Camilo Moncada

Oocyte spindle proteomics analysis leading to rescue of chromosome congression defects in cloned embryos

Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown, we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos.

Splenectomised and spleen intact Aotus monkeys' immune response to Plasmodium vivax MSP-1 protein fragments and their high activity binding peptides.

Two E. coli expressed recombinant polypeptides (rPvMSP-1(14) and rPvMSP-1(20)) contained in the 33kDa fragment, located within Plasmodium vivax merozoite surface protein (PvMSP-1) 42kDa C-terminal region, and a cocktail of high reticulocyte binding synthetic peptides located within these fragments, were evaluated for immunogenicity and protective immune responses in splenectomised and spleen intact Aotus nancymaae monkeys. Thirty splenectomised monkeys who had been previously immunised with either rPvMSP-1(14), rPvMSP-1(20), or a mixture of both recombinant fragments were intravenously challenged with the heterologous P. vivax VCG-1 strain (as determined by DNA sequencing); full protection was observed in five monkeys and low parasitaemia levels were obtained in eight more monkeys. Splenectomised control monkey group rapidly developed high parasitaemia levels, while no significant parasitaemia was obtained in the non-splenectomised control group. Although PvMSP-1 42 and 33kDa fragments were recognised by Western Blot and whole parasites by IFAT when tested with immune monkey sera, no correlation between protection and antibody titres by IFAT and ELISA was observed, suggesting that protection is not being solely mediated by a humoral immune response. This data showed that partial protection against a heterologous strain challenge was best achieved when immunising with a rPvMSP-1(14)-rPvMSP-1(20) mixture (2 were fully protected and 4 with low parasitaemia out of 12) suggesting for the first time, that these fragments could be good candidates for inclusion in a P. vivax multi-stage, multi-antigen vaccine.

Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease

RATIONALE Shifts in the gene expression of nuclear protein in chronic obstructive pulmonary disease (COPD), a progressive disease that is characterized by extensive lung inflammation and apoptosis, are common; however, the extent of the elevation of the core histones, which are the major components of nuclear proteins and their consequences in COPD, has not been characterized, which is important because extracellular histones are cytotoxic to endothelial and airway epithelial cells. OBJECTIVES To investigate the role of extracellular histones in COPD disease progression. METHODS We analyzed the nuclear lung proteomes of ex-smokers with and without the disease. Further studies on the consequences of H3.3 were also performed. MEASUREMENTS AND MAIN RESULTS A striking finding was a COPD-specific eightfold increase of hyperacetylated histone H3.3. The hyperacetylation renders H3.3 resistant to proteasomal degradation despite ubiquitination; when combined with the reduction in proteasome activity that is known for COPD, this resistance helps account for the increased levels of H3.3. Using anti-H3 antibodies, we found H3.3 in the airway lumen, alveolar fluid, and plasma of COPD samples. H3.3 was cytotoxic to lung structural cells via a mechanism that involves the perturbation of Ca(2+) homeostasis and mitochondrial toxicity. We used the primary human airway epithelial cells and found that the antibodies to either the C or N terminus of H3 could partially reverse H3.3 toxicity. CONCLUSIONS Our data indicate that there is an uncontrolled positive feedback loop in which the damaged cells release acetylated H3.3, which causes more damage, adds H3.3 release, and contributes toward the disease progression.

Functional analysis of the -2548G/A leptin gene polymorphism in breast cancer cells

Leptin is overexpressed in human breast tumors and is produced by breast cancer cells in response to obesity‐related stimuli. The leptin promoter polymorphism Lep‐2548G/A can be associated with increased leptin secretion by adipocytes and elevated cancer risk. However, molecular mechanisms underlying the link between Lep‐2548G/A and breast cancer have never been addressed. Lep‐2548G/A is proximal to a binding site for the transcriptional factor Sp1. Furthermore nucleolin, a transcriptional repressor, can bind Sp1 or its consensus site. Consequently, we focused on the impact of Lep‐2548G/A on Sp1‐ and nucleolin‐dependent leptin transcription in breast cancer cells. The Lep‐2548G/A was identified in a homozygous conformation in BT‐474 and SK‐BR‐3 breast cancer cells, in a heterozygous conformation in MDA‐MB‐231 cells, and a wild‐type Lep‐2548G/G sequence was present in MCF‐7 and ZR‐75‐1 cells. The occurrence of Lep‐2548A/A and Lep‐2548G/A coincided with high and intermediate leptin mRNA expression, respectively, while cells containing Lep‐2548G/G expressed low leptin mRNA levels. We demonstrated that the existence of Lep‐2548G/A improved efficient recruitment of Sp1 to DNA under insulin treatment, while Sp1 loading on DNA containing Lep‐2548G/G was not insulin‐dependent. In contrast, nucleolin binding to Lep‐2548G/A was downregulated in response to insulin, while it was not regulated on Lep‐2548G/G. The presence of Lep‐2548G/A was studied in breast cancer epithelial cells by IHC and LCM. Interestingly, all 14 tumors expressing high leptin levels contained Lep‐2548A/A. In conclusion, the occurrence of Lep‐2548G/A can enhance leptin expression in breast cancer cells via Sp1‐ and nucleolin‐dependent mechanisms and possibly contribute to intratumoral leptin overexpression.

