Syntheses of 1-[2-(Phosphonomethoxy)Alkyl]Thymine Monophosphates and an Evaluation of their Inhibitory Activity Toward Human Thymidine Phosphorylase

A series of new monophosphates of 1-[2-(phosphonomethoxy)alkyl]thymines, such as PMPTp_, 3-MeO-PMPTp, HPMPTp, and FPMPTp, were synthesized and tested for their ability to inhibit human thymidine phosphorylase. Kinetic measurements of enzyme activity were performed using thymidine and inorganic phosphate as the substrates. The data show that some monophosphates provide a considerable increase of the multisubstrate inhibitory effect. The highest inhibitory potency was found with (R)-FPMPTp 4c (K _i ^dT = 4.09 ± 0.47 μM, K _i(P_i) = 2.13 ± 0.29 μM) and (R) 3-MeO-PMPTp 4d (K _i ^dT = 5.78 ± 0.71 μM, K _i(P_i) = 2.71 ± 0.37 μM).     

Mathematical modeling of maize starch liquefaction catalyzed by α-amylases from Bacillus licheniformis: effect of calcium, pH and temperature

The first step of starch hydrolysis, i.e. liquefaction has been studied in this work. Two commercial α-amylases from Bacilllus licheniformis, known as Termamyl and Liquozyme have been used for this purpose. Using starch as the substrate, kinetics of both enzymes has been determined at optimal pH and temperature (pH 7, T = 80 °C) and at 65 °C and pH 5.5. Michaelis–Menten model with uncompetitive product inhibition was used to describe enzyme kinetics. Mathematical models were developed and validated in the repetitive batch and fed-batch reactor. Enzyme inactivation was described by the two-step inactivation model. All experiments were performed with and without calcium ions. The activities of both tested amylases are approximately one hundred times higher at 80 °C than at 65 °C. Lower inactivation rates of enzymes were noticed in the experiments performed at 65 °C without the addition of calcium than in the experiments at 80 °C. Calcium ions in the reaction medium significantly enhance amylase stability at 80 °C and pH 7. At other process conditions (65 °C and pH 5.5) a weaker calcium stabilizing effect was detected.     

A Combination of CD28 (rs1980422) and IRF5 (rs10488631) Polymorphisms Is Associated with Seropositivity in Rheumatoid Arthritis: A Case Control Study

IntroductionThe aim of the study was to analyse genetic architecture of RA by utilizing multiparametric statistical methods such as linear discriminant analysis (LDA) and redundancy analysis (RDA).MethodsA total of 1393 volunteers, 499 patients with RA and 894 healthy controls were included in the study. The presence of shared epitope (SE) in HLA-DRB1 and 11 SNPs (PTPN22 C/T (rs2476601), STAT4 G/T (rs7574865), CTLA4 A/G (rs3087243), TRAF1/C5 A/G (rs3761847), IRF5 T/C (rs10488631), TNFAIP3 C/T (rs5029937), AFF3 A/T (rs11676922), PADI4 C/T (rs2240340), CD28 T/C (rs1980422), CSK G/A (rs34933034) and FCGR3A A/C (rs396991), rheumatoid factor (RF), anti–citrullinated protein antibodies (ACPA) and clinical status was analysed using the LDA and RDA.ResultsHLA-DRB1, PTPN22, STAT4, IRF5 and PADI4 significantly discriminated between RA patients and healthy controls in LDA. The correlation between RA diagnosis and the explanatory variables in the model was 0.328 (Trace = 0.107; F = 13.715; P = 0.0002). The risk variants of IRF5 and CD28 genes were found to be common determinants for seropositivity in RDA, while positivity of RF alone was associated with the CTLA4 risk variant in heterozygous form. The correlation between serologic status and genetic determinants on the 1st ordinal axis was 0.468, and 0.145 on the 2nd one (Trace = 0.179; F = 6.135; P = 0.001). The risk alleles in AFF3 gene together with the presence of ACPA were associated with higher clinical severity of RA.ConclusionsThe association among multiple risk variants related to T cell receptor signalling with seropositivity may play an important role in distinct clinical phenotypes of RA. Our study demonstrates that multiparametric analyses represent a powerful tool for investigation of mutual relationships of potential risk factors in complex diseases such as RA.     

Involuntary hospitalizations in the psychiatric hospital Jankomir before and following the alterations and amendments made to ZZODS

Schizophrenia and other psychotic disorders as well as the delirium caused by abstinence from alcohol and accute state of drunkenness appear at the very top of the list of factors, which are positively correlated with involuntary hospitalization of patients. This is at the same time a confirmation of the data found in literature considering psychosis an essential factor of involuntary hospitalization; the same referring to the male sex was not, however, confirmed by the results obtained in the first and second research period. Regarding the positive correlation between schizophrenia and other psychotic disturbances, dementia, delirium and other cognitive impairments including the delirium caused by abstinence from alcohol and an accute state of drunkenness on the one side and the high rate of involuntary hospitalization on the other, there is no statistically significant difference between the period preceding and the period following the alterations and amendments to the Law on the protection of patients with mental disorders.     

