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The converse piezoelectric effect in collagen type II fibrils, the main collagen constituent in cartilage, was investigated using piezoresponse force microscopy. The fibrils exhibited shear piezoelectric behavior similar to that previously reported in collagen type I fibrils and followed the same cantilever-fibril angle dependence present for type I. A uniform polarization directed from the amine to carboxyl termini, as seen for collagen type I, was observed in all type II fibrils studied. The shear piezoelectric coefficient, d15, however, for type II was roughly 28–32% of the value measured for type I fibrils. Possible explanations for the reduced piezoelectric coefficient of type II collagen are provided. [ABSTRACT FROM AUTHOR]

Conventional Kelvin probe force microscopy (KPFM) relies on closed loop (CL) bias feedback for the determination of surface potential (SP). However, SP measured by CL-KPFM has been shown to be strongly influenced by the choice of measurement parameters due to non-electrostatic contributions to the input signal of the bias feedback loop. This often leads to systematic errors of several hundred mV and can also result in topographical crosstalk. Here, open loop (OL)-KPFM modes are investigated as a means of obtaining a quantitative, crosstalk free measurement of the SP of graphene grown on Cu foil, and are directly contrasted with CL-KPFM. OL-KPFM operation is demonstrated in both single and multi-frequency excitation regimes, yielding quantitative SP measurements. The SP difference between single and multilayer graphene structures using OL-KPFM was found to be 63 ± 11 mV, consistent with values previously reported by CL-KPFM. Furthermore, the same relative potential difference between Al2O3-coated graphene and Al2O3-coated Cu was observed using both CL and OL techniques. We observed an offset of 55 mV between absolute SP values obtained by OL and CL techniques, which is attributed to the influence of non-electrostatic contributions to the input of the bias feedback used in CL-KPFM. [ABSTRACT FROM AUTHOR]

Crystal structures of classical cadherins have revealed two dimeric configurations. In the first, N-terminal β-strands of EC1 domains 'swap' between partner molecules. The second configuration (the 'X dimer'), also observed for T-cadherin, is mediated by residues near the EC1-EC2 calcium binding sites, and N-terminal β-strands of partner EC1 domains, though held adjacent, do not swap. Here we show that strand-swapping mutants of type I and II classical cadherins form X dimers. Mutant cadherins impaired for X-dimer formation show no binding in short–time frame surface plasmon resonance assays, but in long–time frame experiments, they have homophilic binding affinities close to that of wild type. Further experiments show that exchange between monomers and dimers is slowed in these mutants. These results reconcile apparently disparate results from prior structural studies and suggest that X dimers are binding intermediates that facilitate the formation of strand-swapped dimers. [ABSTRACT FROM AUTHOR]

Frequency modulation atomic force microscopy (FM-AFM) is rapidly evolving as the technique of choice in the pursuit of high resolution imaging of biological samples in ambient environments. The enhanced stability afforded by this dynamic AFM mode combined with quantitative analysis enables the study of complex biological systems, at the nanoscale, in their native physiological environment. The operational bandwidth and accuracy of constant amplitude FM-AFM in low Q environments is heavily dependent on the cantilever dynamics and the performance of the demodulation and feedback loops employed to oscillate the cantilever at its resonant frequency with a constant amplitude. Often researchers use ad hoc feedback gains or instrument default values that can result in an inability to quantify experimental data. Poor choice of gains or exceeding the operational bandwidth can result in imaging artifacts and damage to the tip and/or sample. To alleviate this situation we present here a methodology to determine feedback gains for the amplitude and frequency loops that are specific to the cantilever and its environment, which can serve as a reasonable “first guess,” thus making quantitative FM-AFM in low Q environments more accessible to the nonexpert. This technique is successfully demonstrated for the low Q systems of air (Q∼40) and water (Q∼1). In addition, we present FM-AFM images of MC3T3-E1 preosteoblast cells acquired using the gains calculated by this methodology demonstrating the effectiveness of this technique. [ABSTRACT FROM AUTHOR]

