Creatine kinase: an enzyme with a central role in cellular energy metabolism

In an attempt to know the effect of sustained hyperinsulinemia on sympathetic function, plasma norepinephrine (NE) and glucose levels were measured in Wistar rats with insulin resistance. Both the basal plasma glucose and the plasma NE levels in insulin-resistant rats were markedly higher than that obtained in normal or streptozotocin (STZ)-induced diabetic rats. Treatment with guanethidine and prazosin reversed these sympathetic hyperactive responses in insulin-resistant rats. Moreover, increase of plasma insulin in rats receiving an intraperitoneal glucose challenge test confirmed the mediation of endogenous insulin in this sympathetic hyperactivity. These results suggest an increase of sympathetic activity in insulin-resistant state that may be related to the hypertension-prone associated with diabetes mellitus in clinics.     

Interferon-gamma activation of a mitogen-activated protein kinase, KFR1, in the bloodstream form of Trypanosoma brucei

KFR1, a mitogen-activated protein (MAP) kinase identified in the African trypanosome, Trypanosoma brucei, is a serine protein kinase capable of phosphorylating the serine residues in histone H-1, myelin basic protein, and beta-casein. It phosphorylates four proteins with estimated molecular masses of 22, 34, 46, and 90 kDa from the T. brucei bloodstream-form lysate in vitro. KFR1 bears significant sequence similarity to the yeast MAP kinases KSS1 and FUS3 but cannot functionally complement the kss1/fus3 yeast mutant. It is encoded by a single-copy gene in the diploid T. brucei, and only one of the two alleles can be successfully disrupted, suggesting an essential function of KFR1 in T. brucei. KFR1 activity is present at a much enhanced level in the bloodstream form of T. brucei when compared with that in the insect (procyclic) form. This enhanced activity can be eliminated in vitro by the treatment with protein phosphatase HVH2 known to act specifically on MAP kinases. It can also be decreased in the bloodstream form of T. brucei by serum starvation but induced specifically by interferon-gamma. The production of interferon-gamma in the mammalian host is known to be triggered by T. brucei infection, and this cytokine, as has been reported, promotes the proliferation of T. brucei in the mammalian blood. Since none of these phenomena can be observed in the procyclic form of T. brucei, activation of KFR1 is most likely involved in mediating the interferon-gamma-induced proliferation of T. brucei in the mammalian host.     

Characterization of a multicatalytic proteinase complex (20S proteasome) from Trypanosoma brucei brucei

African trypanosomes are tsetse-transmitted protozoan parasites that cause sleeping sickness in humans and 'Nagana' in animals. A high relative molecular mass multicatalytic proteinase complex (MCP) was purified and biochemically characterized from the cytosolic fraction of Trypanosoma brucei brucei. The isolation procedure consisted of fractionation of the lysate by high speed centrifugation, chromatography on Q-sepharose molecular sieve filtration on Sephacryl S-300, chromatography on HA-Ultrogel and glycerol density gradient centrifugation (10-40%). The final enzyme preparation yielded a single protein band corresponding to a relative molecular mass of 630 kDa on a non-denaturing polyacrylamide gel. The enzyme hydrolyses a wide range of peptide substrates characteristic of chymotrypsin-like, trypsin-like, peptidylglutamylpeptide-hydrolysing activities determined by fluorogenic peptides, Z-Gly-Gly-Leu-NHMec, Z-Arg-Arg-NHMec and Z-Leu-Leu-Glu-beta NA, respectively. The enzyme was found to have a wide variation in pH optimal activity profile, with optimum activity against Z-Gly-Gly-Leu-NHMec at 7.8, Z-Arg-Arg-NHMec at pH 10.5 and Z-Leu-Leu-Glu-beta NA at pH 8.0, showing that the different activities are distinct. The enzyme hydrolysed oxidized proteins. In addition, the chymotryptic and trypsin-like activities were susceptible to inhibition by peptide aldehyde inhibitors with variable inhibition effects. The study demonstrates the presence of a non-lysosomal proteasome pathway of intracellular protein degradation in the bloodstream form of T. b. brucei. Further, the ability of the enzyme to hydrolyse most oxidized proteins, and the high immunogenicity exhibited suggests a possible involvement of the enzyme in pathogenesis of the disease.     

