PubMed Health⌕ Search

Biomedical subjects

M Farr

Publications and source records attributed to M Farr.

At least 19 recordsLinked to original sources
PubMed2006

Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the software methods.

Automation↗
PubMed2002

Vaccination against hepatitis B infection in patients with end stage renal disease.

BACKGROUND: Experience of hepatitis B vaccination in a contemporary renal replacement programme is reported. METHODS: A total of 406 patients were involved: 214 on haemodialysis, 97 on continuous ambulatory peritoneal dialysis, 67 predialysis (serum creatinine >400 micromol/l), and 28 with a failing transplant. Primary vaccination comprised recombinant hepatitis B vaccine (Engerix B) 40 microg intramuscularly at 0, 1, 2, and 3 months. Booster doses were administered three monthly if anti-HBs titre was <100 IU/l. RESULTS: Uptake of vaccine was 61% (haemodialysis 70%, continuous ambulatory peritoneal dialysis 62%, predialysis 31%, transplant 61%, p<0.0001). Primary seroconversion occurred in 64% of vaccinated patients (anti-HBs; 10-100 U/l, 33%; >100 U/l, 31%). Booster doses led to further improvement in immunity in 66/115 (57%) patients after a first and 8/20 (40%) patients after a second booster dose, but uptake was again poor (first booster 74%, second 31%). Seroprotection declined unexpectedly rapidly; after a mean of 16 months 71/115 patients (62 %) had a significant fall in their anti-HBs titres; 30/115 (26%) lost detectable antibody. CONCLUSIONS: Routine hepatitis B vaccination of patients with end stage renal failure is logistically difficult to administer on a large scale; primary seroconversion is relatively poor, but improves after repeated booster doses; protective anti-HBs titres decline rapidly, and yearly antibody checks with selective booster doses will be required to maintain seroprotection. The cost effectiveness of a vaccination programme will vary greatly depending on the prevalence of hepatitis B in the population at risk.

Female↗
PubMed2001

MMP-TIMP interaction depends on residue 2 in TIMP-4.

Extracellular matrix remodeling and degradation are of great importance in both physiological and pathological situations. Matrix metalloproteinases (MMPs) and their natural occurring inhibitors - tissue inhibitors of metalloproteinases (TIMPs) - are involved in matrix turnover. Among the TIMPs there is only little specificity for inhibiting individual MMPs. In this report we describe the mutational analysis of the interaction of human TIMP-4 with several MMPs. The effects of different substitutions of residue 2 (Ser(2)) in the inhibitory domain of TIMP-4 were determined by kinetic measurements. Size, charge and polarity of residue 2 in the TIMP structure are key factors in MMP inhibition.

Amino Acid Substitution↗
PubMed2001

Direct interactions between immunocytes and neurons after axotomy in Aplysia.

Axon growth during development and after injury has processes in common, but also differs in that regeneration requires the participation of cells of the immune system. To investigate how neuron-immunocyte interactions might influence regeneration, we developed an in vitro model whereby neurons and hemocytes from Aplysia californica were cocultured. The hemocytes, which behave like vertebrate macrophages, migrated randomly throughout the dish. When a neuron was encountered, some hemocytes exhibited an avoidance response, whereas others formed stable contacts. Hemocytes did not distinguish between neurons from different animals. Stable contacts occurred on neurites and growth cones, but not the cell soma, and were benign in that the hemocytes did not impede neurite growth. When hemocytes attached to the cell body, it presaged the destruction of the neuron. Destruction was a dynamic process that was initiated when groups of one to three hemocytes adhered to various regions of the cell soma. Each group was then joined by other hemocytes. They did not contact the neuron, but interconnected the initial groups, forming a network around the neuron. The network then contracted to dismember the cell. Once a neuron was destroyed, hemocytes removed the debris by phagocytosis. Both damaged neurons and those without apparent damage were targets for destruction. Severing neurites with a needle resulted in the destruction of only one of six cells. Our studies suggest that hemocytes, and by extrapolation, vertebrate macrophages, exhibit highly complex interactions with neurons that can exert a variety of influences on the course of nerve regeneration.

Animals↗
PubMed2001

Characterization of C-terminally truncated human tissue inhibitor of metalloproteinases-4 expressed in Pichia pastoris.

