efficient-site-specific-antibody-labeling-strain-promoted-azide

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

genetic-incorporation-biosynthesized-l-dihydroxyphenylalanine-dopa

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine (DOPA) and Its Application to Protein Conjugation

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical ...

Sogang University

sogang university

Emphasis has been placed on understanding the mental health disparities that exist within minority populations in the United States. Disparities are of particular importance when examining access to services. This paper reports findings from a NINR-funded qualitative study that examined the barriers to mental health care service use in an African American low-income population. Barriers were identified at three levels--Individual, Environmental, and Institutional. Fourteen barrier categories were identified within those levels with three categories common to the majority of participants. The identified categories provide insight into the process of seeking services within this population and provide direction for future investigation.

Barriers to mental health care access in an African American population.

The purpose of this study was to examine factors that influence use of mental health service (MHS) by African Americans (AA) first-time voluntary users who accessed public MHS. Using a grounded theory approach, 24 participants were interviewed. Three major enabling factors - family, valued others, and beliefs - were identified that focused on the ability to secure needed MHS. Findings indicate an under identified and underused strategy for meeting first-time users' perceived needs. Findings have implications for culturally competent outreach and future research.

Enabling factors of mental health service use among African Americans.

To assess workplace tobacco use prevention and cessation policies in manufacturing facilities and explore factors associated with tobacco policies and practices in the tobacco-growing state of Kentucky.

Tobacco use prevention and cessation policies in manufacturing facilities in the tobacco-growing state of Kentucky.

This correlational pilot study measured limitations of prostate cancer screening, using a revised Knowledge of Prostate Cancer Questionnaire. Knowledge in 81 low-income men is reported. The Knowledge About Prostate Cancer Screening Questionnaire consists of 12 questions, with scores ranging from 0 to 12. Concepts measured include limitations, symptoms, risk factors, and screening age guidelines. The Total Knowledge Score had a mean of 6.60, with a standard deviation of 3.00, indicating that knowledge was low. Half of the men knew that "some treatments for prostate cancer can make it harder for men to control their urine." More than half of the men knew that, "some treatments for prostate cancer can cause problems with a man's ability to have sex." Married men, low-income men, and Caucasian men had significantly lower Total Knowledge Scores than unmarried, higher income, and African American men. Implications for practice and research are discussed.

Knowledge of the limitations associated with prostate cancer screening among low-income men.

The purpose of this study was to examine the attitudes toward mental health service (MHS) use within a sample of African-American and White adults (N = 739) and to identify correlates associated with those expressed attitudes. African-Americans (n = 132) and Whites (n = 607) were interviewed using the Louisville Metropolitan Survey that included the Attitudes Toward Seeking Professional Psychological Help Scale. Findings from this study indicated that responses regarding seeking mental health services were positively correlated with educational attainment and gender In addition, further findings also suggested that while race was significantly associated with attitudes toward seeking mental health services, it was also associated with prior familiarity with mental health services African-Americans reported both less willingness to seek mental health services and less familiarity with mental health services. The unexpected finding of the association between familiarity and attitudes toward mental health services use has value in furthering scientific inquiry. Investigation into the role of familiarity with mental health services and the decision-making process leading to mental health services use in diverse populations holds potential.

A comparison of influences on attitudes towards mental health service use in an African-American and White community.

The genetic incorporation of unnatural amino acids (UAAs) into proteins has been a useful tool for protein engineering. However, most UAAs are expensive, and the method requires a high concentration of UAAs, which has been a drawback of the technology, especially for large-scale applications. To address this problem, a method to recycle cultured UAAs was developed. The method is based on recycling a culture medium containing the UAA, in which some of essential nutrients were resupplemented after each culture cycle, and induction of protein expression was controlled with glucose. Under optimal conditions, five UAAs were recycled for up to seven rounds of expression without a decrease in expression level, cell density, or incorporation fidelity. This method can generally be applied to other UAAs; therefore, it is useful for reducing the cost of UAAs for genetic incorporation and helpful for expanding the use of the technology to industrial applications.

Genetic incorporation of recycled unnatural amino acids.

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance.

Direct protein-protein conjugation by genetically introducing bioorthogonal functional groups into proteins.

Periplasmic binding proteins (PBPs) are members of a widely distributed protein superfamily found in bacteria and archaea, and are involved in the cellular uptake of solutes. In this report, a leucine-binding PBP was engineered to detect l-Leu based on a fluorescence resonance energy transfer (FRET) change upon ligand binding. A fluorescent unnatural amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (CouA), was genetically incorporated into the protein as a FRET donor, and a yellow fluorescent protein (YFP) was fused with its N-terminus as a FRET acceptor. When CouA was incorporated into position 178, the sensor protein showed a 2.5-fold increase in the FRET ratio. Protein engineering significantly improved its substrate specificity, showing minimal changes in the FRET ratio with the other 19 natural amino acids and d-Leu. Further modification increased the sensitivity of the sensor protein (14-fold) towards l-Leu, and it recognized l-Met as well with moderate binding affinity. Selected mutant sensors were used to measure concentrations of l-Leu in a biological sample (fetal bovine serum) and to determine the optical purity of Leu and Met. This FRET-based sensor design strategy allowed us to easily manipulate the natural receptor to improve its binding affinity and specificity and to recognize other natural molecules, which are not recognized by the wild-type receptor. The design strategy can be applied to other natural receptors, enabling engineering receptors that sense biochemically interesting molecules.

Engineering a periplasmic binding protein for amino acid sensors with improved binding properties.

l-Dihydroxyphenylalanine (DOPA) was biosynthesized by a tyrosine-phenol lyase from catechol, pyruvate, and ammonia in Escherichia coli, and the biosynthesized amino acid was directly incorporated into proteins. Three biochemical experiments with mutant proteins containing DOPA confirmed the genetic incorporation of biosynthesized DOPA, and revealed its potential for various biochemical applications.

Sanggil Kim

Department of Chemistry

Sogang University

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine (DOPA) and Its Application to Protein Conjugation