Search

Human apurinic/apyrimidinic endonuclease APE1 catalyzes endonucleolytic hydrolysis of phosphodiester bonds on the 5′ side of structurally unrelated damaged nucleotides in DNA or native nucleotides in RNA. APE1 additionally possesses 3′-5′-exonuclease, 3′-phosphodiesterase, and 3′-phosphatase activities. According to structural data, endo- and exonucleolytic cleavage of DNA is executed in different complexes when the excised residue is everted from the duplex or placed within the intrahelical DNA cavity without nucleotide flipping. In this study, we investigated the functions of residues Arg177, Arg181, Tyr171 and His309 in the APE1 endo- and exonucleolytic reactions. The interaction between residues Arg177 and Met270, which was hypothesized recently to be a switch for endo- and exonucleolytic catalytic mode regulation, was verified by pre–steady-state kinetic analysis of the R177A APE1 mutant. The function of another DNA-binding–site residue, Arg181, was analyzed too; it changed its conformation when enzyme–substrate and enzyme–product complexes were compared. Mutation R181A significantly facilitated the product dissociation stage and only weakly affected DNA-binding affinity. Moreover, R181A reduced the catalytic rate constant severalfold due to a loss of contact with a phosphate group. Finally, the protonation/deprotonation state of residues Tyr171 and His309 in the catalytic reaction was verified by their substitution. Mutations Y171F and H309A inhibited the chemical step of the AP endonucleolytic reaction by several orders of magnitude with retention of capacity for (2 R ,3 S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran-containing-DNA binding and without changes in the pH dependence profile of AP endonuclease activity, indicating that deprotonation of these residues is likely not important for the catalytic reaction. • Endo- and exonuclease cleavage of a DNA by mutant forms of human APE1 was analyzed. • Process of protein-substrate interaction was analyzed by pre-steady state kinetics. • Conformational changes in APE1 and substrates were observed during their interactions. • The role of certain important amino acids during enzyme pathway was specified. [ABSTRACT FROM AUTHOR]

Background Contemporary EUS-guided FNA techniques involve the use of a needle, with an air column within the lumen, with or without suction. We describe a novel technique with an aim to improve the quality of the aspirate. Objective To compare a novel “wet suction” technique (WEST) with the conventional FNA technique (CFNAT) of EUS-guided FNA using a 22-gauge FNA needle. Design Prospective, single-blind, and randomized trial. Setting Two large tertiary-care hospitals. Patients All consecutive adult patients presenting for EUS with possible FNA of solid lesions were offered the chance to participate in the study. Methods All lesions were sampled with the same needle by using alternating techniques. Patients were randomized to the WEST versus the CFNAT for the first pass. If the first pass was made with the WEST, the second pass was made with the CFNAT, and subsequent passes were made in an alternating manner by using the same sequence. All FNAs were performed using 22-gauge needles. Main Outcome Measurements Specimen adequacy, cellularity, and blood contamination of EUS-guided FNA aspirates graded on a predefined scale. Results The WEST yielded significantly higher cellularity in a cell block compared with the CFNAT, with a mean cellularity score of 1.82 ± 0.76 versus 1.45 ± 0.768 ( P < .0003). The WEST cell block resulted in a significantly better specimen adequacy of 85.5% versus 75.2% ( P < .035). There was no difference in the amount of blood contamination between the 2 techniques. Limitations Lack of cross check and grading by a second cytopathologist. Conclusion The novel WEST resulted in significantly better cellularity and specimen adequacy in cell blocks of EUS-guided FNA aspirate of solid lesions than the CFNAT. [ABSTRACT FROM AUTHOR]

Abstract: A spectrophotometric assay for plasmin and plasminogen-derived activity in dairy products was optimised and extended to determine plasmin and plasminogen-derived activity in turbid samples of dairy products. The method was validated by assessing reproducibility, repeatability, level of detection and recovery of plasmin activity in different sample matrices. Plasmin activity in raw milk was not affected by skimming, but decreased by 30% in pasteurised and homogenised whole milk, leading to an underestimation of plasmin activity. The effects of dissociation of plasmin and caseins by ε-aminocaproic acid (EACA) plus NaCl on the plasmin activity were investigated. Comparison of pasteurised milk with a micellar casein solution showed that the dissociation of plasmin and caseins on adding EACA and NaCl decreases interference by caseins, but increases inhibition of plasmin with serum-based inhibitory components. The level of detection and repeatability of this method for plasmin activity analysis were improved compared with previous spectrophotometric assays. [Copyright &y& Elsevier]

