Folding, calcium binding, and structural characterization of a concatemer of the first and second ligand-binding modules of the low-density lipoprotein receptor

The ligand-binding domain of the low-density lipoprotein (LDL) receptor is comprised of seven tandemly repeated ligand-binding modules, each being approximately 40 amino acids long and containing six conserved cysteine residues. We have expressed and characterized a concatemer of the first two modules (LB1 and LB2) of the human LDL receptor. Oxidative folding of the recombinant concatemer (rLB(1-2)), in the presence of calcium ions, gave a single dominant isomer with six disulfide bonds. Peptic cleavage of the short Linker region that connects the last cysteine residue of LB1 and the first cysteine residue of LB2 yielded two discrete fragments, thus excluding the presence of intermodule disulfide bonds. The N-terminal module, LB1, reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDL receptor. From this, we concluded that the first module was correctly folded, with the same set of disulfide bonds as LB1 of the LDL receptor. The disulfide bond connections of LB2 were identified from mass spectral analysis of fragments formed by digestion of the C-terminal peptic fragment with elastase. These data showed that the disulfide bonds of LB2 connected Cys(I) and Cys(III), Cys(II) and Cys(V), and Cys(IV) and Cys(VI). This pattern is identical to that found for recombinant LB1 and LB2. The concatemer has two high-affinity calcium-binding sites, one per module. An analysis of the secondary chemical shifts of C alpha protons shows that the conformations of LB1 and LB2 in the concatemer are very similar to those of the individual modules, with no evidence for strong interactions between the two modules.

Analysis of pore-fluid pressure gradient and effective vertical-stress gradient distribution in layered hydrodynamic systems

A theoretical analysis is carried out to investigate the pore-fluid pressure gradient and effective vertical-stress gradient distribution in fluid saturated porous rock masses in layered hydrodynamic systems. Three important concepts, namely the critical porosity of a porous medium, the intrinsic Fore-fluid pressure and the intrinsic effective vertical stress of the solid matrix, are presented and discussed. Using some basic scientific principles, we derive analytical solutions and explore the conditions under which either the intrinsic pore-fluid pressure gradient or the intrinsic effective vertical-stress gradient can be maintained at the value of the lithostatic pressure gradient. Even though the intrinsic pore-fluid pressure gradient can be maintained at the value of the lithostatic pressure gradient in a single layer, it is impossible to maintain it at this value in all layers in a layered hydrodynamic system, unless all layers have the same permeability and porosity simultaneously. However, the intrinsic effective vertical-stress gradient of the solid matrix can be maintained at a value close to the lithostatic pressure gradient in all layers in any layered hydrodynamic system within the scope of this study.

Kinetic analysis of vascular marker distribution in perfused rat livers after regeneration following partial hepatectomy

Background/Aims: Liver clearance models are based on information (or assumptions) on solute distribution kinetics within the microvasculatory system, The aim was to study albumin distribution kinetics in regenerated livers and in livers of normal adult rats, Methods: A novel mathematical model was used to evaluate the distribution space and the transit time dispersion of albumin in livers following regeneration after a two-thirds hepatectomy compared to livers of normal adult rats. Outflow curves of albumin measured after bolus injection in single-pass perfused rat livers were analyzed by correcting for the influence of catheters and fitting a long-tailed function to the data. Results: The curves were well described by the proposed model. The distribution volume and the transit time dispersion of albumin observed in the partial hepatectomy group were not significantly different from livers of normal adult rats. Conclusions: These findings suggest that the distribution space and the transit time dispersion of albumin (CV2) is relatively constant irrespective of the presence of rapid and extensive repair. This invariance of CV2 implies, as a first approximation, a similar degree of intrasinusoidal mixing, The finding that a sum of two (instead of one) inverse Gaussian densities is an appropriate empirical function to describe the outflow curve of vascular indicators has consequences for an improved prediction of hepatic solute extraction.

Analysis of small signal stability margins using genetic optimization

Power system small signal stability analysis aims to explore different small signal stability conditions and controls, namely: (1) exploring the power system security domains and boundaries in the space of power system parameters of interest, including load flow feasibility, saddle node and Hopf bifurcation ones; (2) finding the maximum and minimum damping conditions; and (3) determining control actions to provide and increase small signal stability. These problems are presented in this paper as different modifications of a general optimization to a minimum/maximum, depending on the initial guesses of variables and numerical methods used. In the considered problems, all the extreme points are of interest. Additionally, there are difficulties with finding the derivatives of the objective functions with respect to parameters. Numerical computations of derivatives in traditional optimization procedures are time consuming. In this paper, we propose a new black-box genetic optimization technique for comprehensive small signal stability analysis, which can effectively cope with highly nonlinear objective functions with multiple minima and maxima, and derivatives that can not be expressed analytically. The optimization result can then be used to provide such important information such as system optimal control decision making, assessment of the maximum network's transmission capacity, etc. (C) 1998 Elsevier Science S.A. All rights reserved.