The place of nurse prescribing and the preparation for this role is an educational challenge that has been heavily debated in New Zealand and overseas for the past 10 years. Nurse prescribing is relatively new in New Zealand and is related to the expanding roles and opportunities for nurses in health care. Opposition to nurse prescribing in New Zealand has been marked and often this has been linked to concerns over patient safety with the implication that nurses could not be adequately prepared for safe prescribing. The educational framework used to teach pharmacology to nurses by one university in New Zealand is presented, along with early findings on the effectiveness of this approach. Further research is required to confirm that nurse prescribers in New Zealand are well prepared and able to utilise effective decision-making processes for safe prescribing. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To identify genetic effects potentially shared between systemic lupus erythematosus (SLE) and autoimmune thyroiditis (AITD). METHODS: Families from the Lupus Multiplex Registry and Repository were studied in which there was at least 1 member who had both SLE and AITD (Graves' disease or Hashimoto thyroiditis). Genome scan genotyping findings in these pedigrees were evaluated for evidence of genetic linkage, by the maximum-likelihood parametric method. Nineteen pedigrees were used in the initial genome scan. Subsequently, an independent sample of 16 pedigrees was used to replicate findings. RESULTS: Studies of the first set of 19 pedigrees yielded a 2-point parametric logarithm of odds (LOD) of 4.97, which was independently confirmed in the replication sample of 16 pedigrees (LOD 2.89). For all 35 pedigrees together, the 2-point LOD was 7.86, under a dominant model used for screening with 90% penetrance and a disease allele frequency of 10%. The multipoint locus homogeneity LOD in the 35 pedigrees was 6.90 (alpha = 1.0) at 5q14.3-15 between D5S1725 and D5S1453, a 12-cM interval, with the peak at D5S1462 at 96.64 cM (nonparametric linkage P = 0.00002). Fine mapping further confirmed the genetic linkage effect and narrowed the region likely to contain the gene to approximately 5 Mb. CONCLUSION: These results suggest that stratifying SLE pedigrees by the presence of other autoimmune disorders may facilitate the discovery of genes related to SLE and that 5q14.3-15 harbors a susceptibility gene shared by SLE and AITD. [ABSTRACT FROM AUTHOR]

Administration of BCX-1777 to primates results in a rapid elevation of plasma 2′-deoxyguanosine (up to 0.4 μg/ml, 1.5 μM). Maximum 2′-deoxyguanosine Cmax, 0.4 μg/ml, was achieved with the lowest IV dose of BCX-1777 and increasing the IV dose of BCX-1777 did not increase the 2′-deoxyguanosine Cmax. However, plasma 2′-deoxyguanosine remained elevated longer as the dose of BCX-1777 increased. In contrast, increases in the oral dose of BCX-1777 did increase the plasma Cmax of 2′-deoxyguanosine. This was in spite of the observation that overall oral bioavailability of BCX-1777 was only 8.2%. This suggests that the BCX-1777 was absorbed slowly producing a sustained low concentration of BCX-1777, resulting in prolonged plasma concentrations of 2′-deoxyguanosine. After IV dosing, the BCX-1777 was cleared relatively quickly and the plasma 2′-deoxyguanosine tracked slightly behind the BCX-1777. IV administration of 5 mg/kg of BCX-1777 twice daily maintains the plasma 2′-deoxyguanosine concentrations at around 0.3 μg/ml (1.1 μM). These data indicate that oral and IV administration of BCX-1777 induce a rapid rise in 2′-deoxyguanosine and that oral dosing at 8.8 and 17.6 mg/kg are at least equivalent to 4.4 mg/kg IV in effecting the accumulation of 2′-deoxyguanosine. Finally, 2′-deoxyguanosine plasma concentration was maintained longer in the three highest oral doses in comparison to all IV doses. [Copyright &y& Elsevier]

