Abdur Rehman

A tissue specific approach for analysis of membrane and secreted protein antigens from Haemonchus contortus gut and its application to diverse nematode species

General methods to conduct tissue specific analysis are largely lacking for nematodes. An approach is described that focused on isolation of membrane and secreted protein genes from the gut of the parasitic nematode Haemonchus contortus. The approach capitalized on a monoclonal antibody that recognizes multiple membrane and secreted worm proteins. Polyclonal antisera made against these proteins were used to screen expression cDNA libraries made either from adult worm gut or whole worm. The genes identified encode predicted or known membrane and secreted proteins from gut, including a cysteine protease, a zinc metallopeptidase and a previously described GA1 protein. Another gene, Hc40, was isolated from the whole worm cDNA library and is nearly identical to a vaccine patent sequence pBTA879. Tissue analysis demonstrated the intended focus on membrane and secreted proteins from parasite gut was achieved. Proteins related to each of those described were identified from other nematode species through data base analysis. Additionally, this analysis led to (1) identification of homologues of each gene in C. elegans; (2) deduction of a dimorphic structure in the Hc40 protein; (3) recognition of both monomorphic and dimorphic families of Hc40-related proteins; and (4) detection of two apparent classes of transcripts (mep1a and mep1b) that would each encode a divergent version of the putative zinc metallopeptidase MEP1. The tissue specific approach and information base described should generally contribute to investigations on nutrient digestion and related secretory processes in nematode gut.

Multiple lethal effects induced by a benzimidazole anthelmintic in the anterior intestine of the nematode Haemonchus contortus

A mechanism of benzimidazole efficacy against parasitic nematodes is postulated to involve inhibition of intestinal secretory vesicle transport via depolymerization of microtubules. We show that fenbendazole (FBZ) treatment of lambs causes pathology localized to the anterior intestine in the parasitic nematode Haemonchus contortus. The pathology included gross disintegration of the anterior intestine, DNA fragmentation in anterior intestinal nuclei with characteristics of an apoptosis-like process, and inhibition of host erythrocyte digestion. These lethal effects were associated with inhibited transport of apical secretory vesicles in the anterior intestine, and then generalized dispersal of these vesicles contents throughout the intestinal cytoplasm and worm body. Cytoplasmic accumulation of secretory vesicles and undigested erythrocytes preceded DNA fragmentation and vesicle content dispersal. Both DNA fragmentation and vesicle content dispersal were detected in disintegrated intestine and intestine that had not yet undergone disintegration. These pathologic effects in the anterior intestine appear sufficient to explain the efficacy of FBZ against adult H. contortus. The results implicate mechanisms in the anterior intestine that govern dispersal of apical secretory vesicle contents, DNA fragmentation and tissue disintegration as effectors of this pathology.

Detection of mouse-adapted human influenza virus in the olfactory bulbs of mice within hours after intranasal infection

Influenza pneumonitis causes severe systemic symptoms in mice, including hypothermia and excess sleep. The association of extrapulmonary virus, particularly virus in the brain, with the onset of such disease symptoms has not been investigated. Mature C57BL/6 male mice were infected intranasally with mouse-adapted human influenza viruses (PR8 or X-31) under inhalation, systemic, or no anesthesia. Core body temperatures were monitored continuously by radiotelemetry, and tissues (lung, brain, olfactory bulb, spleen, blood) were harvested at the time of onset of hypothermia (13 to 24 h post infection [PI]) or at 4 or 7 h PI. Whole RNA from all tissues was examined by one or more of three reverse transcriptase-polymerase chain reaction (RT-PCR) procedures using H1N1 nucleoprotein (NP) primers for minus polarity RNA (genomic or vRNA) or plus polarity RNA (replication intermediates). Selected cytokines were assayed at 4, 7, and 15 h in the olfactory bulb (OB). Minus and plus RNA strands were readily detected in OBs as early as 4 h PI by nested RT-PCR. Anesthesia was not required for viral invasion of the OB. Cytokine mRNAs were also significantly elevated in the OB at 7 and 15 h PI in infected mice. Controls receiving boiled virus expressed only input vRNA and that only in lung. Immunohistochemistry demonstrated localization of H1N1 and NP antigens in olfactory nerves and the glomerular layer of the OB. Therefore a mouse-adapted human influenza virus strain, not known to be neurotropic, was detected in the mouse OB within 4 h PI where it appeared to induce replication intermediates and cytokines.

Sleep deprivation increases the activation of nuclear factor kappa B in lateral hypothalamic cells

Sleep deprivation increases sleep propensity in rats and mice as well as the production of several sleep-regulatory substances. Nuclear factor kappa B (NF-kappa B) is a transcription factor implicated in the activation of many of these sleep-promoting substances. A unique population of neurons immunoreactive for the p65 subunit of NF-kappa B was previously localized within the caudal dorsolateral hypothalamus of rats. Therefore, we evaluated the effect of sleep deprivation on NF-kappa Bp65-immunoreactivity (IR) in cells of this region in rats as well as its nuclear translocation in a kappa B-lacZ transgenic mouse line. In rats after 6 h of sleep deprivation beginning at light onset, the number of neurons with NF-kappa Bp65-IR increased significantly in the caudal lateral hypothalamus, specifically the magnocellular lateral hypothalamus adjacent to the subthalamus. Sleep deprivation also significantly increased the number of cells expressing NF-kappa B-dependent beta-galactosidase in the magnocellular lateral hypothalamus, zona incerta dorsal, as well as the adjacent subthalamus in the transgenic mice. These results suggest that NF-kappa B expressing cells within the lateral hypothalamus may be important in the maintenance of the sleep-wake cycle.

