Characterization of recombinant rat cathepsin B and nonglycosylated mutants expressed in yeast. New insights into the pH dependence of cathepsin B-catalyzed hydrolyses.

The cysteine proteinase rat cathepsin B was expressed in yeast in an active form and was found to be heterogeneously glycosylated at the consensus sequence for N-linked oligosaccharide substitution. Purified enzyme fractions containing the highest levels of glycosylation were shown to have reduced activity. A glycosylation minus mutant constructed by site-directed mutagenesis (by changing the Ser to Ala in the consensus sequence) was still secreted by the yeast and was shown to be functionally identical with purified rat liver cathepsin B. Recombinant cathepsin B was used to further characterize the pH dependence of cathepsin B-catalyzed hydrolyses using 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA) substrates with arginine as the P1, and either arginine or phenylalanine as the P2 residue. The AMC and pNA groups give insights into the leaving group binding site (P') of cathepsin B. These studies show for the first time that at least seven dissociable groups are involved in substrate binding and hydrolysis in cathepsin B activity. Two of these groups, with pKa values of 6.9 and 7.7 in the recombinant enzyme, are in the leaving group binding site and are most likely His110 and His111. The same groups in rat liver cathepsin B have higher pKa values than in recombinant cathepsin B, but have identical function in the two enzymes. Two other groups are probably the active site Cys29 and His199 with pKa values of 3.6 and 8.6, respectively. A group with a pKa of 5.1 interacts with substrates containing Arg at P2, and the group is most likely Glu245. The remaining two groups, one with a pKa of about 4.9 and the other about 5.3, are most likely carboxyl residues possibly interacting with Arg at P1 in the substrate. The possible candidates on the basis of the x-ray structure are Asp22, Asp69, Glu171, and Glu122, all found within a 13 A radius from the active site thiol of Cys29

Frequency of Different Ligament Tears in Knee Injury On Magnetic Resonance Imaging

Background: MRI had been useful in the diagnosis of ligament injuries and the tears were detected by non-invasive procedure. Objective: To determine the frequency of different ligament tears in knee injury on Magnetic Resonance Imaging. Methods: A descriptive cross-sectional was conducted with the sample size of 206 patients of both genders by selecting the convenient sampling from Ghurki Trust Teaching Hospital, Lahore. Out of 206 patients, 157 were males while 49 were females. Data was analyzed with the help of SPSS version 24. The results were derived by mean, frequency and standard deviation. Results: Findings shows that among 206 patients, with in age limit of 12 years to 70 years. 157(76.2%) were males and 49(23.7%) were female while 96(46.6%) were presented with ACL tear, 19(9.2%) were presented with PCL tears, 51(25.7%) were presented with MCL tears, 33(16.0%) patients were presented with LCL tears and all of these 206 patients were suffering from pain. Conclusion: We concluded that males are more prevalent than females and in this population the incidence of ACL tears is more than other ligament tears. Hence, ACL is proved to be the most injured ligament. Keywords: Anterior cruciate ligament, Posterior cruciate ligament, Medial collateral ligament, Lateral collateral ligament and Magnetic resonance imaging. DOI: 10.7176/JHMN/71-06 Publication date: February 29th 202

Characterization of cathepsin B specificity by site-directed mutagenesis. Importance of Glu245 in the S2-P2 specificity for arginine and its role in transition state stabilization.

The pH dependence of cathepsin B-catalyzed hydrolyzes is very complex. At least seven dissociable groups are involved in the binding and hydrolysis of 7-amido-4-methyl coumarin and p-nitroaniline (pNA)-based substrates containing a P1 Arg and either a Phe or Arg at the P2 position. By site-directed mutagenesis we show that a previous suggestion, that Arg202 is one of the groups which influences the pH dependence of cathepsin B-catalyzed hydrolysis of the Z-Arg-Arg-pNA substrate, is not valid. However, it was found that Glu245, which has a pKa of 5.1 in rat cathepsin B, is responsible for the S2-P2 specificity for Arg-containing substrates and controls the pH dependence of their hydrolysis. Furthermore, the data indicate that Glu245, which forms a hydrogen bond with the guanidinium group of the substrate's P2 Arg, contributes about 1.8 kcal/mol to transition state stabilization in the protonated state and about 0.6 kcal/mol in the deprotonated state. Mutation of Glu245 to Gln results in a 16-fold decrease in kcat but does not affect Km. While cathepsin B has a 7-fold preference for Phe over Arg at the P2 position of a substrate, binding of the aromatic side chain does not appear to be influenced by Glu245

Crystallization of recombinant rat cathepsin B.