Renal disease occurs in 40-75% of systemic lupus erythematosus (SLE) patients and significantly contributes to morbidity and mortality. We used two pedigree stratification strategies to explore the impact of the ACR renal criterion for SLE classification upon genetic linkage with SLE. In both we used SLE as the phenotype. First, we evaluated genome scan data from > 300 microsatellite markers in the 75 pedigrees that had at least one SLE affected with the SLE renal criterion. A maximum-likehood parametric model approach produced a maximum screening LOD score of 3.16 at 10q22.3 in the European-American (EA) pedigrees. The African-American (AA) pedigrees obtained a maximum screening LOD score of 2.58 at 11p15.6. A multipoint sib-pair regression analysis produced P = 0.0000008 in EA at 10q22.3 (SLEN1) and P = 0.000001 in AA at 2q34-35 (SLEN2). A second stratification strategy explored the renal criterion in 35 pedigrees with two or more SLE patients with renal disease and produced a LOD score of 3.34 at 11p15.6 in AA (SLEN3). Sib-pair analysis in these 35 pedigrees revealed P = 0.00003 at 4q13.1 in EA, P = 0.00003 at 11p13 and 0.00007 at 3q23 in AA. Thus, multiple genetic linkages are related to the renal criterion in SLE. Of the significant genetic linkages with SLE described herein, those at 10q22.3 in the EA pedigrees (SLEN1) and at 2q34-35 in the AA pedigrees (SLEN2) have not been previously described. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is an autoimmune disease with complex genetics. We evaluated pedigrees multiplex for SLE that had an affected with antinucleolar antibodies to increase the homogeneity for genetic linkage analysis. We found a significant linkage effect on chromosome 11q14 at marker D11S2002 in African-American Pedigrees. This effect produced a maximum LOD score of 5.62 using a dominant inheritance model with 95% penetrance in males and 99% penetrance in females. The results were supported by multipoint linkage analysis. Fine mapping of the region with two additional markers within 6 cM of D11S2002 further provided evidence of linkage in this region. Linkage at D11S2002, named SLEH1, was previously found in some of these same African-American pedigrees multiplex for SLE, but who were stratified by hemolytic anemia (Kelly et al, submitted). In conclusion, an important SLE susceptibility gene, SLEH1 at 11q14, is identified in African-Americans when stratifying pedigrees by antinucleolar autoantibodies. [ABSTRACT FROM AUTHOR]

Anti-double-stranded DNA (anti-dsDNA) is arguably one of the most specific autoantibodies in systemic lupus erythematosus (SLE). This antibody is associated with more severe SLE and with glomerulonephritis. From 196 pedigrees multiplex for SLE, we selected those that had any SLE affected positive for anti-dsDNA by the Crithidia luciliae kinetoplast imunofluorescence assay. This stratification strategy tested the hypothesis that anti-dsDNA would identify a more genetically homogeneous group of pedigrees, in which previously undetected linkage effects could be established. A genome screen data for linkage to SLE was available at 307 microsatellite markers for this selected group of 71 pedigrees: 37 European-American, 29 African-American, and five others. The most significant results were obtained at 19p13.2 (LOD[sub max] = 4.93), named SLED1, in the 37 European-American pedigrees using a dominant model with mixed penetrances (92% for females and 49% for males) at 100% homogeneity (θ = 0). A second linkage effect, SLED2, was established in the 29 African-American pedigrees at 18q21.1 (LOD[sub max] = 3.40) using a recessive model with 100% penetrance (θ = 0.1). Parametric and non-parametric multipoint analyses were performed, which provided further evidence and support of susceptibility genes residing in these regions. In conclusion, two powerful linkages have been detected with SLE based on the presence of anti-dsDNA. These findings show SLE to be a richly complicated disease phenotype that is now ripe for important new discovery through a genetic approach. [ABSTRACT FROM AUTHOR]

