New insights into the genetics of lissencephaly

The cause of chronic inflammatory bowel disease remains unknown. A genetic origin has been suggested by studies in twins. The gene or genes involved in Crohn's disease is probably situated on chromosome 16 and the genes involved in ulcerative colitis on chromosomes 2 and 7. It will undoubtedly take some time before the exact loci are precisely identified, but current research suggests that clinical applications are not far off.     

New insights into the pathogenesis of distal renal tubular acidosis

The diagnosis and classification of distal renal tubular acidosis (distal RTA) have traditionally been made on the basis of the renal response to physiologic maneuvers, providing only indirect information on the underlying pathophysiology. In the past several years significant advances have been made in our understanding of the molecular basis of distal H+/HCO3- secretion and absorption, at the level of individual transporters. With these advances, a new era of classifying disorders of distal acidification at the molecular level has arrived. In this article we review the cellular and molecular basis of normal acidification mechanisms in the distal nephron. We also review the recent information on the molecular basis of derangements in these mechanisms which lead to distal RTA.     

New insights into the role of follicle-stimulating hormone in reproduction

Follicle-stimulating hormone (FSH) has been considered essential for normal ovarian follicular development and spermatogenesis. Although this concept remains valid, the recent reports of defects in genes encoding FSH and its receptor (FSHR) have altered the concepts, particularly with regard to the role of FSH in spermatogenesis. Complete FSH resistance caused by a mutation of the FSHR gene has demonstrated that normal FSH action is an absolute requirement for female fertility, but spermatogenesis and male fertility are possible without FSH action. Thus, while normal ovarian function is critically dependent on normal FSH action, male fertility is relatively independent of this hormone. The finding contradicts the potential of inhibition of FSH secretion or action as a means of male contraception.     

New insights into the catalytic cycle of flavocytochrome b2

Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction in the mitochondrial intermembrane space. The catalytic cycle for this process can be described in terms of five consecutive electron-transfer events. L-Lactate dehydrogenation results in the two-electron reduction of FMN. The two electrons are individually passed to b2-heme (intramolecular electron transfer) and then onto cytochrome c (intermolecular electron transfer). At 25 degrees C, I 0.10, in the presence of saturating concentrations of ferricytochrome c and L-lactate, the catalytic cycle progresses with rate constant 104 (+/- 5) s-1 [per L-lactate oxidized; Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. Stopped-flow spectrophotometry has been used to show that the major rate-limiting step in the catalytic cycle is electron transfer from flavin semiquinone to b2-heme. This conclusion is based on the observation that pre-steady-state flavin oxidation by ferricytochrome c takes place at 120 s-1. Although flavin oxidation involves several other electron transfer steps, these are considered too fast to contribute significantly to the rate constant. It was also shown that the reaction product, pyruvate, is able to inhibit pre-steady-state flavin oxidation (Ki = 40 +/- 17 mM) consistent with reports that it acts as a noncompetitive inhibitor in the steady state at high concentrations [Ki = 30 mM; Lederer, F. (1978) Eur. J. Biochem, 88, 425-431]. This novel way of measuring the electron transfer rate constant is directly applicable to the catalytic cycle and has enabled us to derive a self-consistent model for it, based also on data collected for enzyme reduction [Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192] and its interaction with cytochrome c [Daff, S., Sharp, R. E., Short, D. M., Bell, C., White, P., Manson, F. D. C., Reid, G. A., & Chapman, S. K. (1996) Biochemistry 35, 6351-6357]. Rapid-freezing quenched-flow EPR has been used to confirm the model by demonstrating that during steady-state turnover of the enzyme approximately 75% of the flavin is in the semiquinone oxidation state.     

