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Biomedical subjects

D E Goldberg

Publications and source records attributed to D E Goldberg.

At least 19 recordsLinked to original sources
PubMed2005

Hemoglobin degradation.

Hemoglobin degradation by Plasmodium is a massive catabolic process within the parasite food vacuole that is important for the organism's survival in its host erythrocyte. A proteolytic pathway is responsible for generating amino acids from hemoglobin. Each of the enzymes involved has its own peculiarities to be exploited for development of antimalarial agents that will starve the parasite or result in build-up of toxic intermediates. There are a number of unanswered questions concerning the cell biology, biochemistry and metabolic roles of this crucial pathway.

Animals↗
PubMed2003

Long term visual outcome of patients with cytomegalovirus retinitis treated with highly active antiretroviral therapy.

BACKGROUND: Healed cytomegalovirus (CMV) retinitis in the setting of highly active antiretroviral therapy (HAART) is complicated by inflammatory sequelae and vision loss. AIM: To determine the long term visual outcome of AIDS patients with CMV retinitis who received HAART. METHODS: 90 eyes of 63 consecutive AIDS patients with extramacular CMV retinitis were studied prospectively. RESULTS: Immune recovery status was related to time to onset of epiretinal membrane (p=0.05) and cystoid macular oedema (p=0.06) as well as to the incidence of cataract (p=0.001) and moderate vision loss (p<0.0001). Severe vision loss was associated with retinal detachment (p<0.001). CONCLUSION: AIDS patients with extramacular CMV retinitis lose vision while on HAART. HAART related immune recovery is associated with increased frequencies of epiretinal membrane, cystoid macular oedema, cataract, and retinal detachment with resultant vision loss in AIDS patients with healed CMV retinitis.

AIDS-Related Opportunistic Infections↗
PubMed2001

Localization of the Plasmodium falciparum PfNT1 nucleoside transporter to the parasite plasma membrane.

Nutrient transporters play critical roles in parasite metabolism, but the membranes in which they reside have not been clearly defined. The transport of purine nutrients is crucial to the survival of the malaria parasite Plasmodium falciparum, and nucleoside transport activity has been associated with a number of different membrane components within the parasitized erythrocyte. To determine the location of the PfNT1 nucleoside transporter, the first component of the nucleoside permeation pathway to be studied at the molecular level in P. falciparum (Carter, N. S., Ben Mamoun, C., Liu, W., Silva, E. O., Landfear, S. M., Goldberg, D. E., and Ullman, B. (2000) J. Biol. Chem. 275, 10683-10691), polyclonal antisera against the NH2-terminal 36 amino acids of PfNT1 were raised in rabbits. Western blot analysis of parasite lysates revealed that the antibodies were specific for PfNT1 and that the level of PfNT1 protein in the infected erythrocyte is regulated in a stage-specific fashion. The amount of PfNT1 polypeptide increases dramatically during the early trophozoite stage and reaches its maximal level in the late trophozoite and schizont stages. Deconvolution and immunoelectron microscopy using these monospecific antibodies revealed that PfNT1 localizes predominantly, if not exclusively, to the plasma membrane of the parasite and not to the parasitophorous vacuolar or erythrocyte membranes.

Animals↗
PubMed2001

Malaria parasite exit from the host erythrocyte: a two-step process requiring extraerythrocytic proteolysis.

