Purification, kinetic characterization and involvement of tryptophan residue at the NADPH binding site of xylose reductase from Neurospora crassa

Xylose reductase (XR) from Neurospora crassa was purified to homogeneity and was found to be specific to NADPH (nicotinamide adenine dinucleotide phosphate). The purified enzyme showed M(r) of 60 and 29 kDa by gel filtration and SDS-PAGE indicating the presence of two subunits. The kinetic mechanism of xylose reductase is 'iso-ordered bi bi'. Inactivation of XR by N-bromosuccinimide (NBS) was found to be biphasic with second-order rate constants of 2.5 x 10(2) and 80 M-1S-1 for the fast (kf) and slow phase (ks), respectively. NADPH protected 90% of XR activity against inhibition by NBS. The fluorescence and circular dichroism (CD) studies revealed that inactivation was not due to gross conformational change in the enzyme. Analysis of the modified Stern-Volmer plot indicated that 49% of the tryptophanyl fluorescence was available for quenching which was completely abolished in the presence of NADPH confirming the involvement of tryptophan at the coenzyme binding site. Experimental evidence presented here serves to implicate the involvement of a tryptophan residue at the low-affinity NADPH binding site and the nature of this site has been assessed by using the hydrophobic probe ANS.     

Scanning laser photostimulation: a new approach for analyzing brain circuits

A new technique for understanding the organization of complex circuits in the vertebrate brain, scanning laser photostimulation, is described. This approach is based on the photolysis of a caged form of the excitatory neurotransmitter glutamate. Computer-controlled photostimulation and whole cell recording in brain slices allow the construction of detailed maps of the position, strength, sign and number of inputs converging on a single postsynaptic neuron. Scanning laser photostimulation offers many advantages over current techniques: spatial resolution is superb, fibers of passage are not activated, and thousands of presynaptic locations can be stimulated. This review describes the technique of photostimulation, outlines the instrumentation, necessary to implement it, and discusses the interpretation of photostimulation-derived data. Several examples of applications, ranging from mapping circuits in the mammalian visual cortex to determining receptor distributions on single neurons are considered. Although still in its early stages, scanning laser photostimulation offers neuroscientists a powerful tool for determining the organization and function of local brain circuits.     

Dual recognition of lipid A and DNA by human antibodies encoded by the VH4-21 gene: a possible link between infection and lupus

The VH4-21 (V4-34) gene segment, a member of the VH4 family, is expressed early in B-cell maturation and is utilized by approximately 6% of normal adult B lymphocytes. This prevalence indicates an importance of VH4-21 in the B-cell repertoire. The gene also encodes certain autoantibodies being mandatory for pathological IgM anti-red cell antibodies directed against the I/i antigen, and also capable of encoding anti-DNA antibodies. Recognition of I/i antigen or DNA appears to be via two distinct sites on VH, with I/i binding mediated by sequences in the framework region, and DNA binding correlating with the presence of positively charged amino acids in complementarity-determining region 3. However, these positively charged residues appear to suppress the ability of the framework region to interact with I/i, rendering a single sequence monospecific for I/i or DNA. The IgM anti-DNA antibodies also recognize bacterial lipid A, whereas the anti-I/i antibodies do not, indicating that CDR3 may be involved in binding the negatively charged lipid A. Structural similarities between the DNA backbone and lipid A provide a possible explanation for this cross-reactivity. This dual recognition of bacterial antigen and autoantigen provides a potential link between infection and autoimmunity.     

Evaluation of the effectiveness of continuous renal replacement therapy in critical patients after cardiovascular surgery

The efficacy of continuous methods of renal substitute therapy (RST) in patients with multiple organ failure is assessed. The patients were divided in 2 groups administered different types of PST. Group 1 were 16 patients subjected to RST by peritoneal dialysis, in group 2 (n = 16) GP and/or GDP were used. Hemodynamics, hematological and biochemical values, and clearance of inflammation mediators were monitored and hemohydrobalance and complications of therapy assessed in the course of RST. Both RST methods proved to be highly effective. The possibility of differentiated use of peritoneal dialysis and GP/GDP permits an individual approach to treatment, and equally high efficacy of both methods solves the problem of treating total renal insufficiency in the majority of patients with multiple organ failure following cardiovascular surgery.     