New insights into the epidemiology of malaria relevant for disease control

Despite over 100 years of scientific investigation, malaria remains the leading cause of death among children living in sub-Saharan Africa. Our understanding of the epidemiology of clinical malaria has, until recently, been hampered by a paucity of empirical data from endemic settings. A striking feature of Plasmodium falciparum malaria is that, compared to infection and mild disease, severe complications and death are rare. Perhaps the single most important factor which ameliorates the risk of asymptomatic infection progressing to life-threatening pathology is the development of clinical immunity. Examination of recent epidemiological evidence suggests that the speed with which clinical immunity is acquired is dependent upon the frequency of parasite exposure from birth. Consequently, the age at which disease presentation peaks, the clinical spectrum of disease and the life-time risks of disease appear to be a function of the intensity of transmission within a given community. These observations are discussed in relation to control measures aimed at reducing P. falciparum exposure and the need to understand better the processes by which children naturally acquire clinical immunity before more rational statements can be made about their wide-spread use in Africa.     

New insights into the structural basis of alpha 1-antitrypsin deficiency

The serpin superfamily of serine proteinase inhibitors contains many members but the best-characterized is the plasma protein alpha 1-antitrypsin. its genetic deficiency is associated, in the homozygote, with hepatic damage that may progress to cirrhosis and hepatocellular carcinoma. Low levels of circulating alpha 1-antitrypsin fail to protect the lungs against proteolytic attack and predispose the homozygote to early onset pan-lobular emphysema, bronchiectasis and asthma. The major cause of alpha 1-antitrypsin deficiency, the Z mutation (Glu342Lys), results in the accumulation of protein in the endoplasmic reticulum of the liver. Using a structural approach, we have shown that the hepatic inclusions result from a protein-protein interaction between the reactive centre loop of one molecule and the beta-pleated sheet of a second. This loop-sheet polymerization is now also recognized to be the basis of deficiencies associated with mutations of C1-inhibitor, antithrombin and alpha 1-antichymotrypsin. Our recent solution of a crystal structure of a thermostable mutant of alpha 1-antitrypsin shows the detailed interactions that result in loop-sheet linkage and helps to explain the mechanism of action of this family of proteinase inhibitors.     

New insights into p53 function from structural studies

Recent structural analysis of p53 has greatly enhanced our understanding of the biochemical activities of this protein by presenting us with a detailed picture of the chemical groups in the protein that are involved in protein stability, conformation and functional interactions. The current structures form the basis for the design of potential therapeutics which could, for example, revert a DNA-binding mutant back to a DNA-binding competent conformation. The structure of the tet domain forms the basis for designing an active therapeutic p53 with an oligomerization domain which would not cross react with a DNA-binding mutant p53. However, as useful as these structures have been in providing insight into the structure/function relationship for p53, a complete understanding of this protein awaits more detailed information on the full-length protein. In this respect, one of the most useful roles for future structural studies will be to help identify the nature of the conformational transition between latent and active p53, and how it can be modulated.     

New insights into cyclosporine A nephrotoxicity by proteome analysis

Using two-dimensional gel electrophoresis (2-DE), we recently discovered an association between decreased calcium-binding protein, calbindin-D 28 kDa, urinary calcium wasting and intratubular corticomedullary calcifications in rat kidney. This observation prompted us to investigate kidney tissues of other species, including man. In this paper we show that in dogs and monkeys, which are generally devoid of cyclosporine A (CsA)-mediated nephrotoxicity, renal calbindin levels were not affected by the CsA treatment whereas in CsA-treated human kidney-transplant recipients with renal vascular or tubular toxicity, a marked decrease in renal calbindin-D 28 kDa protein level was found in most of the kidney biopsy sections. The present results strongly suggest that calbindin is a marker for CsA-nephrotoxicity. The discovery of calbindin-D 28 kDa being involved in CsA toxicity has evolved from the application of 2-DE and has not been reported previously, proving that proteomics can provide essential information in mechanistic toxicology. Considering the current improvements in proteome methods it is expected that high throughput proteomics will become an indispensable tool in preclinical safety testing.     

