Associations between Cognition,Gender and Monocyte Activation among HIV Infected Individuals in Nigeria

The potential role of gender in the occurrence of HIV-related neurocognitive impairment (NCI) and associations with markers of HIV-related immune activity has not been previously examined. In this study 149 antiretroviral-naïve seropositive subjects in Nigeria (SP, 92 women and 57 men) and 58 seronegative (SN, 38 women and 20 men) were administered neuropsychological testing that assessed 7 ability domains. From the neuropsychological test scores was calculated a global deficit score (GDS), a measure of overall NCI. Percentages of circulating monocytes and plasma HIV RNA, soluble CD163 and soluble CD14 levels were also assessed. HIV SP women were found to be younger, more educated and had higher CD4+ T cell counts and borderline higher viral load measures than SP men. On the neuropsychological testing, SP women were more impaired in speed of information processing and verbal fluency and had a higher mean GDS than SN women. Compared to SP men, SP women were also more impaired in speed of information processing and verbal fluency as well as on tests of learning and memory. Numbers of circulating monocytes and plasma sCD14 and sCD163 levels were significantly higher for all SP versus all SN individuals and were also higher for SP women and for SP men versus their SN counterparts. Among SP women, soluble CD14 levels were slightly higher than for SP men, and SP women had higher viral load measurements and were more likely to have detectable virus than SP men. Higher sCD14 levels among SP women correlated with more severe global impairment, and higher viral load measurements correlated with higher monocyte numbers and sCD14 and sCD14 levels, associations that were not observed for SP men. These studies suggest that the risk of developing NCI differ for HIV infected women and men in Nigeria and, for women, may be linked to effects from higher plasma levels of HIV driving activation of circulating monocytes.     

Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase SERCA2a pump activity on cytosolic calcium in living cells

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.     

Cell adhesion molecule 1 (CADM1) is ubiquitously present in the endothelium and smooth muscle cells of the human macro- and micro-vasculature

Cell adhesion molecule 1 (CADM1) is a member of the immunoglobulin cell adhesion molecule family. Recently, we identified CADM1 to be a novel risk factor for venous thrombosis in a large, protein C deficient, thrombophilic family and showed, for the first time, the expression of CADM1 in endothelial cells (Hasstedt et al. in Blood 114:3084–3091, 2009). To further investigate its role in venous thrombosis, as well as other vasculopathies, we undertook a systematic confocal microscopic investigation for the presence of CADM1 in the vasculature of 28 different human tissues. Paraffin embedded tissue sections were dual immunostained with an antibody against CADM1, together with an antibody against either von Willebrand factor (to identify endothelial cells), or α-smooth muscle actin (to identify smooth muscle cells). The results showed that CADM1 was ubiquitously present in endothelial cells and smooth muscle cells in the vasculature from all 28 tissues, though its representation in the various classes of vessels was tissue dependent.     

A possible photosensitizer: Tobacco-specific nitrosamine, 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), induced mutations, DNA strand breaks and oxidative and methylative damage with UVA

We discovered the directly acting mutagenicity of the tobacco-specific nitrosamine, 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), with UVA light (320-400nm) in Ames bacteria and phage M13mp2 in the absence of metabolic activation. We have investigated the spectrum of mutations caused by UVA-activated NNK. The majority (57%) of induced sequence changes were comprised of GC to CG, GC to TA and GC to AT. This suggested that modification of guanine residues was responsible for these mutations. Hence, we explored the formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and O(6)-methylguanine (O(6)meG) in the DNA. When calf thymus DNA was treated with NNK and UVA, the amount of 8-oxodG/dG and O(6)meG/G in the DNA increased up to 20-fold and 100-fold, respectively, compared with the untreated control. DNA strand breaks were observed following NNK and UVA treatment, and the strand breaks were suppressed in the presence of scavengers for oxygen and NO radical. The formation of NO was also observed in NNK solutions irradiated with UVA. We analyzed the photodynamic spectrum of mutation induction, 8-oxodG formation and NO formation using monochromatic radiation. The patterns of the action spectra were comparable to the absorption spectrum of NNK. We conclude that NNK may act as a photosensitizer in response to UVA to produce NO and other oxidative and alkylative intermediates following the formation of 8-oxodG and O(6)meG in DNA, which may lead to mutations and DNA strand breaks.     

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G₀/G₁ cell cycle arrest and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G₀/G₁ cell cycle phase arrest and increased levels of p27^kip1 in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G₀ state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.     

The effect of metoclopramide on inhibition induced by purine nucleotides, noradrenaline, and theophylline ethylenediamine on intestinal muscle and on peristalsis in vitro.

Metoclopramide (N-(diethylaminoethyl)-2-methoxy-4-amino-5-chlorobenzamide) (Mcp) at concentrations of 0.1 and 1.0 muM partially and significantly reduced the relaxations induced by adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP), and adenosine, was without effect on theophylline ethylenediamine whilst significantly potentiating noradrenaline on the atropine-pretreated (0.1 muM) taenia coli, rabbit ileum, and rat duodenum. Mcp (1.0 muM) decreased the inhibitory effects of ATP, ADP, and adenosine on peristalsis induced in the isolated guinea-pig ileum by a constant increase in intraluminal pressure, did not affect inhibition due to theophylline ethylenediamine, whilst it potentiated inhibition of peristalsis due to noradrenaline. It is proposed that this effect of Mcp may be a specific antagonistic action on receptors sensitive to the putative purinergic transmitter, ATP and ADP, and may be partly responsible for its observed facilitatatory action on peristalsis.