A glycosylation-minus mutant of rat cathepsin B expressed in yeast has been purified and crystallized. X-ray diffraction data have been collected and molecular replacement for solving the structure is in progress. The space group for the recombinant rat cathepsin B was determined to be P2(1) with unit cell dimensions alpha = 62.2 A, b = 90.19 A, c = 47.07 A, and beta = 97.43 degrees. A unit cell contains 4 molecules and 2 molecules per asymmetric unit

Competitive science and technology intelligence

As industrial economies head irreversibly towards globalization, access to and applications of, new technological information from worldwide sources become critical. The National Research Council of Canada (NRC) has carried out an experiment to determine whether researchers from a national scientific institution can make a contribution to accessing technological information from foreign sources, analyzing that information, generating competitive intelligence and selectively disseminating that intelligence to Canadian firms. A trial copy of an S&T intelligence bulletin was prepared by NRC in order to determine the essential features of a competitive S&T intelligence service. The intelligence in the bulletin was generated from information gathered from worldwide sources by NRC researchers in the course of their normal activities. The bulletin was sent to a small number of technology-intensive firms and the usefulness of the bulletin was evaluated by follow-up interviews. A description of the process of generation and dissemination of intelligence and the formal evaluation of that experiment is presented.Peer reviewed: YesNRC publication: Ye

Investigating the Role of Employer Branding on Employees Performance with the moderating effect of Talent Management

The goal of this study is to ascertain how employer branding affects employee performance. The second goal of this research is to look at how talent management affects the relationship between employer branding and employee performance. The study's primary data was gathered using structured and closed-ended questions. Likert scales with 5-point likert scale, ranging from 1 to 5, have been used to gauge the level of reaction. This study's statistical analysis is based on a random sampling approach that used a questionnaire to collect answers from 208 respondents. The partial least squares (PLS) technique of structural equation modelling (SEM) was utilised to assess the predicted relationships between the study variables. The researcher also examined the structural model's direct and indirect relationships between the variables. The research's findings demonstrate that employer branding considerably affects workers' performance. It is a strong predictor of higher levels of performance, and talent management significantly moderate the relationship

Characterization of cathepsin B specificity by site-directed mutagenesis: Biol.Chem.

NRC publication: Ye

Paradoxical Reaction to Alprazolam in an Elderly Woman with a History of Anxiety, Mood Disorders, and Hypothyroidism

With less than 1% of patients who use benzodiazepines being affected, paradoxical responses to benzodiazepines are rare. In this case report, we outline the course of an 80-year-old female who developed a paradoxical response to benzodiazepines. Significant medical and psychiatric history includes anxiety, mood disorder, hypothyroidism, bilateral mastectomy, goiter removal, and triple bypass. The patient presented with mental status changes, anxiety, motor restlessness, and paranoia. Over time, a temporal relationship between the severity of the patient’s motor agitation and intake of alprazolam was observed. As doses of alprazolam were decreased, her motor agitation became less severe. In addition to motor agitation, the patient also demonstrated increased aggressiveness, a subjective feeling of restlessness, and increased talkativeness. As her dose of alprazolam decreased, many of the patient’s symptoms were observed to decrease. This case report also discusses theories regarding the pathophysiology of paradoxical reactions to benzodiazepines, known risk factors, and appropriate treatment

Purification, properties, and N-terminal amino acid sequence of certain 50S ribosomal subunit proteins from the archaebacterium Halobacterium cutirubrum

NRC publication: Ye