New insights into influencing variables of water atomisation of iron

Abstract

Trace amounts of surfactants have an acute influence on measured surface tension of melts and may influence viscosity. A water atomisation experiment was performed to investigate if variations of these elements could affect quality. Effects of water pressure, melt superheat, and sulphur content, iron scrap oxygen content, and aluminium content were studied. Responses studied were particle size distribution, apparent density, flow, powder chemistry, morphology, green density, and dimensional change. A large sulphur addition reduced the particle size, as a result of a reduction of surface tension, but the largest effect came from changing water pressure. Higher water pressures also resulted in powders with lower apparent density, lower flowrate, and reduced swelling during sintering. An empirical water atomisation model is proposed. Aluminium additions reduced the powder size standard deviation and increased the carbon content of the powder. A reduced powder size standard deviation was seen also for melts with raised superheating.     

New insights from spectroscopy into the structure/function relationships of lipoxygenases

Spectroscopic properties of the redox-active iron in the active site of plant and mammalian lipoxygenases can now be combined with recent crystal structure determinations to obtain new insights into lipoxygenase reaction mechanisms.     

New insights into the pathogenesis of coagulation dysfunction in acute promyelocytic leukemia

Patients with acute promyelocytic leukemia (APL) are at high risk for the development of life-threatening thrombotic and hemorrhagic complications, particularly during induction chemotherapy. This propensity has been attributed to the release of tissue factor (TF)-like procoagulants from the leukemic cells leading to disseminated intravascular coagulation (DIC). However, recent data suggest that the pathogenesis of the coagulopathy is more complicated and may involve activation of the generalized proteolytic cascade resulting in either clotting and/or excessive fibrinolysis. Furthermore, controversy exists regarding the mechanism(s) responsible for the activation of either clotting or fibrinolysis. The malignant promyelocyte may act directly to activate coagulation and/or fibrinolysis. Alternatively, reactive inflammatory cells, which express procoagulant and/or profibrinolytic activities may play an essential role. A third possibility may involve endothelial cell expression of mediators with procoagulant/profibrinolytic properties. Putative profibrinolytic mechanisms include the release of urokinase-type and tissue-type plasminogen activators, decreases in plasminogen activator inhibitor-1 and 2, and decreases in alpha-2 plasmin inhibitor. Putative procoagulant mechanisms include the release of tissue factor, Cancer Procoagulant, or cytokines such as interleukin-1, tumor necrosis factor and vascular permeability factor. Putative anticoagulant mediators include annexins, a group of proteins in human tissue which bind phospholipids and have anticoagulant activity, which have been reported in patients with APL. The current treatment of APL is rapidly evolving because of the efficacy of all-trans retinoic acid (ATRA). All-trans retinoic acid promotes terminal differentiation of leukemic promyelocytes leading to complete remission in the majority of patients with APL with rapid resolution of the coagulopathy. Although the mechanism by which this occurs has not been established, preliminary data suggest that ATRA blocks the downregulation of the thrombomodulin gene and the up-regulation of the tissue factor gene induced by tumor necrosis factor. Since APL is a relatively uncommon disorder, the collaboration of cooperative oncology groups will be important to study patients receiving ATRA or conventional chemotherapy to further elucidate the mechanism(s) of the coagulopathy.     

New insights into the immunoultrastructural organization of cutaneous basement membrane zone molecules

