Judy A. Sakanari

Bio: Judy A. Sakanari is an academic researcher from University of California, San Francisco. The author has contributed to research in topics: Proteases & Protease. The author has an hindex of 28, co-authored 76 publications receiving 2614 citations. Previous affiliations of Judy A. Sakanari include United States Department of Veterans Affairs & University of California, Santa Cruz.

Cysteine protease inhibitors as chemotherapy: Lessons from a parasite target

Abstract: Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite’s lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

Molecular phylogenetics and diagnosis of Anisakis, Pseudoterranova, and Contracaecum from northern Pacific marine mammals.

Abstract: Individual specimens of Anisakis, Pseudoterranova, and Contracaecum collected from marine mammals inhabiting northern Pacific waters were used for comparative diagnostic and molecular phylogenetic analyses. Forty-eight new sequences were obtained for this study of 14 Anisakis taxa, 8 Pseudoterranova taxa, 4 Contracaecum taxa, and 4 outgroup species. Partial 28S (LSU) and complete internal transcribed spacer (ITS-1, 5.8S, ITS-2) ribosomal DNA was amplified by the polymerase chain reaction and sequenced. Sequences of ITS indicated that Pseudoterranova specimens from Zalophus californianus (California sea lion), Mirounga angustirostris (northern elephant seal), Phoca vitulina (harbor seal), Enhydra lutris (sea otter), and Eumetopias jubatus (Steller's sea lion) exactly matched P. decipiens s. str., extending the host and geographic range of this species. Anisakis from northern Pacific marine mammals were most closely related to members of the A. simplex species complex. Comparison of Anisakis ITS sequences diagnosed the presence of A. simplex C in 2 M. angustirostris hosts, which is a new host record. Anisakis specimens from Phocoena phocoena (harbor porpoise), Lissodelphis borealis (Pacific rightwhale porpoise), and E. jubatus included 3 ITS sequences that did not match any known species. Contracaecum adults obtained from Z. californianus were most closely related to C. ogmorhini s.l. and C. rudolphii, but ITS sequences of these Contracaecum specimens did not match C. ogmorhini s. str. or C. margolisi. These novel Anisakis and Contracaecum ITS sequences may represent previously uncharacterized species. Phylogenetic analysis of LSU sequences revealed strong support for the monophyly of Anisakinae, Contracaecum plus Phocascaris, Pseudoterranova, and Anisakis. Phylogenetic trees inferred from ITS sequences yielded robustly supported relationships for Pseudoterranova and Anisakis species that are primarily consistent with previously published phenograms based on multilocus electrophoretic data.

WormAssay: a novel computer application for whole-plate motion-based screening of macroscopic parasites.

Abstract: Lymphatic filariasis is caused by filarial nematode parasites, including Brugia malayi. Adult worms live in the lymphatic system and cause a strong immune reaction that leads to the obstruction of lymph vessels and swelling of the extremities. Chronic disease leads to the painful and disfiguring condition known as elephantiasis. Current drug therapy is effective against the microfilariae (larval stage) of the parasite, but no drugs are effective against the adult worms. One of the major stumbling blocks toward developing effective macrofilaricides to kill the adult worms is the lack of a high throughput screening method for candidate drugs. Current methods utilize systems that measure one well at a time and are time consuming and often expensive. We have developed a low-cost and simple visual imaging system to automate and quantify screening entire plates based on parasite movement. This system can be applied to the study of many macroparasites as well as other macroscopic organisms.

Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes

Abstract: Serine proteases are one of the biologically most important and widely distributed families of enzymes. Isolation of serine protease genes from organisms of widely diverged phylogenetic groups would provide a basis for studying their biological function, the relationship between structure and function, and the molecular evolution of these enzymes. Serine proteases for which little structural information is known are those that are important in the pathogenesis of parasitic nematode and protozoan diseases. Identification and isolation of protease genes from these organisms is a critical first step in understanding their function for the parasite and possibly suggesting innovative approaches to arresting parasitic diseases. Serine protease gene fragments were isolated from genomic DNA of the parasitic nematode Anisakis simplex by using degenerate oligonucleotide primers and the polymerase chain reaction. Primers were designed based upon the consensus sequence of amino acids flanking the active site serine and histidine residues of eukaryotic serine proteases. Four serine protease gene fragments from this parasite were sequenced and one is 67% identical to the rat trypsin II gene. Alignment of these two genes revealed that the intron-exon junctions are conserved between nematode and rat suggesting that this Anisakis serine protease is structurally and functionally similar to rat trypsin. The generality of this approach to identify serine protease genes from genomic DNA of two very divergent species, a parasitic protozoan and a mammal, was also confirmed. Genes for other enzymes or any protein with conserved structural motifs can be identified and isolated using this technology. Using a similar strategy, a cathepsin B-like cysteine (thiol) protease gene fragment was isolated from Caenorhabditis elegans DNA.

Serine proteases from nematode and protozoan parasites: Isolation of sequence homologs using generic molecular probes (molecular evolution/trypsin/cysteine/active sites/polymerase chain reaction)

Abstract: Serine proteases are one of the biologically most important and widely distributed families of enzymes. Isolation of serine protease genes from organisms of widely diverged phylogenetic groups would provide a basis for study- ing their biological function, the relationship between structure and function, and the molecular evolution of these enzymes. Serine proteases for which little structural information is known are those that are important in the pathogenesis of parasitic nematode and protozoan diseases. Identification and isolation of protease genes from these organisms is a critical first step in understanding their function for the parasite and possibly suggesting innovative approaches to arresting para- sitic diseases. Serine protease gene fragments were isolated from genomic DNA of the parasitic nematode Anisakis simplex by using degenerate oligonucleotide primers and the polymer- ase chain reaction. Primers were designed based upon the consensus sequence of amino acids flanking the active site serine and histidine residues of eukaryotic serine proteases. Four serine protease gene fragments from this parasite were sequenced and one is 67% identical to the rat trypsin II gene. Alignment of these two genes revealed that the intron-exon junctions are conserved between nematode and rat suggesting that this Anisakis serine protease is structurally and function- ally similar to rat trypsin. The generality of this approach to identify serine protease genes from genomic DNA of two very divergent species, a parasitic protozoan and a mammal, was also confirmed. Genes for other enzymes or any protein with conserved structural motifs can be identified and isolated using this technology. Using a similar strategy, a cathepsin B-like cysteine (thiol) protease gene fragment was isolated from Caenorhabditis elegans DNA. Serine proteases are one of the most important families of enzymes found in nature. Members of this ubiquitous class of proteases hydrolyze peptide bonds and are involved in a broad range of biological processes including intra- and extracellular protein metabolism, digestion, blood coagula- tion, clot dissolution, immunological response, developmen- tal regulation, and fertilization (1-4). The three-dimensional structure of serine proteases, in addition to biophysical, molecular biological, and enzymological studies, provides very useful models to understand the mechanism of enzyme action, the basis of substrate specificity, and the molecular evolution of the enzymes themselves (5-15). Aside from their role in the physiology and metabolism of organisms, serine proteases have also been implicated in the pathogenesis of a number of infectious diseases. Among the most prevalent of these are parasitic diseases, such as schis- tosomiasis and onchocerciasis (African river blindness), which The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ?1734 solely to indicate this fact. represent some of the world's greatest health problems (16- 18). Parasite proteases may facilitate invasion of host tissue, metabolism of host proteins, and evasion of the host immune response. A generic molecular technology for isolation of serine protease genes from diverse sources would be of immense value in expanding the data base for serine proteases and to further our understanding of the function of these enzymes in a variety of organisms. In the first step of developing such a generic molecular strategy, we report the isolation of four serine protease genes from a parasitic nematode using degenerate oligonucleotide primers and genomic DNA. These primers were designed based upon mammalian serine protease consensus sequences encoding the active site amino acids and were used to initiate the polymerase chain reaction (PCR) on genomic DNA from the nematode. The applicability of this technique to a wider spectrum of the eukaryotic serine protease family was con- firmed when these primers and the PCR were used also to identify serine proteases from two widely diverged phyloge- netic groups, mammals and protozoa. Analysis of amplified fragments revealed that the catalytic triad of the serine protease active site was conserved among these organisms. Anisakiasis is a human disease caused by the ingestion of the larval nematode Anisakis found in raw seafood dishes such as sushi and sashimi (19). This parasite can be invasive and penetrate the wall of the stomach or intestine. We have found that secretions of tissue-invasive larvae contain a trypsin-like serine protease that may facilitate the invasion of host tissue (20). Based upon the enzymological characteriza- tion of this enzyme by substrate gel electrophoresis, peptide substrate assays, and inhibition studies, we hypothesized that it was structurally related to the trypsin family of serine proteases and not the subtilisin family. Western blot analysis of Anisakis extract with rat trypsin antisera identified a protein (Mr, 25,000) that shared similar epitopes with a mammalian trypsin (unpublished data). All known members of the eukaryotic serine protease family have serine, histidine, and aspartic acid at the active site of the enzyme at amino acid positions 195, 57, and 102, respectively (21). The serine and histidine residues are known to be required for enzymatic activity based on chemical modification experiments using organophosphates for Ser- 195 (22) and chloromethyl ketone affinity reagents for His-57 (23). X-ray crystallography and site-directed mutagenesis were used to verify the role of Asp-102 in catalysis (24, 25). Alignment of representative serine protease sequences re- veals that amino acids flanking these active site residues are also conserved (26). Use of oligonucleotide probes based on these consensus sequences to screen cDNA or genomic libraries has not been a successful approach to isolation of serine protease cDNA or genes, because the corresponding

“Bioinformatics” 특집을 내면서

Abstract: BIOE 402. Medical Technology Assessment. 2 or 3 hours. Bioentrepreneur course. Assessment of medical technology in the context of commercialization. Objectives, competition, market share, funding, pricing, manufacturing, growth, and intellectual property; many issues unique to biomedical products. Course Information: 2 undergraduate hours. 3 graduate hours. Prerequisite(s): Junior standing or above and consent of the instructor.

Development and Validation of A

Abstract: Objectives: Lao Juan (LJ, 劳倦) is a syndrome described in Chinese medicine (CM) that manifests with : Lao Juan (LJ, 劳倦) is a syndrome described in Chinese medicine (CM) that manifests with fatigue, fever, spontaneous sweating, indigestion, work-induced pain, weakness of the limbs, and shortness of breath. fatigue, fever, spontaneous sweating, indigestion, work-induced pain, weakness of the limbs, and shortness of breath. The present study was conducted to examine the reliability and validity of a Lao Juan Questionnaire (LJQ). The present study was conducted to examine the reliability and validity of a Lao Juan Questionnaire (LJQ). Methods: A total of 151 outpatients and 73 normal subjects were asked to complete the LJQ. Seventy-three normal subjects A total of 151 outpatients and 73 normal subjects were asked to complete the LJQ. Seventy-three normal subjects were additionally asked to complete the Chalder Fatigue Scale (CFS). Twelve clinicians determined whether the were additionally asked to complete the Chalder Fatigue Scale (CFS). Twelve clinicians determined whether the 151 outpatients exhibited LJ or not. The internal consistency and construct validity for the LJQ were estimated using 151 outpatients exhibited LJ or not. The internal consistency and construct validity for the LJQ were estimated using data from the outpatient subjects. The CFS data were used to examine the concurrent validity of the LJQ. Total LJQ data from the outpatient subjects. The CFS data were used to examine the concurrent validity of the LJQ. Total LJQ scores and the clinicians' diagnoses of the outpatients were used to perform receiver operating characteristics (ROC) scores and the clinicians' diagnoses of the outpatients were used to perform receiver operating characteristics (ROC) curve analyses and to defi ne an optimum cut-off score for the LJQ. curve analyses and to defi ne an optimum cut-off score for the LJQ. Results: The 19-item LJQ had satisfactory internal : The 19-item LJQ had satisfactory internal consistency (α=0.828) and concurrent validity, with signifi cant correlations between the LJQ and the CFS subscales. consistency (α=0.828) and concurrent validity, with signifi cant correlations between the LJQ and the CFS subscales. In the test of construct validity using principal component analysis, a total of six factors were extracted, and the overall In the test of construct validity using principal component analysis, a total of six factors were extracted, and the overall variance explained by all factors was 59.5%. In ROC curve analyses, the sensitivity, specifi city, and area under the variance explained by all factors was 59.5%. In ROC curve analyses, the sensitivity, specifi city, and area under the curve were 76.0%, 59.2%, and 0.709, respectively. The optimum cut-off score was defi ned as six points. curve were 76.0%, 59.2%, and 0.709, respectively. The optimum cut-off score was defi ned as six points. Conclusions: Our results suggest that the LJQ is a reliable and valid instrument for evaluating LJ. Our results suggest that the LJQ is a reliable and valid instrument for evaluating LJ. KEYWORDS Chinese medicine, chronic fatigue syndrome, Chinese medicine-pattern Chinese medicine, chronic fatigue syndrome, Chinese medicine-pattern

Comparative Protein Structure Modeling Using MODELLER

Abstract: Functional characterization of a protein sequence is a common goal in biology, and is usually facilitated by having an accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described.

Comparative protein structure modeling using Modeller.

Abstract: Functional characterization of a protein sequence is one of the most frequent problems in biology. This task is usually facilitated by accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described.