PubMed Health⌕ Search

Biomedical subjects

Steven H Hinrichs

Publications and source records attributed to Steven H Hinrichs.

At least 19 recordsLinked to original sources
PubMed2006

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay for organophosphorus toxicants bound to human albumin at Tyr411.

Our goal was to determine whether chlorpyrifos oxon, dichlorvos, diisopropylfluorophosphate (DFP), and sarin covalently bind to human albumin. Human albumin or plasma was treated with organophosphorus (OP) agent at alkaline pH, digested with pepsin at pH 2.3, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Two singly charged peaks m/z 1718 and 1831, corresponding to the unlabeled peptide fragments containing the active site Tyr411 residue, were detected in all samples. The sequences of the two peptides were VRYTKKVPQVSTPTL and LVRYTKKVPQVSTPTL. The peptide-OP adducts of these peptides were also found. They had masses of 1854 and 1967 for chlorpyrifos oxon, 1825 and 1938 for dichlorvos, 1881 and 1994 for DFP, and 1838 and 1938 for sarin; these masses fit a mechanism whereby OP bound covalently to Tyr411. The binding of DFP to Tyr411 of human albumin was confirmed by electrospray tandem mass spectrometry and analysis of product ions. None of the OP-albumin adducts lost an alkoxy group, leading to the conclusion that aging did not occur. Our results show that OP pesticides and nerve agents bind covalently to human albumin at Tyr411. The presence of Tyr411 on an exposed surface of albumin suggests that an antibody response could be generated against OP-albumin adducts.

Amino Acid Sequence↗
PubMed2006

Performance of a commercial immunoglobulin M antibody capture assay using analyte-specific reagents to screen for interfering factors during a West Nile virus epidemic season in Nebraska.

In 2003, the Nebraska Public Health Laboratory tested more than 10,371 serum and 516 cerebral spinal fluid specimens. Results showed that without performing the interfering factors screen for specimens in the low positive index value range of >1.1 to <or=3.5, a false positivity rate of 6.5% would have been realized.

Antibodies, Viral↗
PubMed2006

First report of Mycobacterium nebraskense as a cause of human infection.

Newly described nontuberculous Mycobacterium species have emerged as causes of opportunistic infection in compromised patients. This report describes the first case of Mycobacterium nebraskense isolated from a patient with a history of emphysema and details the need for adequate diagnostic capabilities to manage patients with infections caused by slow-growing pathogens.

Aged, 80 and over↗
PubMed2006

Geographic factors contributing to a high seroprevalence of West Nile virus-specific antibodies in humans following an epidemic.

Sera of 624 blood donors were evaluated to determine seroprevalence of West Nile virus (WNV) antibodies following the 2003 WNV epidemic in Nebraska. Geographic factors contributing to differences in WNV seropositivity were evaluated. The overall prevalence of WNV in Nebraska was higher than reported previously in other U.S. locations (9.5% WNV immunoglobulin G seroprevalence rate), with the highest prevalence identified in the western part of the state (19.7%), followed by the central (13.8%) and the eastern (4.2%) parts. Regions of the state with the highest WNV-positive mosquito rates correlated with the highest human WNV seroprevalence rates. The results showed that both the western and central parts of the state, where mosquito positivity rates were highest, had significantly higher seroprevalence rates than the eastern region. Additional studies are needed to determine whether the high prevalence rates in Nebraska will be reflected in other states and what impact environmental and geographical factors may have on future outbreaks of WNV infection.

Animals↗
PubMed2006

Staphylococcus aureus helicase but not Escherichia coli helicase stimulates S. aureus primase activity and maintains initiation specificity.

Bacterial primases are essential for DNA replication due to their role in polymerizing the formation of short RNA primers repeatedly on the lagging-strand template and at least once on the leading-strand template. The ability of recombinant Staphylococcus aureus DnaG primase to utilize different single-stranded DNA templates was tested using oligonucleotides of the sequence 5'-CAGA (CA)5 XYZ (CA)3-3', where XYZ represented the variable trinucleotide. These experiments demonstrated that S. aureus primase synthesized RNA primers predominately on templates containing 5'-d(CTA)-3' or TTA and to a much lesser degree on GTA-containing templates, in contrast to results seen with the Escherichia coli DnaG primase recognition sequence 5'-d(CTG)-3'. Primer synthesis was initiated complementarily to the middle nucleotide of the recognition sequence, while the third nucleotide, an adenosine, was required to support primer synthesis but was not copied into the RNA primer. The replicative helicases from both S. aureus and E. coli were tested for their ability to stimulate either S. aureus or E. coli primase. Results showed that each bacterial helicase could only stimulate the cognate bacterial primase. In addition, S. aureus helicase stimulated the production of full-length primers, whereas E. coli helicase increased the synthesis of only short RNA polymers. These studies identified important differences between E. coli and S. aureus related to DNA replication and suggest that each bacterial primase and helicase may have adapted unique properties optimized for replication.