The epidermal basement membrane zone (BMZ) is composed of various molecules, each of which plays an important role in dermo-epidermal adhesion. Genetic abnormality of certain BMZ molecules leads to an inherited group of skin diseases collectively referred to as epidermolysis bullosa, whose hallmark is skin fragility of varying degrees. Furthermore, development of autoantibodies to certain BMZ molecules leads to the onset of a number of acquired autoimmune blistering diseases in which dermo epidermal separation occurs, including bullous pemphigoid and epidermolysis bullosa acquisita. The ultrastructural location of each BMZ molecule has been studied using a range of immunoelectron microscopy (immuno-EM) techniques. Recent technical advances in immuno-EM and in molecular engineering for production of epitope-specific antibodies have enabled a more correct and precise elucidation of the native ultrastructural molecular organization of the respective molecules and their relationship to each other. These recent studies have also revealed several misinterpretations in the previously established model of the immunoultrastructural organization of BMZ molecules. In response to these findings, this review focuses on three major BMZ-related molecules, type VII collagen, BPAG2 and laminin 5, for which recent immuno-EM studies have produced a revision in the accepted dogma on their ultrastructural distribution at the BMZ.     

Suppressor mutation analysis of the sensory rhodopsin I-transducer complex: insights into the color-sensing mechanism

The molecular complex containing the phototaxis receptor sensory rhodopsin I (SRI) and transducer protein HtrI (halobacterial transducer for SRI) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. One-photon excitation of the complex by orange light elicits attractant responses, while two-photon excitation (orange followed by near-UV light) elicits repellent responses in swimming cells. Several mutations in SRI and HtrI cause an unusual mutant phenotype, called orange-light-inverted signaling, in which the cell produces a repellent response to normally attractant light. We applied a selection procedure for intragenic and extragenic suppressors of orange-light-inverted mutants and identified 15 distinct second-site mutations that restore the attractant response. Two of the 3 suppressor mutations in SRI are positioned at the cytoplasmic ends of helices F and G, and 12 suppressor mutations in HtrI cluster at the cytoplasmic end of the second HtrI transmembrane helix (TM2). Nearly all suppressors invert the normally repellent response to two-photon stimulation to an attractant response when they are expressed with their suppressible mutant alleles or in an otherwise wild-type strain. The results lead to a model for control of flagellar reversal by the SRI-HtrI complex. The model invokes an equilibrium between the A (reversal-inhibiting) and R (reversal-stimulating) conformers of the signaling complex. Attractant light and repellent light shift the equilibrium toward the A and R conformers, respectively, and mutations are proposed to cause intrinsic shifts in the equilibrium in the dark form of the complex. Differences in the strength of the two-photon signal inversion and in the allele specificity of suppression are correlated, and this correlation can be explained in terms of different values of the equilibrium constant (Keq) for the conformational transition in different mutants and mutant-suppressor pairs.     

New insights into positional alcohol nystagmus using three-dimensional eye-movement analysis

BACKGROUND: It is generally accepted that 0 to 4 degreesC is a suitable temperature for organ preservation. The reason for this is based on the premise that at temperatures below 0 degreesC, intracellular ice is likely to form, with subsequent damage to cellular structures. However, it cannot be assumed that subzero temperatures will freeze the cell. In this study, we attempted to confirm the specific freezing point of rat liver and to preserve it at a temperature just above that point. METHODS: Rat livers were stored for 24, 48, 72, and 96 h either at 4 degreesC (Group N) or at -0.8 degreesC (just above the temperature ascertained to be the specific freezing point of rat liver; Group H). After cold storage, the livers were perfused for 60 min using an isolated perfused liver model for assessment of liver function. RESULTS: ATP and TAN (total adenine nucleotides) in reperfused liver tissues were significantly higher in Group H than in Group N for all preservation periods. ADP was significantly higher in Group H than in Group N for 24-, 72-, and 96-h preservation periods. Energy charge was significantly higher in Group H than in Group N for 24-, 48-, and 96-h preservation periods. CONCLUSIONS: Regarding the content of ATP, ADP, and TAN and the adenylate energy charge, our results indicate that preservation at -0.8 degreesC is advantageous. This novel preservation technique seems to prolong the period that organs can be stored.     