Intraerythrocytic malaria parasites replicate by the process of schizogeny, during which time they copy their genetic material and package it into infective merozoites. These merozoites must then exit the host cell to invade new erythrocytes. To better characterize the events of merozoite escape, erythrocytes containing Plasmodium falciparum schizonts were cultured in the presence of the cysteine protease inhibitor, l-transepoxy-succinyl-leucylamido-(4-guanidino)butane (E64). This treatment resulted in the accumulation of extraerythrocytic merozoites locked within a thin, transparent membrane. Immunomicroscopy demonstrated that the single membrane surrounding the merozoites is not erythrocytic but rather is derived from the parasitophorous vacuolar membrane (PVM). Importantly, structures identical in appearance can be detected in untreated cultures at low frequency. Further studies revealed that (i) merozoites from the PVM-enclosed merozoite structures (PEMS) are invasive, viable, and capable of normal development; (ii) PEMS can be purified easily and efficiently; and (iii) when PEMS are added to uninfected red blood cells, released merozoites can establish a synchronous wave of infection. These observations suggest that l-transepoxy-succinyl-leucylamido-(4-guanidino)butane (E64) causes an accumulation of an intermediate normally present during the process of rupture. We propose a model for the process of rupture: merozoites enclosed within the PVM first exit from the host erythrocyte and then rapidly escape from the PVM by a proteolysis-dependent mechanism.

Animals↗
PubMed2001

Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets.

All parasitic protozoa contain multiple proteases, some of which are attracting attention as drug targets. Aspartic proteases are already the targets of some clinically useful drugs (e.g. chemotherapy of HIV infection) and a variety of factors make these enzymes appealing to those seeking novel antiparasite therapies. This review provides a critical analysis of the current knowledge on Plasmodium aspartic proteases termed plasmepsins, proposes a definitive nomenclature for this group of enzymes, and compares these enzymes with aspartic proteases of humans and other parasitic protozoa. The present status of attempts to obtain specific inhibitors of the parasite enzymes that will be useful as drugs is outlined and suggestions for future research priorities are proposed.

Amino Acid Sequence↗
PubMed2001

Co-ordinated programme of gene expression during asexual intraerythrocytic development of the human malaria parasite Plasmodium falciparum revealed by microarray analysis.

Plasmodium falciparum is a protozoan parasite responsible for the most severe forms of human malaria. All the clinical symptoms and pathological changes seen during human infection are caused by the asexual blood stages of Plasmodium. Within host red blood cells, the parasite undergoes enormous developmental changes during its maturation. In order to analyse the expression of genes during intraerythrocytic development, DNA microarrays were constructed and probed with stage-specific cDNA. Developmental upregulation of specific mRNAs was found to cluster into functional groups and revealed a co-ordinated programme of gene expression. Those involved in protein synthesis (ribosomal proteins, translation factors) peaked early in development, followed by those involved in metabolism, most dramatically glycolysis genes. Adhesion/invasion genes were turned on later in the maturation process. At the end of intraerythrocytic development (late schizogony), there was a general shut-off of gene expression, although a small set of genes, including a number of protein kinases, were turned on at this stage. Nearly all genes showed some regulation over the course of development. A handful of genes remained constant and should be useful for normalizing mRNA levels between stages. These data will facilitate functional analysis of the P. falciparum genome and will help to identify genes with a critical role in parasite progression and multiplication in the human host.

Animals↗
PubMed2001

Plasmodium protein phosphatase 2C dephosphorylates translation elongation factor 1beta and inhibits its PKC-mediated nucleotide exchange activity in vitro.

The elongation step of protein synthesis involves binding of aminoacyl-tRNA to the ribosomal A site, formation of a peptide bond and translocation of the newly formed peptidyl-tRNA to the P site. The nucleotide exchange factor EF-1beta plays a major role in the regulation of this process by regenerating a GTP-bound EF-1alpha necessary for each elongation cycle. EF-1beta has been shown to be phosphorylated and its phosphorylation is critical for optimal activity. We have previously identified a serine/threonine protein phosphatase 2C (PP2C) from the human malaria parasite Plasmodium falciparum. In the current work, we performed Far-Western analysis to identify PfPP2C substrates. Several components of the translation and transcription machinery were identified, including translation elongation factor 1-beta (PfEF-1beta). PfEF-1beta is efficiently phosphorylated by protein kinase C and this phosphorylation results in a 400% increase in its nucleotide exchange activity. PKC-phosphorylated PfEF-1beta is readily and selectively dephosphorylated by recombinant and native PfPP2C, which downregulates the nucleotide exchange activity to its basal level. The identification of a translation elongation component as substrate for PP2C suggests an important regulatory function for this enzyme and suggests that it may be a good target for drug design in the fight against malaria.