Bioelectrical impedance analysis predicts outcome in patients with suspected bacteremia

Fluid shifts from intracellular to extracellular water (ICW to ECW) are a feature of sepsis, caused by increased vascular permeability and cell catabolism. Changes in ECW and total body water (TBW) were assessed in a prospective observational study of patients with bacteremia by a bedside technique, and its prognostic impact determined; In 78 hospital patients with fever, the resistance ratio (Rinf/RO) and estimated ECW/TBW ratio from multifrequency bioelectrical impedance analysis, and serum albumin concentration were measured. Rinf/RO and ECW/TBW ratios decreased from day 0 to 2 in patients with significant bacteremia (n = 31), but not in patients with doubtful or negative blood cultures (n = 22 and 25), Increased Rinf/RO at baseline, and further increase of ECW/TBW from day 0 to 2, were associated with lower rate of recovery after 1 week and with higher mortality. Baseline Rinf/RO above the median (0.75) had positive and negative predictive values of 0.31 and 0.95 for death. This prognostic effect was independent of underlying disease and blood culture result in a multivariate model. Hypoalbuminemia at baseline was predictive of outcome, but changes in albumin from day 0 to 2 were unrelated to blood culture results or outcome. In patients with bacteremia,fluid shifts from intracellular to extracellular,vater occur early are rapidly reversible by antibiotic treatment but are associated with adverse prognosis. Bioelectrical impedance deserves further study as a tool for bedside monitoring of patients with bacteremia.

Zinc potentiation of the glycine receptor chloride channel is mediated by allosteric pathways

Molecular mechanisms of zinc potentiation were investigated in recombinant human alpha 1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [H-3]strychnine binding assays. In the wild-type (WT) GlyR, 1 mu M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1-M2 and M2-M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2-M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.

Erosion/productivity modelling of maize farming in the Philippine uplands part III: Economic analysis of alternative farming methods

Two previous papers in this series (Nelson et al., this issue) described the use of the Agricultural Production Systems Simulator (APSIM) to simulate the effect of erosion on maize yields from open-field farming and hedgerow intercropping in the Philippine uplands. In this paper, maize yields simulated with APSIM are used to compare the economic viability of intercropping maize between leguminous shrub hedgerows with that of continuous and fallow open-field farming of maize. The analysis focuses on the economic incentives of upland farmers to adopt hedgerow intercropping, discussing farmers' planning horizons, access to credit and security of land tenure, as well as maize pricing in the Philippines. Insecure land tenure has limited the planning horizons of upland farmers, and high establishment costs reduce the economic viability of hedgerow intercropping relative to continuous and fallow open-field farming in the short term, In the long term, high discount rates and share-tenancy arrangements in which landlords do not contribute to establishment costs reduce the economic viability of hedgerow intercropping relative to fallow open-field farming, (C) 1998 Elsevier Science Ltd. All rights reserved.

Analysis of immunosuppressive proteins in serum of liver-transplanted rats by using an anti-LSF-1 affinity column

Liver suppressor factor one (LSF-1) is a 40-kDa immunosuppressive protein in the serum of rats 60 days after orthotopic liver transplantation (OLT) between the nonrejector combination of DA donors into PVG; recipients. In the present study, the purification of proteins from rat OLT serum taken 60 days after transplantation Mras performed by affinity chromatography using the anti-LSF-1 polyclonal antibody (pAb). The assessment of column eluates using anti-LSF-1 and OLT serum was studied using rat heart and liver transplantation models. Rejection was not suppressed by the administration of OLT serum in heart or liver allografts. However, heart allografts treated with peak eluates (450 mu g single shot im, dissolved in Intralipos) taken from the affinity OLT serum survived significantly longer than untreated rats (median = 36.5 days; n = 7 vs 6.5 days; n = 5, respectively, P = 0.011). The same treatment with anti-LSF-1 column eluates also prolonged liver allografts significantly (>200 days) than those in either the untreated group (median = 11 days; n = 7) or those which received only Intralipos (median = 10.5 days; n = 5, P = 0.019). Subsequent analysis of the N-terminal sequences of some of the proteins which were eluted from the affinity column revealed that the homology of a 30-kDa protein was identical to hemoglobin alpha-chain, a 59-kDa protein to granulocyte inhibitory factor, a 70-kDa and a 90-kDa to albumin and its precursor, respectively. Although the specific immunosuppressive component has not been isolated, our results suggested that the anti-LSF-1 column can extract immunosuppressive moiety of LSF-1 from OLT serum. (C) 1998 Academic Press.

Thermogravimetric analysis of carbon deposition over Ni/gamma-Al2O3 catalysts in carbon dioxide reforming of methane

Carbon formation on Ni/gamma-Al2O3 catalysts and its kinetics during methane reforming with carbon dioxide was studied in the temperature range of 500-700 degrees C using a thermogravimetric analysis technique. The activation energies of methane cracking, carbon gasification in CO2, as well as carbon deposition in CO2-CH4 reforming were obtained. The results show that the activation energy for carbon gasification is larger than that of carbon formation in methane cracking and that the activation energy of coking in CO2-CH4 reforming is also larger than that of methane decomposition to carbon. The dependencies of coking rate on partial pressures of CH4 and CO2 indicate that methane decomposition is the main route for carbon deposition. A mechanism and kinetic model for carbon deposition is proposed.