Objectives: Varying levels of evidence exist for the contribution of indoor air pollution and environmental tobacco smoke as a risk factor for tuberculosis (TB). Despite a similar mechanism of action, the influence of outdoor air pollution exposure as an independent contributor to TB disease has yet to be explored. This area of inquiry is of increasing importance given the level of pollution in the rising economies of many TB-endemic nations. Los Angeles' unique physical environs and traffic patterns mirror other global megacities with a greater burden of TB therefore allowing for preliminary correlative studies. This preliminary study hypothesizes that individuals who reside proximal to elevated pollutant exposures are likely to have a greater burden of disease -- as evidenced by sputum smear-positive TB. Study design: Retrospective medical records review. Methods: Medical records of non-homeless individuals (n = 196) diagnosed with culture positive TB at Los Angeles County and University of Southern California Medical Center Hospital were analyzed from January 2007 to December 2008. The study population was grouped according to acid-fast bacilli (AFB) smear-positive (n = 111) and smear-negative (n = 85) status. Air pollutant exposure was captured using measurements of ozone (03) and particulate matter with an aerodynamic diameter of less than 2.5 (PM2.5). Individual assignment to O3 and PM2.5 exposures were based on residential proximity to the nearest US Environmental Protection Agency's monitoring station. Proximity of home residences to traffic-related pollutants occurred by measurement of distance to the nearest freeway and major non-freeway road. Results: Single factorial models yielded a significant correlation of smear-positive status and residential exposure to PM2.5 Residential distance to freeways and major arterial roads did not yield an association. Conclusions: This is the first report linking ambient pollution exposure as a risk factor for TB. PM2.5 may have the potential to impact TB lung pathology as evidenced by the linkage of fine particulate matter levels and smear-positive TB. [ABSTRACT FROM AUTHOR]

The effect of enzymatic activity in direct steam infusion heat treated milk with ultra-short holding times (>150 °C for <0.2 s) on age gelation during storage was investigated. Preheating at either 72 or 95 °C for 180 s was performed. Milk pre-heated at 72 °C showed extensive proteolysis and exhibited bitter off flavour and contained <40% intact αS- and β-caseins after 6 weeks storage at 20 °C. No proteolysis of κ-casein was detected. Plasmin was identified as active protease and activation of plasminogen was observed as an increase in the rate of casein hydrolysis. Proteolysis in the stored samples correlated with a decrease in pH and with changes in colour. Gelation occurred after 10 weeks along with an increase in viscosity and extensive proteolysis of αS- and β-caseins. In conclusion, plasmin activity was involved in age gelation and bitterness caused by proteolysis was the shelf-life limiting factor. [ABSTRACT FROM AUTHOR]

Human apurinic/apyrimidinic endonuclease APE1 is one of participants of the DNA base excision repair pathway. APE1 processes AP-sites and many other types of DNA damage via hydrolysis of the phosphodiester bond on the 5′ side of the lesion. APE1 also acts as an endoribonuclease, i.e., can cleave undamaged RNA. Using pre-steady-state kinetic analysis we examined the role of certain catalytically important amino acids in APE1 enzymatic pathway and described their involvement in the mechanism of the target nucleotide recognition. Comparative analysis of the cleavage efficiency of damaged DNAs containing an abasic site, 5,6-dihydrouridine, or α-anomer of adenosine as well as 3′-5′-exonuclease degradation of undamaged DNA and endonuclease hydrolysis of RNA substrates by mutant APE1 enzymes containing a substitution of an active-site amino acid residue (D210N, N212A, T268D, M270A, or D308A) was performed. Detailed pre–steady-state kinetics of conformational changes of the enzyme and of DNA substrate molecules during recognition and cleavage of the abasic site were studied. It was revealed that substitution T268D significantly disturbed initial DNA binding, whereas Asn212 is critical for the DNA-bending stage and catalysis. Substitution D210N increased the binding efficacy and blocked the catalytic reaction, but D308A decreased the binding efficacy owing to disruption of Mg2+ coordination. Finally, the substitution of Met270 also destabilized the enzyme–substrate complex but did not affect the catalytic reaction. It was found that the tested substitutions of the active-site amino acid residues affected different stages of the complex formation process as well as the catalytic reaction. • Target nucleotide recognition and cleavage of a DNA or RNA by human APE1 was analyzed • Efficiency of enzymatic hydrolysis of different substrates was compared • Process of protein-substrate interaction was analyzed by pre-steady state kinetics • Conformational changes in APE1 and substrates were observed during their interactions • The role of certain important amino acids during enzyme pathway was specified [ABSTRACT FROM AUTHOR]