Amino Acid Sequence↗
PubMed2001

A purine-sensitive mechanism regulates the molecular program for axon growth.

Axon growth is characterized by a distinctive program of gene expression. We present evidence here that this program is regulated through a purine-sensitive mechanism, and that it can be re-activated in mature CNS neurons to induce extensive axon growth in vitro and in vivo. In dissociated goldfish retinal ganglion cells, the purine nucleoside inosine acts intracellularly to stimulate axon outgrowth by inducing the expression of GAP-43, Talpha-1 tubulin, and other growth-associated proteins. The purine analog 6-thioguanine (6-TG) acts in the opposite fashion, blocking axon growth and the underlying program of molecular changes. Prior studies in PC12 cells have shown that 6-TG selectively inhibits the activity of N-kinase, a 47-49 kDa serine-threonine kinase. Inosine acts as a competitor of 6-TG, suggesting that it acts as an N-kinase agonist, and that this kinase is part of a modular signal transduction pathway controlling axon growth. Following unilateral transections of the corticospinal tract in mature rats, inosine applied to the intact sensorimotor cortex stimulated layer 5 pyramidal cells to upregulate GAP-43 expression and to sprout axon collaterals that crossed the midline and reinnervated regions of the cervical spinal cord which had lost their normal afferents. It will now be important to identify the molecular changes that lie upstream and downstream of N-kinase, and to explore the clinical potential of activating this pathway in patients who have sustained CNS injury.

Animals↗
PubMed2000

Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum.

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is incapable of de novo purine synthesis, and thus, purine acquisition from the host is an indispensable nutritional requirement. This purine salvage process is initiated by the transport of preformed purines into the parasite. We have identified a gene encoding a nucleoside transporter from P. falciparum, PfNT1, and analyzed its function and expression during intraerythrocytic parasite development. PfNT1 predicts a polypeptide of 422 amino acids with 11 transmembrane domains that is homologous to other members of the equilibrative nucleoside transporter family. Southern analysis and BLAST searching of The Institute for Genomic Research (TIGR) malaria data base indicate that PfNT1 is a single copy gene located on chromosome 14. Northern analysis of RNA from intraerythrocytic stages of the parasite demonstrates that PfNT1 is expressed throughout the asexual life cycle but is significantly elevated during the early trophozoite stage. Functional expression of PfNT1 in Xenopus laevis oocytes significantly increases their ability to take up naturally occurring D-adenosine (K(m) = 13.2 microM) and D-inosine (K(m) = 253 microM). Significantly, PfNT1, unlike the mammalian nucleoside transporters, also has the capacity to transport the stereoisomer L-adenosine (K(m) > 500 microM). Inhibition studies with a battery of purine and pyrimidine nucleosides and bases as well as their analogs indicate that PfNT1 exhibits a broad substrate specificity for purine and pyrimidine nucleosides. These data provide compelling evidence that PfNT1 encodes a functional purine/pyrimidine nucleoside transporter whose expression is strongly developmentally regulated in the asexual stages of the P. falciparum life cycle. Moreover, the unusual ability to transport L-adenosine and the vital contribution of purine transport to parasite survival makes PfNT1 an attractive target for therapeutic evaluation.

Adenosine↗
PubMed2000

Origin of the anomalous Fe-CO stretching mode in the CO complex of Ascaris hemoglobin.