Adenosine↗
PubMed2005

C-erb-B2 (HER2/neu) expression in synovial sarcoma of the head and neck.

BACKGROUND: Synovial sarcoma is a malignant mesenchymal tumor composed of varying proportions of spindle and epithelial cell components. Because of the histologic and immunohistochemical similarity of synovial sarcoma to epithelial carcinomas, we hypothesized that the human epithelial growth factor receptor 2 (C-erb-B2, also termed HER2/neu) may contribute to the tumor phenotype and provide a new therapeutic target for this soft tissue tumor. METHODS: Three head and neck, one chest wall, and seven extremity synovial sarcomas were evaluated for C-erb-B2 (HER2/neu) expression by immunohistochemistry, Western immunoblotting, and fluorescence in situ hybridization (FISH). RESULTS: The head and neck cases demonstrated immunohistochemically strong positive staining, whereas tumors from other anatomic locations showed neither positive nor cytoplasmic restricted staining. Antigen-targeted antibody therapy (trastuzumab) was initiated in two patients. CONCLUSIONS: These results demonstrate that C-erb-B2 (HER2/neu) may play a role in the tumorigenesis of synovial sarcoma; and, therefore, antigrowth factor therapies may provide a previously unrecognized pharmaceutical approach to soft tissue tumors. The data also suggest that although synovial sarcoma of the head and neck and synovial sarcoma of the extremities have similar morphologic features, they may be clinically and mechanistically distinct entities.

Adult↗
PubMed2005

Computational approach involving use of the internal transcribed spacer 1 region for identification of Mycobacterium species.

The rapid and reliable identification of clinically significant Mycobacterium species is a challenge for diagnostic laboratories. This study evaluates a unique sequence-dependent identification algorithm called MycoAlign for the differential identification of Mycobacterium species. The MycoAlign system uses pan-Mycobacterium-specific primer amplification in combination with a customized database and algorithm. The results of testing were compared with conventional phenotypic assays and GenBank sequence comparisons using the 16S rRNA target. Discrepant results were retested and evaluated using a third independent database. The custom database was generated using the hypervariable sequences of the internal transcribed spacer 1 (ITS-1) region of the rRNA gene complex from characterized Mycobacterium species. An automated sequence-validation process was used to control quality and specificity of evaluated sequence. A total of 181 Mycobacterium strains (22 reference strains and 159 phenotypically identified clinical isolates) and seven nonmycobacterial clinical isolates were evaluated in a comparative study to validate the accuracy of the MycoAlign algorithm. MycoAlign correctly identified all referenced strains and matched species in 94% of the phenotypically identified Mycobacterium clinical isolates. The ITS-1 sequence target showed a higher degree of specificity in terms of Mycobacterium identification than the 16S rRNA sequence by use of GenBank BLAST. This study showed the MycoAlign algorithm to be a reliable and rapid approach for the identification of Mycobacterium species and confirmed the superiority of the ITS-1 region sequence over the 16S rRNA gene sequence as a target for sequence-based species identification.

Base Sequence↗
PubMed2004

Thermally denaturing high-performance liquid chromatography analysis of primase activity.

Prokaryotic primase, a DNA-dependent RNA polymerase, is a target of interest for the development of novel antibiotics. A new assay was developed to evaluate the inhibition of primase activity while avoiding the limitations of existing assays that require the incorporation of radiolabeled nucleotides into the growing primer followed by electrophoretic separation and autoradiography or scintillation counting. These existing technologies are either time consuming or unable to give detailed information on the kinetics, size, and nature of the primers synthesized. To address these issues in a nonradioactive manner, a thermally denaturing high-performance liquid chromatography (HPLC) assay was developed that was able to (1) measure the two modes of primase activity (de novo and overlong primer synthesis), (2) quantitate de novo primer synthesis kinetics yielding a rate constant of 0.00251 s(-1), and (3) determine that dNTPs inhibited primase activity with an IC50 of 9.5 microM. In addition, the differential elution properties of short DNA and RNA oligonucleotides on an alkylated nonporous polystyrene-divinylbenzene copolymer microsphere bead column were determined. The thermally denaturing HPLC assay provides rapid quantitative analysis of primase function and qualitative analysis of activity with regard to the nature of the primers synthesized.