The tight regulation of extracellular matrix remodeling and degradation is of great importance in physiological processes like development and morphogenesis, as well as in pathological situations like tumor invasion and metastasis. Tissue inhibitors of metalloproteinases (TIMPs) are the naturally occuring inhibitors of matrix metalloproteinases, which are involved in matrix turnover. In this report we describe the cloning of human TIMP-4 from a human adenocarcinoma and an osteosarcoma cell line and the expression of the inhibitory domain in the methylotrophic yeast Pichia pastoris. The inhibition of MMP-8, -9, -12, -13 and -14 by the N-terminal domain of TIMP-4 was analysed. Using a fluorescent MCA-peptide, Ki values for each subclass of MMPs were determined. With dissociation constants in the nanomolar range, TIMP-4 seems to be a good inhibitor for all classes of MMPs without remarkable preference for special MMPs.

Amino Acid Sequence↗
PubMed2000

Pregnancy-associated plasma protein-E (PAPP-E).

A full-length cDNA encoding a novel human protein was cloned from placenta cDNA. The corresponding 1542 amino acid protein sequence was termed 'pregnancy-associated plasma protein-E' (PAPP-E) as it shows a 62% homology to the human pregnancy-associated plasma protein-A (PAPP-A) that is a diagnostic marker for trisomies, especially Down syndrome. The conserved domain structure contains five motifs related to the short consensus repeats of complement proteins and selectins, three motifs related to the lin-notch motifs of proteins regulating early tissue differentiation, and a putative zinc-binding motif and active site of the metzincin-superfamily of metalloproteases. The PAPP-E gene was localized to chromosome 1q23-25. Northern blot analysis showed that PAPP-E is predominantly expressed in placenta.

Amino Acid Sequence↗
PubMed1999

The N-terminus of collagenase MMP-8 determines superactivity and inhibition: a relation of structure and function analyzed by biomolecular interaction analysis.

Tissue inhibitors of metalloproteinases (TIMPs) are the physiological, specific inhibitors of matrix metalloproteinases (MMPs) forming tight, noncovalent complexes. Therefore they control the proteolytic activity of MMPs toward the extracellular matrix. To analyze the inhibition of the "activated" and "superactivated" variants of human neutrophil collagenase (MMP-8) by TIMP-2, we determined complex dissociation constants using biomolecular interaction analysis (BIA). As it is known that the association rate constants can exceed the limits of the BIA instruments, the biomolecular interaction analysis was used to examine the equlibrium situation. The dissociation constants were determined by fitting the parameters of the mathematical term for the binding of collagenase onto the TIMP-coupled sensor chip surface to the saturation curve derived from individual sensorgrams. The resulting values are in the nanomolar range and correlate with the results of fluorescence kinetics. These data reveal that TIMP-2 (the recombinant inhibitory domain of human TIMP-2 and bovine TIMP-2 isolated from seminal plama) is a better inhibitor of the activated neutrophil collagenase than of the superactivated variant (the recombinant catalytic domain of human MMP-8). It has been demonstrated by X-ray analysis that the N-terminal heptapeptide only of superactivated MMP-8 is attached by a salt bridge and hydrophobic interaction to the C-terminal helix. Because these interactions have to be disrupted in the complex formation with TIMP we assume that the activated variant enables higher flexibility and a tighter induced fit in the complex formation. Therefore superactivation of MMP-8 correlates with weaker inhibition by TIMP-2.

Animals↗
PubMed1995

Streptomyces ghanaensis plasmid pSG5: nucleotide sequence analysis of the self-transmissible minimal replicon and characterization of the replication mode.

The naturally temperature-sensitive plasmid pSG5 of Streptomyces ghanaensis DSM 2932 is the basis replicon of the "pGM-vectors." The nucleotide sequence of the pSG5 minimal replicon was determined. Only one single open reading frame (rep) with high coding probability is located on the minimal replicon. The deduced Rep protein consists of 378 aa and contains motifs characteristic of initiation proteins for rolling-circle-type replication. Sequence similarity indicated that the Rep protein of pSG5 is related to the Rep proteins of the pC194 plasmid family. Accumulation of large amounts of single-stranded plasmid DNA was shown for all small pGM vectors. A minus origin for the lagging strand synthesis was localized outside of the pSG5 minimal replicon. Although the sequenced pSG5 fragment is self-transmissible, it seems not to carry further genes in addition to the rep gene. This suggests that the transfer mechanism of plasmid pSG5 differs from that of other Streptomyces plasmids, which all encode specific transfer genes.

Amino Acid Sequence↗
PubMed1995

How and when should combination therapy be used? The role of an anchor drug.

