Urinary stone proteins: an update

The discovery of an organic component in kidney stones dates back to 1684. More than 150 years elapsed before the incrustation of this organic component, which is now called the matrix, was proposed as the mechanism of stone formation. The composition of the matrix remained largely unknown until the development of electron microscopy and the advances in biochemistry combined in the 1950's to usher in the modern era of renal stone matrix investigation. Composed mainly of selectively incorporated proteins generally characterized by high glutamic and aspartic acid content and the frequent occurrence of gamma-carboxyglutamic acid, the matrix displays a variable and complex composition and shares a few proteins in many stones. The embryonic stone may first appear in the renal tubules where it can acquire the blood and cell membrane proteins recently found by analysis of stone protein extracts. The combination of supersaturation, an appropriate environment, the availability of calcium binding proteins which may be abnormal, and the incorporation of proteins extracted from leukocytes and cell wall membranes may induce stone formation.     

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.     

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.     

Inheritance of male hybrid sterility in the house musk shrew (Suncus murinus, Insectivora, Soricidae)

Two geographic races of the house musk shrew (Suncus murinus) were crossed and intercrossed in the laboratory. Many cases of male sterility were detected among the hybrids. Segregation analysis of the pedigree data showed that the inheritance of male sterility in interracial hybrids of S. murinus can be described within the framework of monogene polyallele model with sterility of a single allele combination. This model is similar if not identical to that proposed by Dobzhansky and Muller.     

The A326S mutant of Gialpha1 as an approximation of the receptor-bound state

Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their alpha subunits. In search of an approximation of the receptor-alpha subunit complex, we have considered the properties of A326S Gialpha1, a mutation discovered originally in Gsalpha (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the alphai1beta1gamma2 heterotrimer by 250-fold. Nevertheless, affinity of mutant Gialpha1 for GTPgammaS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the alpha subunit. The mutation also uncouples two activities of betagamma: stabilization of the GDP-bound alpha subunit (which is retained) and retardation of GDP dissociation from the heterotrimer (which is lost). For wild-type and mutant Gialpha1, beta gamma prevents irreversible inactivation of the alpha subunit at 30 degreesC. However, the mutation accelerates irreversible inactivation of alpha at 37 degreesC despite the presence of beta gamma. Structurally, the mutation weakens affinity for GTPgammaS by steric crowding: a 2-fold increase in the number of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those containing A326S Gialpha1. However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers containing A326S Gialpha1. In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissociation of GDP can be observed in the absence of substantial structural alteration of a Galpha subunit in the GDP-bound state.     

Novel approaches to the discovery of antimicrobial agents

OBJECTIVE: To examine the cost-effectiveness of prenatal carrier screening for cystic fibrosis. METHODS: A cost-benefit equation was developed that was based on the hypothesis that the cost of prenatal diagnosis required to diagnose and prevent one case of cystic fibrosis should be equal to or less than the lifetime cost generated from the birth of a neonate with cystic fibrosis. The formula was adjusted because a woman's positive or negative carrier status remains unchanged, thus eliminating the need for testing in subsequent pregnancies. The formula was manipulated to identify the optimal cost per screening test, as well as the net cost savings per prenatally diagnosed case of cystic fibrosis for various racial or ethnic groups. Sensitivity analyses included some key assumptions regarding the cost per screening test ($50-150), patient screening acceptance rates (25-100%), and therapeutic abortion rates (50-100%). RESULTS: Assuming therapeutic abortion rates of 50-100%, the net savings per prenatally diagnosed case of cystic fibrosis are $58,369-$382,369 among whites. Given the previously reported patient screening acceptance rates of 50-78%, the overall annual cost savings in the United States for whites are $161-251 million. However, the screening program was not found to be cost-effective for blacks, Asians, or Hispanics. CONCLUSION: Under most assumptions and sensitivity analyses, a prenatal cystic fibrosis-carrier screening program appears to be cost-effective.     

Molecular mechanisms of transforming growth factor-beta signaling

The present experiments examined the effects of posttraining intrahippocampal injections of the degradative enzyme-resistant methylcarbamyl analog of the bioactive phospholipid platelet-activating factor (mc-PAF) and the platelet-activating factor (PAF) receptor antagonists BN52021 and BN 50730 on memory in male Long-Evans rats trained in a hidden platform version of the Morris water maze. Following an eight-trial training session, rats received a unilateral intrahippocampal injection of mc-PAF (0.5, 1.0, or 2.0 microgram/0.5 microliter), lyso-PAF (1.0 microgram/0.5 microliter), the cell surface PAF receptor antagonist BN 52021 (0.25, 0.5, or 1.0 micrigram/0.5 microliter/, the intracellular PAF receptor antagonist BN 50730 (2.0, 5.0, or 10.0 microgram/0.5 microliter), or vehicle (50% DMSO in 0.9% saline; 0.5 microliter). On a retention test conducted 24 h after training, the escape latencies of rats administered mc-PAF (1.0 or 2.0 microgram) were significantly lower than those of the vehicle-injected controls, demonstrating a memory-enhancing effect of mc-PAF. Injections of lyso-PAF, a structurally similar metabolite of PAF, had no influence on memory, indicating that the memory-enhancing effect of mc-PAF is not caused by membrane perturbation by the phospholipid. The retention test escape latencies of rats administered BN 52021 (0.5 microgram) and BN 50730 (5.0 or 10 microgram) were significantly higher than those of the controls, indicating a memory impairing effect of both PAF antagonists. When mc-PAF, BN 52021, or BN 50730 was administered 2 h posttraining, no effect on retention was observed, indicating a time-dependent effect of the neuroactive substances on memory storage. The findings suggest a role for endogenous PAF in hippocampal-dependent memory processes.