Chymosin-induced cleavage of κ-casein (κ-CN) occurs during the first enzymatic phase in milk coagulation during cheese manufacturing, where the hydrophilic C-terminal peptide of κ-CN, named caseino-macropeptide (CMP), is released into the whey. The CMP peptide is known for its rather heterogeneous composition with respect to both genetic variation and multiple posttranslational modifications, including phosphorylation and O-linked glycosylation. An approach of liquid chromatography coupled with mass spectrometry was used to investigate (1) the overall protein profile and (2) the release of various forms of CMP after addition of chymosin to individual cow milk samples from 2 breeds, Danish Jersey (DJ) and Danish Holstein-Friesian (DH). The cows were selected to represent distinct homo- and heterozygous types of the κ-CN genetic variants A, B, and E (i.e., genotypes AA, BB, AB, EE, and AE). Initially, investigation of the protein profile showed milk with κ-CN BB exhibited the highest relative content of κ-CN, whereas AE milk exhibited the lowest, and after 40 min of renneting >90% of intact κ-CN was hydrolyzed by chymosin in milk representing all κ-CN genotype. By in-depth analysis of the CMP chromatographic profile, multiple CMP isoforms with 1 to 3 O-linked glycans (1-3 G) and 1 to 3 phosphate groups (1-3 P) were identified, as well as nonmodified CMP isoforms. The number of identified CMP isoforms varied to some extent between breeds (21 CMP isoforms identified in DJ, 26 CMP isoforms in DH) and between κ-CN genetic variants (CMP variant A being the most heterogeneous compared with CMP B and E), as well as between individual samples within each breed. The predominant forms of glycans attached to CMP were found to be the acidic tetrasaccharide {N-acetyl-neuraminic acid α(2-3)galactose β(1-3)[N-acetyl-neuraminic acid α(2-6)]N-acetyl galactose} or trisaccharides {N-acetyl-neuraminic acid α(2-3)galactose β(1-3)N-acetyl galactose and galactose β(1-3)[N-acetyl-neuraminic acid (α2-6)]N-acetyl galactose}. The CMP release was calculated to follow first-order kinetics and was determined by the measurement of CMP content during renneting. The highest rate of release for all CMP isoforms occurred from 0 to 2 min after chymosin addition. Concurring results from both breeds showed that CMP variant A with 1-2 P had the highest reaction rate of CMP release, followed by CMP B 1-2 P and then by CMP E 1-2 P (only in DH). All the identified glycosylated CMP isoforms had lower reaction rates of release compared with that of nonglycosylated CMP, thus glycan modifications seemed to negatively influence the reaction rate of chymosin-induced hydrolysis of κ-CN. [ABSTRACT FROM AUTHOR]

The aim of the present investigation was to study the underlying causes of noncoagulating (NC) milk. Based on an initial screening in a herd of 53 Danish Holstein-Friesians, 20 individual Holstein-Friesian cows were selected for good and poor chymosin-induced coagulation properties; that is, the 10 cows producing milk with the poorest and best coagulating properties, respectively. These 20 selected cows were followed and resampled on several occasions to evaluate possible changes in coagulation properties. In the follow-up study, we found that among the 10 cows with the poorest coagulating properties, 4 cows consistently produced poorly coagulating (PC) or NC milk, corresponding to a frequency of 7%. Noncoagulating milk was defined as milk that failed to form a coagulum, defined as increase in the storage modulus (G') in oscillatory rheometry, within 45 min after addition of chymosin. Poorly coagulating milk was characterized by forming a weak coagulum of low G'. Milk proteomic profiling and contents of different casein variants, ionic contents of Ca, P and Mg, Κ-casein (CN) genotypes, casein micelle size, and coagulation properties of the 4 NC or PC samples were compared with milk samples of 4 cows producing milk with good coagulation properties. The studies included determination of production of caseinomacropeptide to ascertain whether noncoagulation could be ascribed to the first or second phase of chymosin-induced coagulation. Caseinomacropeptide was formed in all 8 milk samples after addition of chymosin, indicating that the first step (cleavage of κ-CN) was not the cause of inability to coagulate. Furthermore, the effect of mixing noncoagulating and well-coagulating milk was studied. By gradually blending NC with well-coagulating milk, the coagulation properties of the well-coagulating samples were compromised in a manner similar to titration. Milk samples from cows that consistently produced NC milk were further studied at the udder quarter level. The coagulation properties of the quarter milk samples were not significantly different from those of the composite milk sample, showing that poor coagulation traits and noncoagulation traits of the composite milk were not caused by the milk quality of a single quarter. The milk samples exhibiting PC or NC properties were all of the κ-CN variant AA genotype, and contained casein micelles with a larger mean diameter and a lower fraction of κ-CN relative to total CN than milk with good coagulation properties. Interestingly, the relative proportions of different phosphorylation forms of α-CN differed between well-coagulating milk and PC or NC milk samples. The PC and NC milk samples contained a lower proportion of the 2 less-phosphorylated variants of α-CN (αS1-CN-8P and αS2-CN-11P) compared with samples of milk that coagulated well. [ABSTRACT FROM AUTHOR]

Tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non- receptor tyrosine kinases can phosphorylate PLCγ in vitro, the specific kinase(s) controlling BCR-dependent PLCγ activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCγ2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCγ2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCγ2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr753 and Tyr759. Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCγ2 carboxyl-terminal sites, Tyr1197 and Tyr1217, was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCγ2 SH2-SH3 linker. [ABSTRACT FROM AUTHOR]