New insights into the phylogeny of eukaryotes based on ciliate Hsp70 sequences

The current framework of the eukaryotic phylogeny is based on the analysis of a comprehensive set of sequences of the small subunit ribosomal RNA. However, phylogenies based on protein-encoding genes are not completely congruent with this picture. Since congruence between different markers is the best tool to determine evolutionary history, we focused on Hsp70 (heat-shock protein of 70 kDa), a chaperone protein which is highly conserved and is a potentially reliable phylogenetic marker. We used a PCR-based approach to sequence Hsp70s in two distinct classes of Ciliates. Seven Hsp70s were identified from Paramecium tetraurelia (Oligohymenophora) and six Hsp70s from Euplotes aediculatus (Hypotricha), encompassing orthologous genes for all major Hsp70 classes of Eukaryotes, i.e., those localized in cytosol, in endoplasmic reticulum, and in mitochondria. Three independent phylogenies of eukaryotes, based on each set of orthologous genes, have been constructed using different tree reconstruction methods. A significant advantage of Hsp70s is the existence of outgroups close to Eukaryotes for these major classes, reducing the long-branch attraction artifact due to the outgroup. The monophyly of Ciliates is supported by good bootstrap proportions in the phylogenetic reconstructions, and this phylum is generally a sister-group of Sporozoa, forming the expected Alveolates clade. The Hsp70 seems to be a suitable phylogenetic marker since it recovers all the monophyletic groups, undoubtedly defined by morphological criteria. The Hsp70 trees are, however, notably different from the rRNA ones and do not show two aspects of the classical topology, i.e., the successive emergence of deeply branching groups and the vast assembly of the major eukaryotic groups, emerging at the tip of the tree, i.e., the "terminal crown". More precisely, the Hsp70 trees do not resolve the relationships between the major groups of Eukaryotes with confidence, in keeping with the hypothesis that all these groups emerged in a great radiation that occurred at the origin of all the extant Eukaryotes.     

New insights into the co-evolution of cytochrome c reductase and the mitochondrial processing peptidase

The mitochondrial processing peptidase (MPP) is a heterodimeric enzyme that forms part of the cytochrome c reductase complex from higher plants. Mitochondria from mammals and yeast contain two homologous enzymes: (i) an active MPP within the mitochondrial matrix and (ii) an inactive MPP within the cytochrome c reductase complex. To elucidate the evolution of MPP, the cytochrome c reductase complexes from lower plants were isolated and tested for processing activity. Mitochondria were prepared from the staghorn fern Platycerium bifurcatum, from the horsetail Equisetum arvense, and from the colorless algae Polytomella, and cytochrome c reductase complexes were purified by a micro-isolation procedure based on Blue-native polyacrylamide gel electrophoresis and electroelution. This is the first report on the subunit composition of a respiratory enzyme complex from a fern or a horsetail. The cytochrome c reductase complexes from P. bifurcatum and E. arvense are shown to efficiently process mitochondrial precursor proteins, whereas the enzyme complex from Polytomella lacks proteolytic activity. An evolutionary model is suggested that assumes a correlation between the presence of an active MPP within the cytochrome c reductase complex and the occurrence of chloroplasts.     

New insights into the compartmentation of glutamate and glutamine in cultured rat brain astrocytes

Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of 14CO2 production from U-[14C]glutamate and U-[14C]glutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation of 1 mM glutamine and glutamate were 26.4 +/- 1.4 and 63.0 +/- 7.4 nmol/h/mg protein, respectively. The addition of 1 mM glutamate decreased the rate of oxidation of glutamine to 26.3% of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of 14CO2 production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate < or = 0.1 mM by approximately 50%, but decreased the oxidation of 0.5-1 mM glutamate by only approximately 20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.     

New insights into proteasome function: from archaebacteria to drug development

The proteasome is not simply a 'garbage disposal unit' but also has functions in the control of the cell cycle and immune responses. The structure of an archaebacterial proteasome has recently been determined to high resolution, and provides insight into the unusual mechanism of proteolytic cleavage by the proteasome.