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Biomedical subjects

J L Harris

Publications and source records attributed to J L Harris.

At least 19 recordsLinked to original sources
PubMed2000

Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and single-chain urokinase-type plasminogen activator as substrates.

Membrane-type serine protease 1 (MT-SP1) was recently cloned, and we now report its biochemical characterization. MT-SP1 is predicted to be a type II transmembrane protein with an extracellular protease domain. This localization was experimentally verified using immunofluorescent microscopy and a cell-surface biotinylation technique. The substrate specificity of MT-SP1 was determined using a positional scanning-synthetic combinatorial library and substrate phage techniques. The preferred cleavage sequences were found to be (P4-(Arg/Lys)P3-(X)P2-(Ser)P1-(Arg)P1'-(Ala)) and (P4-(X)P3-(Arg/Lys)P2-(Ser)P1(Arg) P1'(Ala)), where X is a non-basic amino acid. Protease-activated receptor 2 (PAR2) and single-chain urokinase-type plasminogen activator are proteins that are localized to the extracellular surface and contain the preferred MT-SP1 cleavage sequence. The ability of MT-SP1 to activate PARs was assessed by exposing PAR-expressing Xenopus oocytes to the soluble MT-SP1 protease domain. The latter triggered calcium signaling in PAR2-expressing oocytes at 10 nm but failed to trigger calcium signaling in oocytes expressing PAR1, PAR3, or PAR4 at 100 nm. Single-chain urokinase-type plasminogen activator was activated using catalytic amounts of MT-SP1 (1 nm), but plasminogen was not cleaved under similar conditions. The membrane localization of MT-SP1 and its affinity for these key extracellular substrates suggests a role of the proteolytic activity in regulatory events.

Animals↗
PubMed2000

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries.

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137, 180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.

Combinatorial Chemistry Techniques↗
PubMed2000

Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin.

We have developed a strategy for the synthesis of positional-scanning synthetic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent. To demonstrate the strategy, we synthesized a tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 members of the library were synthesized on solid support with an alkane sulfonamide linker, and then displaced from the solid support by condensation with a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin, which are involved in the blood coagulation pathway. The optimal P4 to P2 substrate specificity for plasmin was P4-Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmin's physiological substrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found in many of the physiological substrates of thrombin. Single-substrate kinetic analysis of plasmin and thrombin was used to validate the substrate preferences resulting from the PS-SCL. By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identified potential determinants of the defined substrate specificity. This method is amenable to the incorporation of diverse substituents at the P1 position for exploring molecular recognition elements in proteolytic enzymes.

Amino Acid Sequence↗
PubMed2000

The structure of the pro-apoptotic protease granzyme B reveals the molecular determinants of its specificity.

Granzyme B is a serine protease of the chymotrypsin fold that mediates cell death by cytotoxic lymphocytes. It is a processing enzyme, requiring extended peptide substrates containing an Asp residue. The determinants that allow for this substrate specificity are revealed in the three-dimensional structure of granzyme B in complex with a macromolecular inhibitor. The primary specificity for Asp occurs through a side-on interaction with Arg 226, a buried Arg side chain of granzyme B. An additional nine amino acids make contact with the substrate and define the granzyme B extended substrate specificity profile. The substrate determinants found in this structure are shared by other members of this protein class and help to reveal the properties that define substrate specificity.

Amino Acid Sequence↗
PubMed2000

Nested cladistic analysis indicates population fragmentation shapes genetic diversity in a freshwater mussel.

Recently developed phylogeographic analyses that incorporate genealogical relationships of alleles offer the exciting prospect of disentangling historical from contemporary events. However, the relative advantages and shortfalls of this approach remain to be studied. We compared the nested cladistic method to the more traditional analysis of variance approach in a study of intraspecific genetic variation in the freshwater mussel, Lampsilis hydiana. We surveyed 257 specimens for nucleotide sequence level variation in a fragment of the mitochondrial 16S rRNA gene. When compared side by side, nested cladistic analysis and analysis of molecular variance (AMOVA) identified fragmentation of Arkansas river populations from remaining populations to the southwest. Nested cladistic analysis identified a second, more recent separation of Ouachita and Upper Saline river populations that was not detected by AMOVA. Differences among analytical methods probably arise from treatment of spatial hierarchical information: hierarchical groups emerge via a parsimony criterion in nested cladistic analysis but must be specified a priori in AMOVA. Both methods identified significant genetic structure among localities within hierarchical groups. Results from AMOVA suggested little gene flow among local populations with an island model. However, inferences about process that gave rise to patterns at this level were not possible in nested cladistic analysis, because an ancestral (interior) haplotype was not observed for a key one-step clade in the parsimony network. Our results suggest that, under some circumstances, nested cladistic analysis has lower power than more traditional analysis of variance to infer processes at the local population level.