The T-cell receptor in primates: identifying and sequencing new owl monkey TRBV gene sub-groups

The New World primate Aotus nancymaae (owl monkey) has been shown to be an excellent experimental model when studying malarial parasites. Characterising the T-cell receptor (TR) αβ repertoire by means of the different variable beta (TRBV) genes displayed contributes to a better understanding of these lymphocytes’ role in the response against several malarial antigens. This study describes identifying and characterising eleven new TRBV gene sub-groups in cDNA from Aotus nancymaae’s peripheral blood lymphocytes; these 11 gene sequences displayed homology to the previously reported human TRBV3, TRBV10, TRBV11, TRBV14, TRBV18, TRBV19, TRBV20, TRBV25, TRBV27, TRBV29 and TRBV30 sub-groups, resulting in 83% overall homology at the amino acid level. An additional Aotus sequence was found having similarity with the human TRBJ-2–7*01 gene. Evolutionary relationships amongst these sequences and the homologous genes from both New and Old World primates have shown that the TRBV repertoire has been maintained in the species being studied, displaying varying association patterns and substitution rates, depending on the sub-group being studied. The degree of identity observed when comparing human and Aotus genes suggests that these species might have a similar TRBV repertoire.

Mechanism and Tissue Specificity of Nicotine-mediated Lung S-Adenosylmethionine Reduction

We previously reported that chronic nicotine infusion blocks development of Pneumocystis pneumonia. This discovery developed from our work demonstrating the inability of this fungal pathogen to synthesize the critical metabolic intermediate S-adenosylmethionine and work by others showing nicotine to cause lung-specific reduction of S-adenosylmethionine in guinea pigs. We had found nicotine infusion to cause increased lung ornithine decarboxylase activity (rate-controlling enzyme of polyamine synthesis) and hypothesized that S-adenosylmethionine reduction is driven by up-regulated polyamine biosynthesis. Here we report a critical test of our hypothesis; inhibition of ornithine decarboxylase blocks the effect of nicotine on lung S-adenosylmethionine. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of S-adenosylmethionine toward polyamine synthesis and away from methylation reactions; these shifts are reversed by inhibition of ornithine decarboxylase. Because the nicotine effect on Pneumocystis is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection, we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ornithine decarboxylase protein in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on Pneumocystis pneumonia. Earlier we reported that the full effect of nicotine requires 3 weeks of treatment, and here we show recovery is symmetrical, also requiring 3 weeks after treatment cessation. Because this time frame is similar to pneumocyte turnover time, the shift in polyamine metabolism may occur as new pneumocytes are produced.

Pneumocystis S-adenosylmethionine transport: a potential drug target.

Pneumocystis pneumonia (PCP) is a life-threatening condition in immunosuppressed patients. Current treatments are inadequate, and new drug leads are needed. This fungus depends on its host for S-adenosylmethionine (AdoMet), a critical metabolic intermediate ordinarily synthesized by individual cells as needed. Pneumocystis contains a gene coding for the AdoMet-synthesizing enzyme methionine ATP transferase (MAT), and the protein is expressed. However, the fungus lacks MAT activity, and infection causes the depletion of host plasma AdoMet. The uptake of Pneumocystis AdoMet was shown to be exquisitely specific, which suggests the transport of AdoMet as a potential drug target. Here we report on the discovery of PcPET8, a Pneumocystis gene with homology to mitochondrial AdoMet transporters. When expressed by Saccharomyces cerevisiae, it locates properly to the mitochondrion and complements a strain of S. cerevisiae lacking its native mitochondrial AdoMet transporter. The importance of AdoMet transport is demonstrated by the ability of the AdoMet analogue sinefungin to block the uptake of Pneumocystis AdoMet and inhibit growth in culture. Because PcPET8 is likely critical for Pneumocystis, the yeast construct has potential as a surrogate for testing compounds against Pneumocystis.