Objectives: (1) To determine the validity of current recommendations for direct arterial BP measurement that suggest that the transducer (zeroed to atmosphere) be placed level with the catheter access regardless of subject positioning: and (2) to investigate the effect of transducer level, catheter access site, and subject positioning on direct arterial BP measurement.Design: Prospective, controlled laboratory study.Setting: Large animal laboratory.Subjects: Five Yorkshire pigs.Interventions: Anesthetized animals had 16F catheters placed at three access sites: aortic root, femoral artery, and distal hind limb. Animals were placed in supine, reverse Trendelenburg 35 degrees, and Trendelenburg 25 degrees positions with a transducer placed level to each access site while in every position.Measurements and Main Results: For each transducer level, five systolic and diastolic pressures were measured and used to calculate five corresponding mean arterial pressures (MAPs) at each access site. When transducers were at the aortic root, MAP corresponding to aortic root pressure was obtained in all positions regardless of catheter access site. When transducers were moved to the level of catheter access, as current recommendations suggest, significant errors in aortic MAP occurred in the reverse Trendelenburg position. The same trend for error was noted in the Trendelenburg position but did not reach statistical significance.Conclusions: (1) Current recommendations that suggest placing the transducer at the level of catheter access regardless of patient position are invalid. Significant errors occur when subjects are in nonsupine positions. (2) Valid determination of direct arterial BP is dependent only on transducer placement at the level of the aortic root, and independent of catheter access site and patient position. [ABSTRACT FROM AUTHOR]

The existence of soluble insulin-anti-insulin immune complexes in the serum of patients with diabetes mellitus was investigated. Formation of such immune complexes in vitro was studied by adding radioiodinated insulin to the sera of patients with anti-insulin antibodies; immune complexes were formed readily, but apparently differed from patient to patient. Immune complexes formed in vitro were precipitated with 5% polyethylene glycol. They eluted in the high-molecular weight fractions when the precipitated material was fractionated by get filtration, and they remained bound at neutral pH when the high-molecular weight Tractions were submitted to affinity chromatography on protein A-Sepharose. When the bound immune complexes were recovered by acid elution and immediately filtered through a Sephadex G-50 column equilibrated with the same acid buffer, free antigen (radiolabelled insulin) and antibody were recovered. This antibody, after neutralization, showed binding capacity when remixed with radioiodinated insulin. When this protocol was applied to a serum that gave positive results in several screening methods for soluble immune complexes, insulin was detected by radioimmunoassay in the high-molecular weight fractions separated from the 5% polyethylene glycol precipitate and in the fractions retained by protein A; however, no free insulin was detected after gel filtration in Sephadex G-50, perhaps due to excessive dilution. The high-molecular weight fraction did have binding capacity for radioiodinated insulin. No insulin-binding protein could be recovered with a similar procedure from a serum negative by all screening tests for soluble immune complexes. These results prove that soluble immune complexes can be formed easily in sera containing anti-insulin antibodies and can be recovered from sera of diabetic patients that show positive results in screening techniques for soluble immune complexes. [ABSTRACT FROM AUTHOR]

We have tried to determine the most favourable conditions for the in vitro induction of specific antibody (Ab) responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH). Human peripheral blood mononuclear cells (PBMNC) were obtained from normal volunteers and stimulated with PWM, TT, KLH, and mixtures of PWM and antigens in the presence or absence of autologous red blood cells (RBC) (1:50 ratio of PBMNC/RBC). The cultures were harvested on day 11: immunoglobulins were determined immunonephelometrically and Ab levels by ELISA with human antibodies used for Calibration. While anti-TT responses were easy to induce with PBMNC from recently boosted individuals, the production of anti-TT from PBMNC obtained from non-recently boosted individuals was only possible when PBMNC were stimulated with TT and PWM in the presence of autologous RBC Similarly, anti-KLH responses were easier to induce with PBMNC from an immune donor; maximal response was observed after stimulation with PWM + KLH in the presence of autologous RBC. Stimulation of primary anti-KLH responses with PBMNC from non-immune donors was only successful when the cells were stimulated with KLH + PWM in the presence of autologous RBC. The potentiation of human B-cell responses with autologous RBC can be abrogated by pre-treatment of PBMNC with anti-CD2 antibodies and is associated with increased expression of IL-2 receptors and increased production of gamma interferon (IFN-y). However, addition of IFN-γ in different doses and at different times to PWM-stimulated PBMNC cultures was not as effective as addition of RBC in enhancing the production of immunoglobulin and antibody. [ABSTRACT FROM AUTHOR]