Base Sequence↗
PubMed2004

Utilization of the relative complexity measure to construct a phylogenetic tree for fungi.

The relative complexity measure (RCM) is a new approach to evaluate relatedness of DNA sequences which eliminates the requirement to align sequences prior to analysis, a step required with standard reference methods. The value of the RCM approach to generate distance matrices for use in phylogenetic analysis of organisms has not been determined. This study compared RCM with the algorithmic and tree searching reference methods for phylogenetic analysis using fungal sequences. Sequences of the cytochrome b gene and the 18S rDNA gene were obtained from the GenBank database to determine feasibility of this method for phylogenetic relatedness. The RCM approach was also used to construct a phylogenetic tree using internal transcribed spacer (ITS) sequences from 23 medically relevant fungal species. The robustness of the RCM and reference approaches was determined by comparing the topology of seven medically relevant fungi within the phylogenetic trees generated after progressive removal of 10, 20, 30, 40 and 50% of the nucleotide bases from either the 5' or 3' end of the three genomic target sequences. The results demonstrated that the RCM method was equivalent to the reference methods for construction of phylogenetic trees from cytochrome b and 18S rDNA gene sequences. The phylogenetic tree constructed using the ITS sequence generated no contradictory topology. The RCM generated trees retained the appropriate topology after removal of up to 50% of the cytochrome b sequence, 40% of the ITS sequence, and 30% of the 18S gene target sequence. Comparatively, the reference methods failed to maintain topology after only a 10% sequence deletion for each genomic target. The results showed the RCM to be a reliable and robust computational approach for use in the construction of fungal phylogenetic trees without the requirement for prior sequence alignment.

Algorithms↗
PubMed2004

Two-stage reimplantation of total joint infections: a comparison of resistant and non-resistant organisms.

The prevalence of resistant organisms has increased throughout the last decade. In order to determine best treatment strategies, studies on reimplantation must address this population. The purpose of this study is to compare the results of delayed reconstruction for total joint infections caused by methicillin-sensitive and methicillin-resistant organisms. This investigation included 46 patients (21 men, 25 women) with an average age 67 years (range, 36-86 years) who had a two-staged reimplantation after periprosthetic total joint infection. Thirty-seven patients cultured methicillin-sensitive organisms and nine cultured methicillin-resistant organisms. Treatment included resection, aggressive debridement, 6 weeks of parenteral antibiotics, and subsequent debridement and reimplantation. Treatment was considered successful if the patient was disease free at last followup (average 5 years, range 2-10 years). The efficacy of two-stage reconstruction was similar for infections with sensitive and resistant bacteria. Only 3 of 46 patients failed delayed reconstruction requiring further treatment. Failure rates with regards to infections with sensitive bacteria were 5.4% (2 of 37 infections) and resistant bacteria 11.1% (1 of 9 infections), which was not significant. The current study shows clinical success of two-stage reimplantation for patients with methicillin-resistant bacteria complicating total joint arthroplasty.

Adult↗
PubMed2004

Mycobacterium nebraskense sp. nov., a novel slowly growing scotochromogenic species.

The characterization of a novel slowly growing, scotochromogenic Mycobacterium species is reported. This previously undescribed mycobacterial species was isolated from five different patients with symptomatic pulmonary infections. All isolates were acid-fast-positive and the mycolic acid profiles were unique and supported placement into the genus Mycobacterium. Phenotypic characteristics of each strain included optimal growth after 3 weeks at a temperature range of 30-35 degrees C, yellow pigmentation after incubation in the dark and production of a heat-stable catalase. The 16S rRNA gene and internal transcribed spacer 1 sequences were identical for all five strains, but distinct from all known mycobacterial species. Phylogenetic analysis based on the 16S rRNA gene sequence placed the novel species within the slowly growing mycobacteria group in close proximity to Mycobacterium malmoense, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium scrofulaceum. These data support the conclusion that the related five described organisms represent a novel Mycobacterium species, for which the name Mycobacterium nebraskense sp. nov. is proposed, with the type strain UNMC-MY1349(T) (=ATCC BAA-837(T)=DSM 44803(T)).

