Objectives: Tissue from Marfan syndrome (MFS) or bicuspid aortic valve (BAV) aneurysm is characterized by vascular smooth muscle cell (VSMC) loss, cystic medial necrosis and elastic tissue destruction. We examined morphological changes in aneurysm tissue using light microscopy (LM) and transmission electron microscopy (TEM). Conclusions: The findings suggest that morphological abnormalities in VSMC precede recognizable alterations of EL, consistent with the hypothesis that the primary defect in the pathogenesis of MFS and BAV aneurysm arises within VSMCs.

Blood-feeding parasites use mechanistically distinct proteases to digest hemoglobin (Hb), often as multienzyme cooperative cascades. We investigated the roles played by 3 distinct proteases from adults of the human hookworm Necator americanus. The aspartic protease Na-APR-1 and the cysteine protease Na-CP-3 were expressed in catalytically active form in yeast, and the metalloprotease Na-MEP-1 was expressed in catalytically active form in baculovirus. Antibodies to all 3 proteases were used to immunolocalize each native enzyme to the intestine of adult N. americanus. Recombinant Na-APR-1 cleaved intact human Hb. In contrast, Na-CP-3 and Na-MEP-1 could not cleave Hb but instead cleaved globin fragments that had been hydrolyzed by Na-APR-1, implying an ordered process of hemoglobinolysis. Seventy-four cleavage sites within Hb alpha- and beta-chains were characterized after digestion with all 3 proteases. All of the proteases demonstrated promiscuous subsite specificities within Hb; noteworthy preferences included aromatic and hydrophobic P1 residues and hydrophobic P1' residues for NaAPR-1 and hydrophobic P1 residues for Na-MEP-1. We conclude that Hb digestion in N. americanus involves a network of distinct proteases, some of which act in an ordered fashion, providing a potential mechanism by which some of these hemoglobinases exert their efficacy as recombinant vaccines against hookworm infection.

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.

Whether purified HIV-1 virion cores are capable of reverse transcription or require uncoating to be activated is currently controversial. To address this question we purified cores from a virus culture and tested for the ability to generate authentic reverse transcription products. A dense fraction (approximately 1.28 g/ml) prepared without detergent, possibly derived from disrupted virions, was found to naturally occur as a minor sub-fraction in our preparations. Core-like particles were identified in this active fraction by electron microscopy. We are the first to report the detection of authentic strong-stop, first-strand transfer and full-length minus strand products in this core fraction without requirement for an uncoating activity.

Thoracic aortic aneurysm (TAA) associated with Marfan syndrome (MFS) and bicuspid aortic valve (BAV) is characterized histologically by vascular smooth muscle cell (VSMC) loss, areas of cystic medial necrosis and patchy elastic tissue destruction in the tunica media. In this study we identified the relationship between morphological changes in VSMC and disorganization of aortic wall structure using light microscopy (LM) and transmission electron microscopy (TEM). No TEM study of MFS or BAV VSMC has been previously reported. Aortic tissue was collected from normal aorta (1M; 4F; age 39 ± 14 yr), MFS (5M; 3F; age 35 ± 11 yr) and BAV (6M; 5F; age 57 ± 16 yr). Aortic tissue and cultured VSMC derived from tissue were processed and embedded in resin. Semi thin sections were cut and stained with toluidine blue for LM and viewed at a magnification of 1000x and ultra thin sections were cut for TEM analysis at a magnification of 25000x. Organelles including the mitochondria, endoplasmic reticulum and Golgi apparatus appeared normal in each group. Apart from the presence of cystic medial necrosis reported previously in MFS and BAV aneurysm, LM study revealed generalized severe structural abnormalities of VSMC that were independent of areas of cystic medial necrosis. VSMC contained numerous vacuoles and structural masses occupying most of the cytoplasmic region (normal=10.1±0.1 %; MFS=34.0±0.1%; BAV=29.1±0.1%). Many VSMC were partly disintegrated lying between intact elastic lamellae (normal=1.4±0.1%; MFS=5.3±0.1%; BAV=10.0±0.1%). TEM study showed a loss of interdigitation between VSMC and elastic lamellae. Calcification of VSMC and extracellular matrix components were common findings in BAV. Cultured VSMC showed bulging intracellular masses, tearing of the cytoplasmic membrane, cellular indentations and disintegration. The findings suggest that morphological VSMC changes were present prior to recognizable alterations of elastic lamellae, and that the primary defect in the pathogenesis of TAA arises within the VSMC.

Giardia duodenalis is a protozoal, intestinal parasite that is a common aetiological agent of infectious diarrhoea in humans worldwide. Chemotherapeutic intervention presently offers a limited range of drugs and these are usually only employed after clinical diagnosis. Moreover, these drugs are ineffective against the infectious cysts, can produce unpleasant side effects, and are expensive with limited availability in developing countries. Frequent reports of drug toxicity, treatment failure and parasite drug resistance have, in some instances, also resulted in the increasing reluctance to over-prescribe synthetic anti-microbials. Alternatively, there is now mounting evidence to suggest that some of the naturally derived, medium-chain, saturated fatty acids (MCSFAs) possess anti-microbial and anti-parasitic properties. We have therefore examined the effects of four different fatty acids on G. duodenalis trophozoites in vitro. Cytotoxicity was determined using fluorescence, scanning and transmission electron microscopic techniques and standard cytotoxicity assays. Our studies have confirmed that the MCSFA, dodecanoic acid (C: 12) (common name: lauric acid), is anti-giardial, with an LD50 concentration comparable to that of metronidazole, the drug of choice in the treatment of giardiasis. Dodecanoic acid appeared to induce trophozoite death by accumulating within the parasite cytoplasm resulting in rupture of the cell membrane. This study has opened fresh avenues for development of natural drug therapy in which food supplementation may augment, or even replace, some of the standard chemotherapeutic agents presently employed in the treatment of giardiasis and possibly other infectious intestinal diseases.

The recently cloned gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wider role in signal transduction. Antibodies prepared against peptides from the predicted protein sequence detected a ∼ 350 kDa protein corresponding to the open reading frame, which was absent in 13/23 A-T homozygotes. Subcellular fractionation, immunoelectronmicroscopy and immunofluorescence showed that the ATM protein is present in the nucleus and cytoplasmic vesicles. This distribution did not change after irradiation. We also provide evidence that ATM protein binds to p53 and this association is defective in A-T cells compatible with the defective p53 response in these cells. These results provide further support for a role for the ATM protein as a sensor of DNA damage and in a more general role in cell signalling, compatible with the broader phenotype of the syndrome.