We report an unusually high frequency (543 cm(-)(1)) for an Fe-CO stretching mode in the CO complex of Ascaris suum hemoglobin as compared to vertebrate hemoglobins in which the frequency of the Fe-CO mode is much lower. A second Fe-CO stretching mode in Ascaris hemoglobin is observed at 515 cm(-1). We propose that these two Fe-CO stretching modes arise from two protein conformers corresponding to interactions of the heme-bound CO with the B10-tyrosine or the E7-glutamine residues. This postulate is supported by spectra from the B10-Tyr --> Phe mutant in which the 543 cm(-1) line is absent. Thus, a strong polar interaction, such as hydrogen bonding, of the CO with the distal B10 tyrosine residue is the dominant factor that causes this anomalously high frequency. Strong hydrogen bonding between O(2) and distal residues in the oxy complex of Ascaris hemoglobin has been shown to result in a rigid structure, rendering an extremely low oxygen off rate [Gibson, Q. H., and Smith, M. H. (1965) Proc. R. Soc. London B 163, 206-214]. In contrast, the CO off rate in Ascaris hemoglobin is very similar to that in sperm whale myoglobin. The high CO off rate relative to that of O(2) in Ascaris hemoglobin is attributed to a rapid equilibrium between the two conformations of the protein in the CO adduct, with the off rate being determined by the conformer with the higher rate.

Animals↗
PubMed2000

Hypertension incidence is predicted by high levels of hopelessness in Finnish men.

Recent studies have reported that hopelessness is an important factor in cardiovascular morbidity and mortality, including ischemic heart disease, acute myocardial infarction, and atherosclerotic progression. This study examined the relationship between hopelessness and incident hypertension in a population-based sample of 616 initially normotensive, middle-aged men from eastern Finland, an area with high rates of cardiovascular disease. Participants completed a medical examination and a series of psychological questionnaires at baseline and at the 4-year follow-up. Hopelessness was measured by 2 items assessing negative expectancy about the future and one's goals. A logistic regression model with adjustments for age, body mass index, baseline resting blood pressure, physical activity, smoking, alcohol consumption, education, parental history of hypertension, and self-reported depressive symptoms revealed that men reporting high levels of hopelessness at baseline were 3 times more likely to become hypertensive (systolic blood pressure > or =165 mm Hg and/or a diastolic blood pressure > or =95 mm Hg or confirmed use of antihypertensive medication) in the intervening 4 years than men who were not hopeless (odds ratio, 3.22; 95% confidence interval, 1. 56, 6.67). Men reporting moderate levels of hopelessness were not at a significantly increased risk of hypertension (odds ratio, 1.27; 95% confidence interval, 0.79, 2.07). This is the first study to identify a significant relationship between hopelessness and incident hypertension. Research is needed to explore the neuroendocrine and central nervous system mechanisms underlying this association.

Adaptation, Psychological↗
PubMed2000

Linkage problem, distribution estimation, and Bayesian networks.

This paper proposes an algorithm that uses an estimation of the joint distribution of promising solutions in order to generate new candidate solutions. The algorithm is settled into the context of genetic and evolutionary computation and the algorithms based on the estimation of distributions. The proposed algorithm is called the Bayesian Optimization Algorithm (BOA). To estimate the distribution of promising solutions, the techniques for modeling multivariate data by Bayesian networks are used. The BOA identifies, reproduces, and mixes building blocks up to a specified order. It is independent of the ordering of the variables in strings representing the solutions. Moreover, prior information about the problem can be incorporated into the algorithm, but it is not essential. First experiments were done with additively decomposable problems with both nonoverlapping as well as overlapping building blocks. The proposed algorithm is able to solve all but one of the tested problems in linear or close to linear time with respect to the problem size. Except for the maximal order of interactions to be covered, the algorithm does not use any prior knowledge about the problem. The BOA represents a step toward alleviating the problem of identifying and mixing building blocks correctly to obtain good solutions for problems with very limited domain information.

Algorithms↗
PubMed1999

Inosine stimulates extensive axon collateral growth in the rat corticospinal tract after injury.

The purine nucleoside inosine has been shown to induce axon outgrowth from primary neurons in culture through a direct intracellular mechanism. For this study, we investigated the effects of inosine in vivo by examining whether it would stimulate axon growth after a unilateral transection of the corticospinal tract. Inosine applied with a minipump to the rat sensorimotor cortex stimulated intact pyramidal cells to undergo extensive sprouting of their axons into the denervated spinal cord white matter and adjacent neuropil. Axon growth was visualized by anterograde tracing with biotinylated dextran amine and by immunohistochemistry with antibodies to GAP-43. Thus, inosine, a naturally occurring metabolite without known side effects, might help to restore essential circuitry after injury to the central nervous system.