Catalase↗
PubMed2004

Temperature-mediated heteroduplex analysis for detection of pncA mutations associated with pyrazinamide resistance and differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by denaturing high- performance liquid chromatography.

The goal of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates and simultaneously differentiate between M. tuberculosis and Mycobacterium bovis. Features that contributed to an optimal assay included the use of two different reference probes for the pncA gene targets from wild-type M. tuberculosis and wild-type M. bovis, optimization of the column temperature, increasing the starting concentration of the elution buffer, and reducing the rate of elution buffer increase (slope). A total of 69 strains were studied, including 48 wild-type M. tuberculosis strains (13 were PZA-resistant strains) and 21 M. bovis strains (8 were BCG strains). In all isolates tested, wild-type M. tuberculosis generated a single-peak pattern when mixed with the M. tuberculosis probe and a double-peak pattern with the M. bovis probe. In contrast, all M. bovis isolates generated a double-peak pattern when mixed with the M. tuberculosis probe and a single-peak pattern with the M. bovis probe. PZA-resistant mutant M. tuberculosis isolates generated characteristic patterns that were easily distinguishable from both wild-type M. tuberculosis and M. bovis isolates. Chromatographic patterns generated by the two reference probes allowed the rapid detection of PZA resistance with the simultaneous ability to distinguish between M. tuberculosis and M. bovis. This approach may allow the detection of drug resistance-associated mutations, with potential application to clinical and epidemiological aspects of tuberculosis control.

Animals↗
PubMed2003

PCR rapidly detects methicillin-resistant staphylococci periprosthetic infection.

Optimal treatment of methicillin-resistant staphylococcal periprosthetic infections is predicated on rapid and reliable detection of these organisms. Culture has served as the gold standard for identification of these organisms despite shortcomings with sensitivity and processing time. The objective of the current study was to investigate a polymerase chain reaction assay aimed at rapid genomic detection of methicillin-resistance in staphylococci (mecA gene). The feasibility of the molecular approach first was validated using a septic arthritis model consisting of 73 synovial fluid samples inoculated with methicillin-resistant staphylococci and four negative controls. MecA polymerase chain reaction then was done on 35 clinical samples from 18 patients obtained at the time of revision arthroplasty. Results of the polymerase chain reaction were compared with culture. MecA polymerase chain reaction successfully predicted the presence of methicillin-resistant staphylococci in the septic arthritis model. In the clinical samples studied, the polymerase chain reaction results were concordant with culture results in 34 of the 35 samples tested. The one discordant result represented a false-positive culture result. The molecular assay was processed in less than 5 hours compared with 2 to 3 days for culture. Detection of methicillin-resistant staphylococci involved in periprosthetic infections by the polymerase chain reaction is a rapid and reliable approach.

Arthritis, Infectious↗
PubMed2003

Specific detection of human BK polyomavirus in urine samples of immunocompromised patients.

A semiquantitative PCR assay for the detection of BK virus in urine was developed using primers for BK virus that specifically amplified BK but not JC virus. DNA was extracted from urine through treatment with proteinase K followed by DNA precipitation with sodium acetate. Semiquantitation was achieved by amplifying serial dilutions (1:1, 1:10, 1:100, and 1:1,000) of the urine specimens. Each assay included both positive (stock BK virus and previously positive patient urine) and negative (no template) controls. A urine sample was interpreted as positive if any of the serial dilutions showed amplification of the DNA fragment of the expected size. For some patient-derived samples, amplification of the expected-size fragment was achieved with a dilute template whereas no amplification was achieved with a concentrated template. This was attributed to interfering substances in the urine. PCR results were compared with urine cytology and shown to be more sensitive. Validation studies were performed at the University of Nebraska Medical Center, utilizing a separate qualitative PCR assay that detects both BK and JC virus and distinguishes between them by restriction enzyme digestion patterns. Of 46 urine samples analyzed using both methods, 22 were positive by both assays, 18 were negative by both assays, 5 were positive only by the Nebraska method, and 1 was positive only by our method. In comparison with the Nebraska PCR, our PCR assay had a sensitivity of 81% and specificity of 95%. For twenty-one (43%) of 49 immunocompromised patients, tests were positive when specimens were submitted because of clinical suspicion of BK virus infection.

BK Virus↗