Both IgM and IgG anti-VSG antibodies initiate a cycle of aggregation-disaggregation of bloodstream forms of Trypanosoma brucei without damage to the parasite

Bloodstream forms of Trypanosoma brucei, when aggregated in the presence of either acute immune plasma, acute immune serum, purified IgM anti-VSG antibodies or purified IgG anti-VSG antibodies, subsequently disaggregated with a t1/2 for disaggregation of 15 min at 37 degrees C as long as the trypanosomes were metabolically active at the beginning of the experiment and maintained during the experiment in a suitable supporting medium. The t1/2 for disaggregation was found to be directly dependent upon temperature and inversely proportional to the antibody concentration. The trypanosomes were always motile and metabolically active during aggregation and after disaggregation and were fully infective for a mammalian host following disaggregation as well as able to grow and divide normally during axenic culture. The disaggregation was strictly energy dependent and was inhibited when intracellular ATP levels were reduced by salicylhydroxamic acid or following addition of oligomycin while respiring glucose. In addition the process of disaggregation was dependent upon normal endosomal activity as evidenced by its sensitivity to a wide variety of inhibitors of various endosomal functions. Disaggregation was not due to separation of immunoglobulin chains by either disulphide reduction or disulphide exchange reactions and gross proteolytic cleavage of the immunoglobulins attached to the surface of the parasite was not detected. In addition, gross cleavage or release of the VSG from the surface of the cell did not occur during disaggregation but proteolytic cleavage of a small proportion of either the VSG or the immunoglobulins could not be eliminated from consideration. Finally the mechanism of disaggregation was found to be a regulated process, independent of Ca2+ movements but dependent upon the activity of protein kinase C or related kinases and inhibited by the activity of protein kinase A as evidenced by the effects of a panel of inhibitors and cAMP analogues on the process of disaggregation. The mechanism of disaggregation displayed by trypanosomes aggregated by anti-VSG antibody is proposed to form part of the parasite's defence against the host immune system and functions to aid survival of trypanosomes in the presence of antibody in the host prior to the occurrence of a VSG switching event.     

Partitioning of large and minichromosomes in Trypanosoma brucei

The Trypanosoma brucei nuclear genome contains about 100 minichromosomes of between 50 to 150 kilobases and about 20 chromosomes of 0.2 to 6 megabase pairs. Minichromosomes contain nontranscribed copies of variant surface glycoprotein (VSG) genes and are thought to expand the VSG gene pool. Varying VSG expression allows the parasite to avoid elimination by the host immune system. The mechanism of inheritance of T. brucei chromosomes was investigated by in situ hybridization in combination with immunofluorescence. The minichromosome population segregated with precision, by association with the central intranuclear mitotic spindle. However, their positional dynamics differed from that of the large chromosomes, which were partitioned by kinetochore microtubules.     

Compartmentation and control of arginine metabolism in Neurospora

The fate of [14-C]arginine derived from the medium or from biosynthesis has been examined in Neurospora growing in arginine-supplemented medium. In both cases the label enters the cytosol, where it is used efficiently for both protein synthesis and catabolism before mixing with the majority of the endogenous [12C]arginine pool. Both metabolic processes appear to use the same cytosolic arginine pool. It is calculated that the nonorganellar cytoplasm contains approximately 20% of the intracellular arginine pool when the cells are growing in arginine-supplemented medium. The results suggest that compartmentation of arginine is a significant factor in controlling arginine metabolism in Neurospora. The significance of these results for studies of amino acid metabolism in other eukaryotic systems is discussed.     

Tyrosine 140 of the gamma-aminobutyric acid transporter GAT-1 plays a critical role in neurotransmitter recognition

The gamma-aminobutyric acid (GABA) transporter GAT-1 is located in nerve terminals and catalyzes the electrogenic reuptake of the neurotransmitter with two sodium ions and one chloride. We now identify a single tyrosine residue that is critical for GABA recognition and transport. It is completely conserved throughout the superfamily, and even substitution to the other aromatic amino acids, phenylalanine (Y140F) and tryptophan (Y140W), results in completely inactive transporters. Electrophysiological characterization reveals that both mutant transporters exhibit the sodium-dependent transient currents associated with sodium binding as well as the chloride-dependent lithium leak currents characteristic of GAT-1. On the other hand, in both mutants GABA is neither able to induce a steady-state transport current nor to block their transient currents. The nontransportable analog SKF 100330A potently inhibits the sodium-dependent transient in the wild type GAT-1 but not in the Y140W transporter. It partly blocks the transient of Y140F. Thus, although sodium and chloride binding are unimpaired in the tyrosine mutants, they have a specific defect in the binding of GABA. The total conservation of the residue throughout the family suggests that tyrosine 140 may be involved in the liganding of the amino group, the moiety common to all of the neurotransmitters.     

Protein kinase C, but not tyrosine kinases or Ras, plays a critical role in angiotensin II-induced activation of Raf-1 kinase and extracellular signal-regulated protein kinases in cardiac myocytes

Angiotensin II (AngII) induces cardiac hypertrophy through activating a variety of protein kinases. In this study, to understand how cardiac hypertrophy develops, we examined AngII-evoked signal transduction pathways leading to the activation of extracellular signal-regulated protein kinases (ERKs), which are reportedly critical for the development of cardiac hypertrophy, in cultured cardiac myocytes isolated from neonatal rats. Inhibition of protein kinase C (PKC) with calphostin C or down-regulation of PKC by pretreatment with a phorbol ester for 24 h abolished AngII-induced activation of Raf-1 and ERKs, and addition of a phorbol ester conversely induced a marked increase in the activities of Raf-1 and ERKs. Pretreatment with two chemically and mechanistically dissimilar tyrosine kinase inhibitors, genistein and tyrphostin, did not attenuate AngII-induced activation of ERKs. In contrast, genistein strongly blocked insulin-induced ERK activation in cardiac myocytes. Although pretreatment with manumycin, a Ras farnesyltransferase inhibitor, or overexpression of a dominant-negative mutant of Ras inhibited insulin-induced ERK activation, neither affected AngII-induced activation of ERKs. Overexpression of a dominant-negative mutant of Raf-1 completely suppressed ERK2 activation by AngII, endothelin-1, and insulin. These results suggest that PKC and Raf-1, but not tyrosine kinases or Ras, are critical for AngII-induced activation of ERKs in cardiac myocytes.     

Possible role of an adrenal parasite reservoir in the pathogenesis of chronic Trypanosoma cruzi myocarditis

A non-parametric estimator of the AIDS survival time (after developing AIDS) is computed for the AIDS data set from the US Air Force (USAF). Survival times are unobservable. They are censored by the screening mechanism. The Armstrong Laboratory's Epidemiologic Research Division maintains data on over 954 active duty US Air Force (USAF) individuals who tested positive for human immunodeficiency virus (HIV) antibodies. Many have been clinically evaluated seven times since 1986. The HIV-positive individual is classified in seven stages of the disease complex as time progresses. Exact times of transition from one stage to the next are unknown. It is known that transition occurred between two consecutive evaluations. The aim of this study is to analyse distributions of the times that individuals spend in each stage of the HIV disease complex. We will discuss methods used to obtain non-parametric estimators of the distribution of times that individuals spend in stage 6. Finally, it is hoped to model the median time spent in each stage of the disease. This, along with incidence and separation data, will allow the prediction of the impact of HIV disease on USAF individuals and medical care systems.     

Epitope glycosylation plays a critical role for T cell recognition of type II collagen in collagen-induced arthritis

Immunization of mice with type II collagen (CII) leads to collagen-induced arthritis (CIA), a model for rheumatoid arthritis. T cell recognition of CII is believed to be a critical step in CIA development. We have analyzed the T cell determinants on CII and the TCR used for their recognition, using twenty-nine T cell hybridomas derived from C3H.Q and DBA/1 mice immunized with rat CII. All hybridomas were specific for the CII(256-270) segment. However, posttranslational modifications (hydroxylation and variable O-linked glycosylation) of the lysine at position 264 generated five T cell determinants that were specifically recognized by different T cell hybridoma subsets. TCR sequencing indicated that each of the five T cell epitopes selected its own TCR repertoire. The physiological relevance of this observation was shown by in vivo antibody-driven depletion of TCR Valpha2-positive T cells, which resulted in an inhibition of the T cell proliferative response in vitro towards the non-modified CII(256-270), but not towards the glycosylated epitope. Most hybridomas (20/29) specifically recognized CII(256-270) glycosylated with a monosaccharide (beta-D-galactopyranose). We conclude that this glycopeptide is immunodominant in CIA and that posttranslational modifications of CII create new T cell determinants that generate a diverse TCR repertoire.     

In situ analysis of a variant surface glycoprotein expression-site promoter region in Trypanosoma brucei

Duchenne muscular dystrophy is frequently associated with a non-progressive cognitive deficit attributed to the absence of 427,000 mol. wt brain dystrophin, or to altered expression of other C-terminal products of this protein, Dp71 and/or Dp140. To further explore the role of these membrane cytoskeleton-associated proteins in brain function, we studied spatial learning and ex vivo synaptic plasticity in the mdx mouse, which lacks 427,000 mol. wt dystrophin, and in the mdx3cv mutant, which shows a dramatically reduced expression of all the dystrophin gene products known so far. We show that reference and working memories are largely unimpaired in the two mutant mice performing a spatial discrimination task in a radial maze. However, mdx3cv mice showed enhanced emotional reactivity and developed different strategies in learning the task, as compared to control mice. We also showed that both mutants display apparently normal levels of long-term potentiation and paired-pulse facilitation in the CA1 field of the hippocampus. On the other hand, an increased post-tetanic potentiation was shown by mdx, but not mdx3cv mice, which might be linked to calcium-regulatory defects. Otherwise, immunoblot analyses suggested an increased expression of a 400,000 mol. wt protein in brain extracts from both mdx and mdx3cv mice, but not in those from control mice. This protein might correspond to the dystrophin-homologue utrophin. The present results suggest that altered expression of dystrophin or C-terminal dystrophin proteins in brain did not markedly affect hippocampus-dependent spatial learning and CA1 hippocampal long-term potentiation in mdx and mdx3cv mice. The role of these membrane cytoskeleton-associated proteins in normal brain function and pathology remains to be elucidated. Furthermore, the possibility that redundant mechanisms could partially compensate for dystrophins' deficiency in the mdx and mdx3cv models should be further considered.     

Does NADH play a central role in energy metabolism in Helicobacter pylori?

The complete genome sequence of Helicobacter pylori reveals an unusual NADH-quinone oxidoreductase (NDH-1 or Complex I) that might lack the NADH-binding domain. H. pylori also lacks various NADH-generating enzymes. What are the consequences for electron transfer to H. pylori NDH-1 and could NADPH be involved?     

