711 units of blood. The blood was judged suitable for use in transfusion because it had a normal level of ALT activity and gave a negative reaction for HBsAg. The guanase activities of the blood used for transfusion were examined. Cases in which an increase of ALT to at least twice the upper limit of normal persisted for at least 3 weeks and the ALT value increased to more than five times the normal upper limit at least once during this period, which also gave a negative reaction for HBsAg, were judged to have posttransfusional non-A, non-B hepatitis. Of 107 recipients, 18 developed posttransfusional non-A, non-B hepatitis. It was detected in 2 of 71 recipients (3%) with blood guanase activities below 3.5 units per liter and in 16 of 36 recipients (44%) with blood guanase activities above 3.6 units per liter. Thus, the incidence of posttransfusional non-A, non-B hepatitis was significantly higher in recipients with blood guanase activities above 3.6 units per liter. The incidence of posttransfusional non-A, non-B hepatitis increased linearly with increase in the level of guanase activity in donor blood. Thus, a high guanase activity in donor blood is considered to be an important predicting factor for posttransfusional non-A, non-B hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS)