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S Graves

Publications and source records attributed to S Graves.

At least 19 recordsLinked to original sources
PubMed2001

Identification and in vivo characterization of the Epiphyas postvittana nucleopolyhedrovirus Ecdysteroid UDP-glucosyltransferase.

The genome of Epiphyas postvittana Nucleopolyhedrovirus (EppoMNPV) contains an ecdysteroid UDP-glucosyltransferase (egt) gene. The egt gene was completely sequenced and surrounding open reading frames identified. EppoMNPV egt is 1479 nucleotides in length encoding a protein with a predicted molecular mass of 55 kDa. Analysis of upstream sequence revealed dual TATA boxes and two CGT upstream activating region motifs. Mapping of the 5' terminus of the egt transcript identified a major transcript produced from an adenine residue 29 nucleotides downstream from the distal TATA box. No transcript was detected from a late promoter motif (GTAAG). Characterization of egt transcripts showed that poly-adenylation occurs at the 3' terminus. EppoMNPV egt transcripts were first detected in infected Epiphyas postvittana larvae by Northern hybridization at 9 h post-infection (hpi) and EGT enzyme activity was detected at 9 hpi in haemolymph from infected larvae. EppoMNPV EGT can conjugate the sugars from both UDP-glucose and UDP-galactose to ecdysone in vitro. Localization assays performed using radiolabelled ecdysone demonstrated that the conjugation of glucose or galactose, from the respective UDP-sugar, led to the prevention of the uptake of ecdysone by SF-21 cells. We propose that EGT functions to prevent the uptake of ecdysone or 20-hydroxyecdysone by metabolite and target cells respectively.

Amino Acid Sequence↗
PubMed2000

Oral administration of dimethylvinyl chloride increases frequency of forestomach papillomas in Tg.AC mice.

This work was initiated to determine the potential for the Tg.AC mouse model to identify chemical carcinogens by an oral route of administration. Tg.AC v-Ha-ras transgenic mice were exposed to dimethyvinyl chloride (DMVC; 1-chloro-2-methylpropene), a structural analog of the human carcinogen vinyl chloride. In the National Toxicology Program 2-yr bioassay, DMVC induced tumors in the oral, nasal, and gastric epithelia of rats and mice. Initial studies were performed in female Tg.AC mice to determine an appropriate oral dose of DMVC to evaluate the potential for stratified gastric or oral epithelia of Tg.AC mice to serve as a target tissue for a transgene-dependent induced tumorigenic response. DMVC was administered to 13- to14-wk-old Tg.AC mice by gavage at doses of 0, 50, 100, and 200 mg/kg five times a week for 20 wk. The forestomachs of DMVC-treated Tg.AC mice had an increasing number of papillomas, which were associated with an increase in the dose of DMVC. The average numbers of papillomas per mouse per dose were 2.4, 7.6, 14.1, and 12.6 for the 0, 50, 100, and 200-mg/kg dose groups, respectively. The optimum papillomagenic dose of 100 mg/kg DMVC was established and administered for 5, 10, and 15/wk to investigate the kinetics of papilloma induction in Tg.AC mice. The average numbers of papillomas per animal were 1.8, 8.8, and 19.0 at 5, 10, and 15 wk, respectively. Reverse transcription-polymerase chain reaction assays determined that the v-Ha-ras transgene was transcriptionally active in all tumor tissues but not in nontumor tissues. In situ hybridization assays performed in conjunction with bromodeoxyuridine in vivo labeling localized the transgene-expressing cells of the forestomach papillomas to the proliferating cellular component of the tumors, as previously seen in skin papillomas of Tg.AC mice. The present results confirm that DMVC is tumorigenic and that oral routes of administration can be used to rapidly elicit a transgene-associated tumor response in the forestomach of Tg.AC mice.

Administration, Oral↗
PubMed2000

Therapeutic monitoring of sirolimus in human whole-blood samples by high-performance liquid chromatography.

BACKGROUND: Sirolimus is a macrolide antibiotic isolated from Streptomyces hygroscopicus that has demonstrated immunosuppressive activity. Human and animal studies have shown a good correlation of trough sirolimus concentrations with immunosuppressive efficacy. OBJECTIVE: This report describes a reverse-phase high-performance liquid chromatography (HPLC) method used for therapeutic drug monitoring of sirolimus. METHODS: A reverse-phase C18 column method was developed using an automated HPLC system and ultraviolet (UV) detection. Whole-blood samples collected in ethylenediamine-tetraacetic acid (EDTA) are first hemolyzed, and an internal standard (desmethoxysirolimus) is added to 1.0 mL of sample, which is then extracted with 1-chlorobutane and, after the organic layer is removed, evaporated to dryness. The residue is reconstituted in a 70% methanol/water mixture. Reconstituted extracts are analyzed by HPLC at a column temperature of 60 degrees C and a flow rate of 1.0 mL/min. Typically, chromatography requires 35 minutes between each sample injection. The UV detector is set at 278 nm with a response sensitivity of 0.010 AUFS (absorbance units full scale). Standards and controls prepared in hemolyzed EDTA-anticoagulated whole blood are extracted and run in parallel. Identification of peaks of interest is by retention time; quantification of sirolimus in controls and clinical samples uses a peak-height ratio (sirolimus/internal standard). RESULTS: The assay's precision (coefficients of variation, 5.7%-14.4%) and sensitivity (2.5 ng/mL) were found to be appropriate for therapeutic monitoring purposes. Analytical recovery of 88.0% to 106.3% was observed throughout the assay's linear range (2.5-150.0 ng/mL). Stability studies at 20 degrees C to 25 degrees C and 2 degrees C to 8 degrees C showed an estimated recovery of sirolimus ranging from 85% to 110% of target concentrations (10-90 ng/mL). In a study comparing the results of 194 samples from kidney transplant recipients assayed by the HPLC-UV assay and by a microparticle enzyme immunoassay, the HPLC-UV method provided approximately 10% lower values. CONCLUSION: The HPLC-UV assay is analytically capable of providing useful data for the clinical assessment of patients receiving sirolimus.

Biotransformation↗
PubMed2000

DNAssist: the integrated editing and analysis of molecular biology sequences in windows.

MOTIVATION: The programs currently available for the analysis of nucleic acid and protein sequences suffer from a variety of problems: Web-based programs often require inconvenient reformatting of sequences when proceeding from one analysis to the next, and commercial-console-based programs are cost prohibitive. Here, we report the development of DNASSIST:, an inexpensive, multiple-document, interface program for the fully integrated editing and analysis of nucleic acid and protein sequences in the familiar environment of Microsoft Windows.

Animals↗
PubMed2000

DNAssist, a C++ program for editing and analysis of nucleic acid and protein sequences on PC-compatible computers running Windows 95, 98, NT4.0 or 2000.

The size of sequence databases, the rapid rate of generating sequence data and the need to routinely construct recombinant DNA molecules in molecular biological research necessitate the frequent handling and analysis of nucleotide and protein sequences on a computer. Although several console or Web-based utility programs are available, the application of these programs generally requires reformatting the data when proceeding from one such program to the next. The acquisition of elaborate, integrated program suites is financially prohibitive to smaller laboratories. Here, we report the development of DNAssist, a shareware program for editing and analysis of nucleic acid and protein sequences. It was developed as a multiple-document interface program--similar to a word processor--where sequences are entered, edited and analyzed in a single integrated environment. DNAssist can calculate the physicochemical properties of a sequence, convert between nucleic acid and protein sequences, translate DNA in multiple frames, identify open reading frames and locate ambiguous sequence patterns allowing gaps and mismatches. DNAssist also performs restriction enzyme and transcription factor-binding site analyses of DNA sequences, the multiple alignment of nucleic acid and protein sequences and the analysis of DNA sequences for nucleosome positioning sites.

Amino Acid Sequence↗
PubMed2000

Clinical optimization of pretargeted radioimmunotherapy with antibody-streptavidin conjugate and 90Y-DOTA-biotin.

UNLABELLED: Pretargeted radioimmunotherapy (PRIT) was evaluated using an antibody-streptavidin conjugate, followed by a biotin-galactose-human serum albumin clearing agent and 90Y-dodecane tetraacetic acid (DOTA)-biotin as the final step for therapy. The objective was to develop a clinical protocol that could show an improved tumor-to-red marrow therapeutic ratio compared with conventional radioimmunotherapy (RIT) and at the same time preserve the efficiency of tumor targeting. METHOD: Forty-three patients with adenocarcinomas reactive to NR-LU-10 murine monoclonal antibody received the 3 components. Doses and timing parameters were varied to develop an optimized schema. In some patients, the conjugate was radiolabeled with 186Re as an imaging tracer to assess biodistribution of the conjugate and effectiveness of the clearing agent. 111In-DOTA-biotin was coinjected with 90Y-DOTA-biotin for quantitative imaging. Safety, biodistribution, pharmacokinetics, dosimetry, and antiglobulin formation were evaluated. RESULTS: The optimal schema was defined as a conjugate dose of 125 microg/mL plasma volume followed at 48 h by a clearing agent in a 10:1 molar ratio of clearing agent to serum conjugate. The therapeutic third step was 0.5 mg radiobiotin administered 24 h later. No significant adverse events were observed after administration of any of the components. The mean tumor-to-marrow absorbed dose ratio when using the optimized PRIT schema was 63:1, compared with a 6:1 ratio reported previously for conventional RIT. Antiglobulin to murine antibody and to streptavidin developed in most patients. CONCLUSION: This initial study confirmed that the PRIT approach is safe and feasible and achieved a higher therapeutic ratio than that achieved with conventional RIT using the same antibody.

Adenocarcinoma↗
PubMed2000

Modular robotic workcell for coagulation analysis.

BACKGROUND: Total laboratory automation (TLA) has been shown to increase laboratory efficiency and quality. However, modular automation is smaller, requires less initial capital, and requires less planning than TLA. We engineered and performed clinical trials on a modular robotic preanalytical workcell for coagulation analysis. METHODS: Timing studies were used to quantify the efficiency of the manual processes and to identify areas in the processing of coagulation specimens where bottlenecks and long waiting periods were encountered. We then designed our modular robotic system to eliminate these bottlenecks. Our robotic modular workcell was engineered to allow a choice of specimen introduction manually, by conveyor, or by mobile robot. Additional timing studies were performed during clinical trials of the robotic system. RESULTS: Prior to automation, the time required for preanalytical processing time was 18-107 min; after automation, it was 45-50 min. Additional improvements in workcell efficiency could be realized when high quality, prelabeled specimens were introduced into the system. CONCLUSION: Compared with manual methods, modular automation provides more predictable variation in specimen processing.

Blood Chemical Analysis↗
PubMed1999

Analysis of various nucleosides in plasma using solid phase extraction and high-performance liquid chromatography with UV detection.

The National Cancer Institute (NCI) has screened many nucleosides for antiviral activity to the HIV-1 virus. Drugs demonstrating antiviral activity are tested in animal models to evaluate their toxicity and pharmacokinetic characteristics. These drugs are subsequently evaluated for efficacy in human clinical trials. Sensitive analytical methodology is needed to quantify nucleosides in plasma and other biological matrices in support of these studies. Battelle has modified and validated a reversed phase high-performance liquid chromatography (HPLC) method for several of these nucleosides that could be easily adapted for similar compounds. Methods have been validated for 6-chloro-2',3'-dideoxyguanosine (6ClddG), 6-chloro-2',3'-dideoxyinosine (6ClddI) and their primary metabolites 2',3'-dideoxyguanosine (ddG) and 2',3'-dideoxyinosine (ddI) in both rat and dog plasma containing EDTA. The method has also been validated for 2'-fluoro-2',3'-dideoxyara-adenosine (betaFlddA) and its primary metabolite 2'-beta-fluorodideoxyinosine (betaFddI) in rat plasma containing heparin. Calibration plasma standards were prepared over ranges of 0.1-10 microg ml(-1) for betaFlddA and betaFddI, 0.1-50 microg ml(-1) for 6ClddG and ddG, and 0.25-50 microg ml(-1) for 6ClddI and ddI in plasma containing 4 microg ml(-1) pentostatin. The addition of pentostatin to the plasma samples inhibits in-vitro deamination of the drug after collection. Quality control (QC) standards were prepared containing the appropriate anticoagulant and 4 microg ml(-1) pentostatin at concentrations within each of the bracketed calibration ranges in plasma. These methods have been successfully applied to plasma samples generated during various animal studies.

Animals↗
PubMed1999

Rickettsia serosurvey in Kimberley, Western Australia.

To determine if antibodies to rickettsiae (scrub typhus, spotted fever, and typhus group rickettsiae) occur among persons living in the Kimberley (northern tropical) region of Western Australia, 920 sera collected in a non-random manner in 1996 from patients in Kununurra, Broome, Fitzroy Crossing, Wyndham, Derby, and Halls Creek were tested by micro-immunofluorescence for antibodies to a panel of rickettsial antigens. Of 920 sera examined, 52 (5.6%) were positive for antibodies to one or more of the three groups of rickettsial microorganisms. The largest group of sera (24; 2.6%) were positive for scrub typhus (Orientia tsutsugamushi). Eleven other sera (1.2%) were positive for scrub typhus and spotted fever group rickettsiae and four (0.4%) were positive for scrub typhus, spotted fever group, and typhus group rickettsiae. In addition 13 sera (1.4%) were positive only for spotted fever group rickettsiae. In this study, only titers > or = 1:256 were considered significant. Thus, there is serologic evidence for scrub typhus and spotted fever group rickettsial infections in the Kimberley region of Western Australia. Because of the method of serum collection, it is not possible to determine the prevalence of seropositivity, but the data support the need for a proper epidemiologic study of rickettsial diseases in this region of Australia.

Antibodies, Bacterial↗
PubMed1999

Reading the future: the increased relevance of laboratory medicine in the next century.

Through intelligent process control and data management, the laboratory may become the most frequently used--and the most important--source of diagnostic information in medicine. The central laboratory of the future is destined to become an esoteric testing center, whereas routine testing--administered at the patient bedside or at home--will become more economical. Point-of-care testing will soon become the most profitable way to provide laboratory services. Novel phlebotomy techniques and noninvasive tests may allow some diagnostic testing to be done through automated robotic companions that serve homebound patients or the elderly.

Autoanalysis↗
PubMed1998

Robotic automation of coagulation analysis.

Laboratory automation systems (LAS) have been installed in over 22 sites across North America providing automation of many preanalytical and analytical tasks in clinical laboratories. Only a few laboratories have automated the analysis of citrated whole blood for the diagnosis of hemostasis disorders. The analysis of coagulation factors in citrated blood requires a large amount of labor in order to provide rapid turnaround; thus automation of this analytical process is attractive. Therefore, we have created an automated coagulation workstation using a systematic approach to automation design and engineering. First, we used discrete event simulation to calculate potential throughput and to identify possible bottlenecks for the proposed coagulation workcell. We then created a three-dimensional animated computer model of the workstation to simplify workstation design. Finally, we constructed a prototype workcell using a mobile robot, an articulated robotic arm, and a coagulation analytical system.

Blood Coagulation Disorders↗
PubMed1998

Mapping and polyhedrin gene analysis of the Epiphyas postvittana nucleopolyhedrovirus genome.

The light brown apple moth, Epiphyas postvittana, is a major insect pest of a variety of fruit crops grown in New Zealand and we are studying a nucleopolyhedrovirus, EppoNPV, isolated from this insect. Restriction endonuclease analysis of EppoNPV DNA shows that this is a single strain of virus with a genome size of approximately 119 kbp and a complete library of the EppoNPV genome has been cloned. A strategy of single-stranded sequencing of the termini of REN fragment clones was employed to map the virus genome. Sequence homologies to NPV gene sequences present in the GenBank database allowed a nearly complete restriction map of the EppoNPV genome to be constructed. The mapping was completed with Southern blotting and restriction analysis. Fifty-five open reading frames (ORFs) with similarity to genes from other NPVs have been identified and placement of these on the restriction map shows that EppoNPV has a nearly identical genome organization to Orgyia pseudotsugata (Op)MNPV. The polyhedrin gene of EppoNPV has been fully sequenced and an ORF of 738 bp encodes a predicted protein of 28.8 kDa. The conserved 12 bp consensus sequence typical of very late baculovirus gene promoters, AATAAGTAATTT, has been located upstream of the ATG initiation codon. An ORF located downstream of the polyhedrin gene shows homology to the 1629-capsid protein from OpMNPV. Phylogenetic comparison to polyhedrin gene sequences from 23 other NPVs shows EppoNPV to be a group I NPV closely related to OpMNPV.