Sequence analysis of rabbit haemorrhagic disease virus (RHDV) in Australia: alterations after its release

Liver samples from rabbits killed by RHDV, collected from five States in Australia in 1996 and 1997 were analysed by RT-PCR. A 398 bp fragment of the capsid protein (VP60) gene was amplified by PCR and directly sequenced. The alignment of the nucleotide and amino acid sequences and their comparison with the original strain of the virus released in Australia indicated genetic changes after two years have been small with 98.2% to 100% identity. The constructed phylogenetic tree suggests slight differences in nucleotide substitutions in various States but there is no clear evidence of clustering of sequences according to their geographic origin. In practical terms, sequencing of viral RNA provides a means of testing the efficacy of further releases and subsequent spread of the virus if such a strategy is employed as a means of enhancing RHD as a biological control of the wild rabbit in Australia.

Molecular development of the olfactory nerve pathway

There are, at least, two major questions concerning the molecular development of the olfactory nerve pathway. First, what are the molecular cues responsible for guiding axons from the nasal cavity to the olfactory bulb? Second, what is the molecular basis of axon targeting to specific glomeruli once axons reach the olfactory bulb? Studies in the primary olfactory pathway have focused on the role of the extracellular matrix and ensheathing cells in establishing an initial substrate for growth of pioneer axons between the periphery and brain. The primary axons also express a multitude of cell adhesion molecules that regulate fasciculation of axons and hence may play a role in fascicle formation in the olfactory nerve. Although the olfactory neuroepithelium principally consists of a morphologically homogeneous class of primary olfactory neurons, there are numerous subpopulations of olfactory neurons expressing chemically distinct phenotypes. In particular, numerous subpopulations have been characterized by expression of unique carbohydrate residues and olfactory receptor proteins. Some of these molecules have recently been implicated in axon guidance and targeting to specific glomeruli.

Predictors of local recurrence after treatment of ductal carcinoma in situ - A meta-analysis

lBACKGROUND. Management of patients with ductal carcinoma in situ (DCIS) is a dilemma, as mastectomy provides nearly a 100% cure rate but at the expense of physical and psychologic morbidity. It would be helpful if we could predict which patients with DCIS are at sufficiently high risk of local recurrence after conservative surgery (CS) alone to warrant postoperative radiotherapy (RT) and which patients are at sufficient risk of local recurrence after CS + RT to warrant mastectomy. The authors reviewed the published studies and identified the factors that may be predictive of local recurrence after management by mastectomy, CS alone, or CS + RT. METHODS. The authors examined patient, tumor, and treatment factors as potential predictors for local recurrence and estimated the risks of recurrence based on a review of published studies. They examined the effects of patient factors (age at diagnosis and family history), tumor factors (sub-type of DCIS, grade, tumor size, necrosis, and margins), and treatment (mastectomy, CS alone, and CS + RT). The 95% confidence intervals (CI) of the recurrence rates for each of the studies were calculated for subtype, grade, and necrosis, using the exact binomial; the summary recurrence rate and 95% CI for each treatment category were calculated by quantitative meta-analysis using the fixed and random effects models applied to proportions. RESULTS, Meta-analysis yielded a summary recurrence rate of 22.5% (95% CI = 16.9-28.2) for studies employing CS alone, 8.9% (95% CI = 6.8-11.0) for CS + RT, and 1.4% (95% CI = 0.7-2.1) for studies involving mastectomy alone. These summary figures indicate a clear and statistically significant separation, and therefore outcome, between the recurrence rates of each treatment category, despite the likelihood that the patients who underwent CS alone were likely to have had smaller, possibly low grade lesions with clear margins. The patients with risk factors of presence of necrosis, high grade cytologic features, or comedo subtype were found to derive the greatest improvement in local control with the addition of RT to CS. Local recurrence among patients treated by CS alone is approximately 20%, and one-half of the recurrences are invasive cancers. For most patients, RT reduces the risk of recurrence after CS alone by at least 50%. The differences in local recurrence between CS alone and CS + RT are most apparent for those patients with high grade tumors or DCIS with necrosis, or of the comedo subtype, or DCIS with close or positive surgical margins. CONCLUSIONS, The authors recommend that radiation be added to CS if patients with DCIS who also have the risk factors for local recurrence choose breast conservation over mastectomy. The patients who may be suitable for CS alone outside of a clinical trial may be those who have low grade lesions with little or no necrosis, and with clear surgical margins. Use of the summary statistics when discussing outcomes with patients may help the patient make treatment decisions. Cancer 1999;85:616-28. (C) 1999 American Cancer Society.