Analysis of Variance↗
PubMed1999

Physiologic basis of antenatal testing.

Hypoxic injury accounts for approximately 20 to 40% of stillbirths in published autopsy series. Antepartum fetal testing modalities can discriminate between the fetus at risk for hypoxic injury and the normally oxygenated fetus. These modalities have resulted in a significant reduction in this category of stillbirths. The physiologic underpinning for the antepartum testing is reviewed with a brief discussion of the two major approaches to testing.

Adaptation, Physiological↗
PubMed1998

Definition and redesign of the extended substrate specificity of granzyme B.

Granzyme B is a protease involved in the induction of rapid target cell death by cytotoxic lymphocytes. Definition of the substrate specificity of granzyme B allows for the identification of in vivo substrates in this process. By using the combinatorial methods of synthetic substrate libraries and substrate-phage display, an optimal substrate for granzyme B that spans over six subsites was determined to be Ile-Glu-Xaa-(Asp downward arrowXaa)-Gly, with cleavage of the Asp downward arrowXaa peptide bond. Granzyme B proteolysis was shown to be highly dependent on the length and sequence of the substrate, supporting the role of granzyme B as a regulatory protease. Arginine 192 was identified as a determinant of P3-Glu and P1-Asp substrate specificity. Mutagenesis of arginine 192 to glutamate reversed the preference for negatively charged amino acids at P3 to positively charged amino acids. The preferred substrate sequence matches the activation sites of caspase 3 and caspase 7 and thus is consistent with the role of granzyme B in activation of these proteases during apoptosis. The caspase substrate poly(ADP)-ribose polymerase is cleaved by granzyme B in a cell-free assay at two sites that resemble the granzyme B specificity determined by the combinatorial methods. Many caspase substrates contain granzyme B cleavage sites and are proposed as potential granzyme B targets, suggesting a redundant function with certain caspases.

Animals↗
PubMed1998

Engineering enzyme specificity.

Protein engineering is the application of knowledge to design and alter protein function and structure. Although powerful methods, from specific to random, have been developed for the redesign of protein architecture, their successful application is dependent on the information known about the protein. This database of information is providing a foundation for establishing rules that govern enzyme-substrate interactions.

Enzymes↗
PubMed1998

Written language comprehension in younger and older adults.

This study examined the effects of text genre and repeated reading on written language comprehension in younger (M = 21 years) and older (M = 72 years) healthy adults (N = 54). Participants verified four text-based statements (i.e., explicit, implicit, contradictory, and elaborated) after reading expository, narrative, and procedural texts. Verification accuracy was comparable for both age groups; however, text genre, statement-type, and repeated reading produced significant effects. Expository passages, explicit and implicit statements, and repeated reading yielded superior results. Procedural passages and contradictory and elaborated statements yielded less accurate results. Statement-types invoked multiple levels of cognitive representation across text genres and age groups. Overall, reading time was significantly faster for younger adults, and reading times were significantly faster for both age groups during the repeated reading trial. Text genre also influenced reading time, with expository passages read faster than narrative and procedural passages. These findings suggest the appreciable influences of text genre and repeated reading on measures of text processing and comprehension in healthy adults, irrespective of age.

Adolescent↗
PubMed1998

Urban African American adolescent parents: their perceptions of sex, love, intimacy, pregnancy, and parenting.

This study examined the perceptions of twenty-two urban African American adolescent mothers and six adolescent fathers (across seven grade levels and a general education equivalency program) regarding sex, love, intimacy, pregnancy, and parenting. Structured interviews and focus groups were used to gather data. Demographic and personal history information was also obtained. Inductive data analysis using constant comparison methods was employed to identify patterns and themes within and across gender groups.

Adolescent↗
PubMed1997

P-4 and RNKP-7, new granzyme-like serine proteases expressed in activated rat lymphocytes.