Animals↗
PubMed1996

Bartonella (Rochalimaea) quintana causing fever and bacteremia in an immunocompromised patient with non-Hodgkin's lymphoma.

A 48-yr-old man with stage IV non-Hodgkin's lymphoma, became neutropenic following chemotherapy and developed a fever. His blood cultures were processed to enhance the yield of fastidious bacteria. A slow-growing, capnophilic Gram-negative rod was isolated. The febrile episode was treated with cefotaxime, imipenem and vancomycin and resolved. The bacterial isolate was identified as Bartonella (Rochalimaea) quintana by 16S-rDNA gene sequencing. The isolate showed 99.8% sequence homology with the type strain. This is the first isolation of Bartonella (Rochalimaea) quintana from a bacteremic patient in Australia. This bacterium is a fastidious Gram-negative rod requiring prolonged culture for its isolation. Patients with culture-negative pyrexia, especially immunocompromised patients, may need to be investigated for infection with this agent.

Bacteremia↗
PubMed1995

Integrity of the first metatarsophalangeal joint: a biomechanical analysis.

Five fresh-frozen cadaver feet obtained from traumatic amputations were tested during hyperdorsiflexion stress of the first metatarsophalangeal joint. Three different types of injury were observed: (1) rupture of the capsule proximal to the sesamoids, (2) rupture of the plantar plate distal to the sesamoids, and (3) rupture of the capsular structures medially, allowing a lateral swing of the sesamoids around the metatarsal head. Incomplete dislocation can be associated with significant damage to the plantar plate and other soft tissues of the foot.

Adult↗
PubMed1994

Psychogenic seizures in children: long-term analysis of 43 cases.

Forty-three patients exhibiting psychogenic seizures with onset before the age of 16 years were studied. All patients underwent intensive electroencephalography and video-electroencephalography monitoring. Thirty-two were female and 11 male. Mean age of the population at seizure onset was 12.4 years (range, 5 to 16 years). Twenty-one patients (48.8%) were taking anticonvulsants. Neurologic past history was abnormal in nine cases. Family history of epilepsy was found in 15 cases (34.9%). Median seizure frequency was one seizure every 5 days. Clinical characteristics of the seizures varied. However, unresponsiveness with generalized violent and uncoordinated movements involving the whole body (n = 19) or with generalized trembling (n = 11) were the most common features. Neuropsychological testing, carried out in 22 cases, failed to show major abnormalities in most of the cases. Significant personal and family distress was found in most of the cases. An important impact on patient's quality of life was evident when the seizures were present as compared to the seizure-free periods. There were no statistically significant predictors of clinical outcome.

Adolescent↗
PubMed1994

Isolation, characterization and functional activity of human marrow stromal progenitors in hemopoiesis.

In conclusion, monoclonal antibody STRO-1 has proven to be an extremely valuable reagent for the identification, isolation and functional characterization of human bone marrow stromal cell precursors. We were able to isolate CFU-F free of contaminating hemopoietic progenitors and are beginning to construct a detailed picture of their cell surface phenotype and have developed assays to examine the conditions required for their growth and differentiation. In the future, we hope to determine whether multipotential stromal stem cells exist in human bone marrow and to establish culture conditions that selectively promote self-renewal or development along any of a number of stromal cell lineages. Such cells would be an ideal target for gene therapy and may provide a means of treating disorders of the hemopoietic system with a suspected stromal etiology, such as aplastic anemia. We have recently succeeded in generating bone cells from STRO-1+ marrow cells (Gronthos et al, in preparation) and thus marrow stromal cell precursors may also find application in the treatment of other disorders such as those of the osteogenic system. For the present, these must remain tantalizing possibilities.

Adult↗