Phosphorylation of moesin by rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates moesin at Thr558 in vitro. Here, using a site- and phosphorylation state-specific antibody, we found that the expression of dominant active RhoA in COS7 cells induced moesin phosphorylation and the formation of microvilli-like structures at apical membranes where the Thr558-phosphorylated moesin accumulated, whereas the expression of dominant negative Rho-kinase inhibited both of these processes. The expression of dominant active Rho-kinase also induced moesin phosphorylation. When COS7 cells expressing moesin or moesinT558A (substitution of Thr by Ala) were cultured under serum-depleted conditions, there were few microvilli-like structures, whereas microvilli-like structures remained in the cells expressing moesinT558D (substitution of Thr by Asp). The expression of moesinT558A inhibited the dominant active RhoA-induced formation of microvilli-like structures. These results indicate that Rho-kinase regulates moesin phosphorylation downstream of Rho in vivo and that the phosphorylation of moesin by Rho-kinase plays a crucial role in the formation of microvilli-like structures.     

Trypanosoma brucei gamma-glutamylcysteine synthetase. Characterization of the kinetic mechanism and the role of Cys-319 in cystamine inactivation

The parasitic protozoan Trypanosoma brucei utilizes a conjugate of glutathione and spermidine, termed trypanothione, in place of glutathione to maintain cellular redox balance. The first committed step in the biosynthesis of glutathione and thereby trypanothione, is catalyzed by gamma-glutamylcysteine synthetase (gamma-GCS). We have determined the kinetic mechanism for T. brucei gamma-GCS. The kinetics are best described by a rapid equilibrium random ter-reactant mechanism, in which the model derived Kd values for the binding of L-Glu, L-alpha-aminobutyrate, and ATP to free enzyme are 2.6, 5.1, and 1.4 mM, respectively. However, significant dependences exist between the binding of some of the substrate pairs. The binding of either ATP or L-Glu to the enzyme increases the binding affinity of the other by 18-fold, whereas the binding of L-Glu or L-alpha-aminobutyrate decreases the binding affinity of the other by 6-fold. Similarly to the mammalian enzyme, cystamine is a time-dependent, irreversible inhibitor of T. brucei gamma-GCS. It has been suggested by several studies that cystamine labels an active site Cys residue essential for catalysis. Among the enzymes reported to be inactivated by cystamine, only one Cys residue is invariant (Cys-319 in T. brucei gamma-GCS). Mutation of Cys-319 to Ala in T. brucei gamma-GCS renders the enzyme insensitive to cystamine inactivation without significantly affecting the enzyme's catalytic efficiency, kinetic mechanism, or substrate affinities. These studies suggest that cystamine inactivates the enzyme by blocking substrate access to the active site and not by labeling an essential active site residue.     

Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation

Phosphoglycerate kinase (PGK), a key enzyme in glycolysis, catalyses the transfer of a phosphoryl-group from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP. Despite extensive kinetic and structural investigations over more than two decades, the conformation assumed by this enzyme during catalysis remained unknown. Here we present the 2.8 A crystal structure of a ternary complex of PGK from Trypanosoma brucei, the causative agent of sleeping sickness. This structure determination relied on a procedure in which fragments containing less than 10% of the scattering mass were successively positioned in the unit cell to obtain phases. The PGK ternary complex exhibits a dramatic closing of the large cleft between the two domains seen in all previous studies, thereby bringing the two ligands, 3-phosphoglycerate and ADP into close proximity. Our results demonstrate that PGK is a hinge-bending enzyme, reveal a novel mechanism in which substrate-induced effects combine synergistically to induce major conformational changes and, to our knowledge, afford the first observation of the PGK active site in a catalytic conformation.     

Stable expression of mosaic coats of variant surface glycoproteins in Trypanosoma brucei

The paradigm of antigenic variation in parasites is the variant surface glycoprotein (VSG) of African trypanosomes. Only one VSG is expressed at any time, except for short periods during switching. The reasons for this pattern of expression and the consequences of expressing more than one VSG are unknown. Trypanosoma brucei was genetically manipulated to generate cell lines that expressed two VSGs simultaneously. These VSGs were produced in equal amounts and were homogeneously distributed on the trypanosome surface. The double-expressor cells had similar population doubling times and were as infective as wild-type cells. Thus, the simultaneous expression of two VSGs is not intrinsically harmful.