Fast identification of folded human protein domains expressed in <Emphasis Type="Italic">E. coli</Emphasis> suitable for structural analysis

Background  

High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination.     

The production and application of single-chain antibody fragments

This review discusses methods for the single-chain antibody fragment ($cFv) generation and scFv expression systems, and describes potential applications of scFv in the therapy of viral diseases and cancer, with emphasis on intracellularly expressed scFvs (intrabodies), application of scFvs in detection and diagnostics, and their use in proteomics.     

Editing autoreactive TCR enables efficient positive selection

Allelic exclusion is inefficient at the TCRalpha locus, allowing a sizeable portion of T cells to carry two functional TCRs. The potential danger of dual TCR expression is a rescue of autoreactive TCRs during selection in the thymus and subsequent development of autoimmunity. In this study, we examine the reason(s) for replacing an autoreactive TCR and for allowing the survival of cells carrying two TCRs. We compared development of TCR transgenic CD4(+)CD8(-) thymocytes in the presence or absence of MHC class II autoantigen that does not induce deletion of thymocytes. Contrary to the expected negative effect of the presence of autoantigen, approximately 100% more CD4(+)CD8(-) thymocytes were found in the presence of MHC class II autoantigen than in the neutral background. A further increase in the strength of autoantigenic signal via expression of a human CD4 transgene led to an additional increase in the numbers of CD4(+)CD8(-) thymocytes. Thus, editing autoreactive TCR results in more efficient positive selection, and this may be both a reason and a reward for risking autoimmunity.     

The role of intrinsic muscle flaps of the foot for bone coverage in foot and ankle defects in diabetic and nondiabetic patients

Local muscle flaps, pioneered by Ger in the late 1960s, were extensively used for foot and ankle reconstruction until the late 1970s when, with the evolution of microsurgery, microsurgical free flaps became the reconstructive method of choice. To assess whether the current underuse of local muscle flaps in foot and ankle surgery is justified, the authors identified from the Georgetown Limb Salvage Registry all patients who underwent foot and ankle reconstruction with local muscle flaps and microsurgical free flaps from 1990 through 1998. By protocol, flap coverage was the reconstructive choice for defects with exposed tendons, joints, or bone. Local muscle flaps were selected over free flaps if the defect was small (3 x 6 cm or less) and within reach of the local muscle flap. During the same time frame, the authors performed 45 free flaps (96 percent success rate) in the same areas when the defects were too large or out of reach of local muscle flaps. Thirty-two consecutive patients underwent local muscle flap reconstruction for 19 diabetic wounds and 13 traumatic wounds. All wounds, after debridement, had exposed bone at their base, with osteomyelitis being present in 52 percent of the diabetic wounds and in 70 percent of the nondiabetic wounds. Wounds were located in the hindfoot (47 percent), midfoot (44 percent), and ankle (9 percent). Vascular disease was more prevalent in the diabetic group, in which 42 percent of the affected limbs required revascularization procedures before reconstruction (versus 7 percent in the nondiabetic group). Subsequently, 83 total operations were required to heal the wounds, of which 46 percent were limited to debridement only. Thirty-four pedicled muscle flaps were used: 19 abductor digiti minimi (56 percent), nine abductor hallucis (26 percent), three extensor digitorum brevis (9 percent), two flexor digitorum brevis (6 percent), and one flexor digiti minimi (3 percent). An additional skin graft for complete coverage was required in 18 patients (53 percent). One patient died and one flap developed distal necrosis, for a 96 percent success rate. The complication rate was 26 percent and included patient death, dehiscence, and partial flap or split-thickness skin graft loss. Twenty-nine of the 32 wounds healed. One patient died in the postoperative period; in two others the wounds failed to heal and required below-knee amputations, for an overall limb salvage rate of 91 percent. Diabetes did not significantly affect healing and limb salvage rates. Diabetes, however, did affect healing times (twofold increase), length of stay (2.7 times as long), and long-term survival (63 percent survival in diabetic patients versus 100 percent in the trauma group). Local muscle flaps provide a simpler, less expensive, and successful alternative to microsurgical free flaps for foot and ankle defects that have exposed bone (with or without osteomyelitis), tendon, or joint at their base. Diabetes does not appear to adversely affect the effectiveness of these flaps. Local muscle flaps should remain on the forefront of possible reconstructive options when treating small foot and ankle wounds that have exposed bone, tendon, or joint.