Deficits in discriminated learning remain despite clearance of long-term persistent viral infection in mice

Mice persistently infected with lymphocytic choriomeningitis virus (LCMV) exhibit impaired learning ability. In this report, we determined whether clearance of the virus was associated with restoration of behavioral function. Neonatal Balb/cByJ mice were persistently infected with LCMV and tested as adults in a nonconditional spatial discrimination task. The presence of viral proteins in neurons was confirmed immunohistochemically and infectious virus was quantified in the blood by plaque assay. LCMV-infected adult mice made more errors in a Y-maze avoidance task compared to sham-inoculated controls. After the initial behavioral analysis, infected and control mice received a dose of cytotoxic T-lymphocytes sufficient to clear virus from these mice. Following complete clearance of the virus, mice were re-tested in the behavioral task, 5 months after the original test. No reversal of the learning deficit was seen following viral clearance; mice that had been cleared of the virus and those that remained persistently infected behaved similarly. These data indicate that persistent LCMV infection of the CNS lasting up to 7 months results in discriminated learning impairments that are not reversed by subsequent anti-viral immunocytotherapy.     

Fluorescence in situ hybridization analysis of chromosome 1p36 deletions in human MYCN amplified neuroblastoma

Artificial illumination is an important factor in the management of layers. In this study, a new monochromatic light system was developed for egg layers. Prelaying pullets (Lohmann) were marked and housed in nine light and temperature control rooms (15 battery cages, 3 hens per cage; n = 45), divided into three light treatments: 0.1 and 0.01 W/m2 light intensity using light emitting diode (LED) lamps and 0.1 W/m2 using mini-fluorescent bulbs (PL) (control). In each of the LED rooms, three wavelengths were tested: 560 (n = 9), 660 (n = 9), 880 (n = 6), and 660 intermitted lighting (15 min light 45 min dark, 660IN) (n = 9). Birds were exposed to 12 h light and 12 h of darkness using PL lamps. At 21 wk of age, the light period was increased to 12.75 h by using 5.5 h of LED lamps and 7.25 of PL light source for Groups 1 and 2, the third group received 12.75 h of PL light. Until 28 wk of age, light hours increased by 0.5 h/w using LED light for Groups 1 and 2 and PL source for the third group, reaching 16 h of light at 28 wk of age. Egg production and feed consumption were recorded daily; egg components were recorded weekly for 10 mo. A significant reduction in egg production was observed in all 880nm groups; no differences in egg production and quality were found in the other groups. Feed consumption was significantly lower by 7% in all 0.01 W/m2 groups. We suggest that an important reduction in rearing costs of laying hens may be obtained by using this system.     

Enzymatic Firming of Processed Red Pepper by Means of Exogenous Pectinesterase

The objective of this investigation was to demonstrate that the firmness of a commercial vegetable product, diced and frozen red pepper (Capsicum annum var. Sendt), could be improved by the use of exogenous pectinesterase in an industrially relevant process. The diced pepper pieces 10 × 10 × 7 mm³ were infused under vacuum with a commercially available pectinesterase. The range of optimal process conditions was: 15-20°C, 45 min infusion time, a 10-25 mM CaCl₂ infusion brine, a w/w ratio of pepper fruit to infusion brine of 1.5:1, and an enzyme dosage of 30-60 pectinesterase units (PEU) per kg pepper fruit. The firmness as measured by back extrusion was improved by a factor of two to three. The effect of firming was robust and conserved after freezing and heating in a simulated household cooking process. The firming process seems easily adaptable to industrial conditions and may be applicable to other vegetable and fruit products.