Lysine 194 is functional in isocitrate lyase from Escherichia coli.

Abstract. Lysine 194 in conserved stretch 1 of tetrameric isocitrate lyase from Escherichia coli has been replaced by using the restriction-enzyme-site elimination method of directed mutagenesis. Expression of subunits of each variant and of wild-type (wt) enzyme was equivalent and all variants assembled into tetrameric proteins. The variants K194R and K194H had kcat values relative to that of wt enzyme taken as 100 of 11 and 7, respectively. Km values for Mg2+-Ds-isocitrate (in mM units) were: 0.13 for wt-enzyme; 0.12 for the K194R variant; and 0.55 for the K194H variant. Substitution at position 194 of Leu or Glu resulted in zero catalytic activity. These results establish that Lys 194 is another functional residue in conserved stretch one of isocitrate lyase from E. coli besides H184, K193, C195, and H197. Because K194 can be specifically replaced by the basic residues His and Arg with resultant lowered activity and by His with an increased Km value, it may contribute to a cation center and facilitate substrate binding as well as orientation of the developing transition state.

Cysteine 195 has a critical functional role in catalysis by isocitrate lyase from Escherichia coli

Abstract. Cysteine 195 in isocitrate lyase from Escherichia coli has been replaced by directed mutagenesis. Substitution by Ser yields enzyme with a kcat that is 0.03% that of wild type, and substitution by Ala, Gly, Thr, or Val yields completely inactive enzyme. The present results are consistent with a functional role of Cys 195.

Serine319 and 321 Are Functional in Isocitrate Lyase from Escherichia coli

Abstract. With site-directed mutagenesis, Ser319 and Ser321 in conserved stretch 3 of tetrameric isocitrate lyase from Escherichia coli were each substituted with alanine, cysteine, asparagine, or threonine in addition to simultaneous alanine/alanine substitutions. Besides their absolute conservation in all aligned isocitrate lyase sequences, the location of these serine residues, which flank a completely conserved proline, had been suggested in the active site in previous research by studies of photoinactivation of the enzyme by vanadate [Ko et al. (1992) J Biol Chem 267:91]. All substitutions for Ser321 and 319 except by threonine appreciably reduced the kcat of E. coli isocitrate lyase relative to that for wild-type (100) as follows: S319A, 0.4; S319C, 0.05; S319N, 0.01; S319T, 32.3; S321A, 2.9; S321C, 0.3; S321N, 0.1; S321T, 0.3; and S319A/S321A, 0, with little or no effect on the Km for the substrate Mg2+-Ds-isocitrate. The most active variant S319T exhibited threefold less activity than the wild-type enzyme; all variants assembled into tetramers. The S319T mutant isocitrate lyase was 100-fold more active than the S321T variant. This observation suggests that the requirement for a β-hydroxymethyl group of serine in catalysis is less important at position 319 than at position 321. Although most singly substituted variants had very low isocitrate lyase activity, all variants harboring mutant isocitrate lyase of very low activity did grow on acetate as a sole carbon source albeit with longer doubling times and lag phases. Substitution of Pro320 by Ala, Asp, Gly, or His was highly detrimental to activity and increased the Km for substrate 3.5- to 8-fold; this suggests that Pro fixes the location of adjacent Ser OH groups and facilitates substrate binding and catalysis. From these collective results, it is proposed that Ser319 and Ser321 are involved in E. coli isocitrate lyase catalysis, perhaps by stabilizing the postulated reaction intermediate succinate trianion in the aci-carboxylate form and the related transition state via hydrogen bonding.

Integration of Genomics and Proteomics in Dissecting p53 Signaling

The discovery of the human genome and subsequent expansion of proteomics research combined with emerging technologies such as sophisticated computational biology are producing unprecedented changes in our understanding of the role of tumor suppressors in cell signaling. p53, a key tumor suppressor gene, is mutated in the majority of human cancers. Successful outcome of chemotherapy and radiotherapy, in many cases, depends on functional p53. Therefore, elucidation of the function, regulation, and molecular interactions of p53 with its targets is of great importance for developing successful cancer therapy. Because p53 is a transcription factor with an expanded repertoire of genes that are known to be directly or indirectly under its control, global analysis of gene expression profiles represents the best approach for studying the p53 response to chemotherapeutic agents. Our laboratory and others have used gene expression profiling with microarrays to isolate p53 target genes in an attempt to understand the molecular signaling of p53 and to determine the molecular consequences that follow from treatment of cancer cells with chemotherapeutic agents. Although these attempts have been successful in isolating novel genes, gene expression profiling alone at the transcription level is not sufficient. Most of pharmacological targets are proteins or DNA, not RNA. Therefore, we need an approach that focuses on the molecular profiling of cancer cells at the DNA, RNA, and proteins. In this chapter, key findings on the current genomics and proteomics approaches for isolating p53 targets and their functional analysis are reviewed, and perspective is provided on the potential of integrating both approaches in the molecular pharmacology of p53 signaling.