Animals↗
PubMed1999

1H NMR investigation of the distal hydrogen bonding network and ligand tilt in the cyanomet complex of oxygen-avid Ascaris suum hemoglobin.

The O(2)-avid hemoglobin from the parasitic nematode Ascaris suum exhibits one of the slowest known O(2) off rates. Solution (1)H NMR has been used to investigate the electronic and molecular structural properties of the active site for the cyano-met derivative of the recombinant first domain of this protein. Assignment of the heme, axial His, and majority of the residues in contact with the heme reveals a molecular structure that is the same as reported in the A. suum HbO(2) crystal structure (Yang, J., Kloek, A., Goldberg, D. E., and Mathews, F. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4224-4228) with the exception that the heme in solution is rotated by 180 degrees about the alpha,gamma-meso axis relative to that in the crystal. The observed dipolar shifts, together with the crystal coordinates of HbO(2), provide the orientation of the magnetic axes in the molecular framework. The major magnetic axis, which correlates with the Fe-CN vector, is found oriented approximately 30 degrees away from the heme normal and indicates significant steric tilt because of interaction with Tyr(30)(B10). The three side chain labile protons for the distal residues Tyr(30)(B10) and Gln(64)(E7) were identified, and their relaxation, dipolar shifts, and nuclear Overhauser effects to adjacent residues used to place them in the distal pocket. It is shown that these two distal residues exhibit the same orientations ideal for H bonding to the ligand and to each other, as found in the A. suum HbO(2) crystal. It is concluded that the ligated cyanide participates in the same distal H bonding network as ligated O(2). The combination of the strong steric tilt of the bound cyanide and slow ring reorientation of the Tyr(30)(B10) side chain supports a crowded and constrained distal pocket.

Animals↗
PubMed1999

Identification and characterization of falcilysin, a metallopeptidase involved in hemoglobin catabolism within the malaria parasite Plasmodium falciparum.

The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in the hemoglobin proteolytic pathway. It is unable to cleave hemoglobin or denatured globin but readily destroys peptide fragments of hemoglobin. Falcilysin cleavage sites along the alpha and beta chains of hemoglobin are polar in character, with charged residues located in the P1 and/or P4' positions. In contrast, plasmepsins I and II and falcipain prefer hydrophobic residues around the scissile bond. The gene encoding falcilysin has been cloned. Its coding sequence exhibits features characteristic of clan ME family M16 metallopeptidases, including an "inverted" HXXEH active site motif. Falcilysin shares primary structural features with M16 family members such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin as well as with data base hypothetical proteins that are potential M16 family members. The characterization of falcilysin increases our understanding of hemoglobin catabolism in P. falciparum and the unusual M16 family of metallopeptidases.

Amino Acid Sequence↗
PubMed1999

Ascaris haemoglobin is a nitric oxide-activated 'deoxygenase'.

The parasitic nematode Ascaris lumbricoides infects one billion people worldwide. Its perienteric fluid contains an octameric haemoglobin that binds oxygen nearly 25,000 times more tightly than does human haemoglobin. Despite numerous investigations, the biological function of this molecule has remained elusive. The distal haem pocket contains a metal, oxygen and thiol, all of which are known to be reactive with nitric oxide. Here we show that Ascaris haemoglobin enzymatically consumes oxygen in a reaction driven by nitric oxide, thus keeping the perienteric fluid hypoxic. The mechanism of this reaction involves unprecedented chemistry of a haem group, a thiol and nitric oxide. We propose that Ascaris haemoglobin functions as a 'deoxygenase', using nitric oxide to detoxify oxygen. The structural and functional adaptations of Ascaris haemoglobin suggest that the molecular evolution of haemoglobin can be rationalized by its nitric oxide related functions.

Anaerobiosis↗