Serine proteases (granzymes) in killer lymphocytes are required for lymphocyte cytotoxic granules to lyse target cells. Herein we report the development of a 3-step PCR cloning technique to amplify novel granzyme genes and two new rat granzymes are described. Degenerate oligonucleotide primers were designed based on sequence motifs selectively expressed in granzymes. These motifs flank "delta" regions that are unique for each granzyme. Total RNA of RNK-16 cells or activated splenocytes was amplified by reverse transcriptase-PCR to obtain cDNA fragments of several new granzymes. Gene-specific primers based on these delta regions were then used for 3'-RACE to obtain clones with the 3' gene ends. Reverse (antisense) delta-based or active site serine primers were used with a granzyme 5'-UTR primer to obtain clones extending to the 5' ends. Using this technique, two new cDNAs, RNKP-4 and RNKP-7, which encode granzymes of 248 and 241 amino acids, respectively, were cloned from activated lymphocytes. RNKP-4 is likely the rat equivalent of mouse granzyme C. RNKP-7 is most closely related to granzymes F and G. Modeling of the predicted proteins suggests large/polar P1 (Gln/Asn) specificity for RNKP-4 and large/hydrophobic P1 (e.g., Phe) specificity for RNKP-7. These specific protease activities were found in cytotoxic RNK-16 lymphocyte granules indicating that the two new genes may be translated and stored as active granzymes.

Amino Acid Sequence↗
PubMed1997

Dental erosion in a wine merchant: an occupational hazard?

This paper reports a case of dental erosion apparently caused by wine tasting. Dental erosion can cause extensive damage to the dentition, thereby creating a variety of complex restorative problems. Within the literature dental erosion has been attributed to many factors, but to date there are no documented cases specifically linking it with wine tasting. The dental professional is ideally placed to implement early preventive regimes which would make the occupation less damaging to the dentition.

Food Handling↗
PubMed1997

Treatment of postprostatectomy urinary incontinence with behavioral methods.

Prostate cancer is the most commonly diagnosed cancer in men. The radical prostatectomy is the treatment of choice for localized cancer; however, this surgery carries with it the potential complication of urinary incontinence postoperatively. The increasing number of radical prostatectomies each year will thus lead to a growing number of patients suffering from postprostatectomy urinary incontinence. According to the medical literature, the treatment of postprostatectomy incontinence is almost always limited to pharmacological or surgical therapy. Because of the failure of the standard treatment options, many men are left to live with their incontinence. The guidelines for the treatment of urinary incontinence recommend the use of behavioral methods as first-line treatment for stress and urge incontinence. The potential treatment of postprostatectomy incontinence with the use of behavioral methods has a promising future for the advanced practice nurse and for the men who are affected.

Aged↗
PubMed1996

Distinguishing detection from identification in subliminal auditory perception: a review and critique of Merikle's study.

Assertions made by Merikle (1988) regarding audio subliminal messages were tested. Seventeen participants were presented subliminal messages embedded in a white-noise cover, and three signal-to-noise (S/N) detection ratios were examined. Participants were asked to guess message presence and message content, to determine subjective/objective thresholds. Results showed that participants were unable to identify target words presented in this audio subliminal stimulus format beyond chance levels.

Adolescent↗
PubMed1992

Upward mobility for LPNs: a vision made reality.

A recurrent theme in the nursing literature is the shortage of realistic, long-term solutions directed toward registered nurse (RN) recruitment and retention. If fragmentation of RN recruitment and retention is to be eliminated, innovative programs involving collaboration and use of existing resources will be required. This article provides an overview of how collaboration among nurses, administrators, educators, and a personnel specialist resulted in an upward mobility program for licensed practical nurses (LPNs) to become RNs. While the program is specific to one setting, there are numerous implications for other hospitals confronted with persistent recruitment and retention problems.

Career Mobility↗
PubMed1992

Sociologic aspects of pregnancy.

Increasingly, social factors are recognized as having a major impact on pregnancy, childbearing, and prenatal care. This review examines some of the more recent societal developments that have influenced health care for pregnant women. Although human gestation is a biologic phenomena, it exists with the social context of personal needs, family, and community of the pregnant women. This social fabric determines the perception, management, and outcome of that gestation. Significant influences to examine include pregnant women in the work force; malpractice concerns of health care providers; issues related to the cost of health care in pregnancy; the legal rights of the pregnant woman versus the legal rights of the fetus; pregnancy and occupational health; and the role of stress in pregnancy outcome.

Career Choice↗