Most patients with rheumatoid arthritis do not achieve a complete response to monotherapy; some achieve a sub-optimal response and others become resistant to therapy and escape from control after an initial good response. It is essential to recognize those with an incomplete response early and to introduce combination therapy promptly so as to induce remission and minimize joint damage and disability. The main concern about combination therapy has been the risk of additive or synergistic toxicity. The aim is therefore to choose drugs that are the least toxic and that also have a rapid and sustained action. Sulphasalazine (SASP) possesses these properties; it has a mild toxicity profile and good long-term tolerability. Most adverse events occur during the first few months of therapy and accumulative toxicity on stable maintenance doses is rare. It also has a rapid onset of action and sustained efficacy. On these grounds we recommend that SASP is used in combination therapy as the 'anchor drug' to which other drug(s) can be added sequentially. This sequential regimen allows those patients who respond to monotherapy to be identified, and gives flexibility of dose control so that the drugs can be tailored to the individual patient. Combination therapy may have real advantages in inducing remission and preventing resistance to therapy. it also has the potential for long-term disease modification.

Antirheumatic Agents↗
PubMed1992

Influence of acetylator status on sulphasalazine efficacy and toxicity in patients with rheumatoid arthritis.

The influence of acetylator status on the therapeutic efficacy and the toxicity of sulphasalazine (SASP) was assessed in 106 patients with rheumatoid arthritis (RA). Changes of indices of disease activity after 6 months, and progression of erosions after 2 years of SASP treatment were similar in fast and slow acetylators. Incidence and nature of withdrawals and side-effects, and requirement for intra-articular steroid injections or combination therapy due to poor response to SASP were almost identical in the two groups. A significant increase of the hepatic enzyme aspartate transaminase was noted mainly in slow acetylators, but was not associated with clinical disease. These results suggest that acetylator status does not relate significantly to either the efficacy or the toxicity of SASP in RA. It is possible that hepatic metabolism is affected by SASP, particularly in slow acetylators, but this does not lead to clinically identifiable problems.

Acetylation↗
PubMed1991

Immunodeficiencies associated with sulphasalazine therapy in inflammatory arthritis.

Abnormalities in serum immunoglobulin levels (Igs) are documented in a series of 350 patients with rheumatoid arthritis (RA) and other inflammatory joint diseases treated with sulphasalazine (SASP) for up to 10 years. Low Ig levels occurred in just over 10% of patients after therapy. Three per cent developed selective IgA deficiency between 8 and 20 weeks after starting SASP. Low IgG levels occurred in 2% at 4-52 weeks and low IgM levels in 5% after 3-7 months. One per cent developed panhypogammaglobulinaemia (hypo gamma) 3-7 months after commencing therapy. Most immunodeficiencies were not accompanied by other toxic reactions and SASP was continued in all but one patient with a rash and thrombocytopenia. A good clinical response was observed in most patients particularly those with selective IgA deficiency and hypo gamma. Two patients with hypo gamma developed chest infections which responded to antibiotics. A low level of individual Igs is not usually an indication to stop SASP unless accompanied by other reactions. Panhypo gamma is potentially serious and should be monitored carefully and replacement therapy should be considered in these patients if infections occur.

Adolescent↗
PubMed1991

Assessment of rheumatoid arthritis.

Accurate assessment of disease activity and progression is essential in diseases with marked chronicity such as rheumatoid arthritis. It is not surprising that rheumatologists continue to seek improvement in old indices and to develop new ones. In the field of clinical indices, selection of data that contribute independently to the overall value of a compound assessment index will simplify trial methodology considerably. The trend is to reduce the number of tests done, but there is still a need to include functional indices and perhaps psychologic ones, because disability and self-image clearly affect prognosis. In the laboratory area, the trend is to develop methods to specifically assess different aspects of inflammation rather than to rely on overall nonspecific measures such as acute-phase proteins. Radiographs remain the simplest method for detecting scars of previous disease and the potential exists to improve and perhaps mechanize the reading of such films. Magnetic resonance imaging has the potential for assessment of the current state of inflammation if newer methodologies are used. Radionuclides may also have much to offer in joint assessment in the 1990s.

Arthritis, Rheumatoid↗
PubMed1990

Sulphasalazine in psoriatic arthritis: a double-blind placebo-controlled study.

Sulphasalazine (SASP) is now accepted as an effective slow-acting antirheumatic drug for treating active rheumatoid arthritis (RA), but has not been previously evaluated in psoriatic arthritis. An earlier open study suggested that it was well tolerated and potentially beneficial. The present double-blind placebo-controlled trial of 30 patients has now confirmed its efficacy. Greater improvement occurred in those patients on active treatment than on placebo, with more benefit being detected in those patients with the symmetrical polyarticular but seronegative pattern of arthritis associated with a high acute-phase response. SASP was stopped in 26% because of side-effects but these were mild. No exacerbation or remission of psoriasis was observed. Further studies are in progress to determine the degree of efficacy of SASP in different clinical subgroups of psoriatic arthritis.

Arthritis↗