VP22-mediated intercellular transport correlates with enhanced biological activity of MybEngrailed but not (HSV-I) thymidine kinase fusion proteins in primary vascular cells following non-viral transfection

BACKGROUND: The intercellular transport properties of the herpes simplex virus (HSV) protein VP22 have been harnessed to enhance the effectiveness of viral gene transfer. We investigated the intercellular transport and biological effects of VP22 fused with the dominant negative c-Myb chimera, MybEngrailed (MybEn) and HSV-I thymidine kinase (TK), in primary vascular smooth muscle cells (VSMC) following non-viral transfection. MATERIALS AND METHODS: Porcine VSMC transfected with plasmids encoding MybEn, TK and their respective N- and C-terminal VP22 fusion proteins were assayed for the extent and distribution of transgene expression (by immunohistochemistry), culture growth and apoptosis. RESULTS: The N-terminal MybEn fusion with VP22 (MybEnVP22) and both TK fusions, but not VP22MybEn, exhibited intercellular spread from primary transfected to up to 200 surrounding cells. pMybEnVP22-transfected cultures exhibited growth inhibition and apoptosis rates that were 10.6 +/- 3.6 and 3.2 +/- 1.0 fold higher than in pMybEn-transfected cultures; pVP22MybEn-transfected cultures showed no difference in these parameters. pTK-transfected cultures underwent 60-70% cell death in the presence of ganciclovir despite <2% primary transfection, which was not increased in cultures transfected with plasmids encoding VP22-TK fusions. CONCLUSIONS: The close correlation between immunocytochemical and biological assays suggests that intercellular transport is crucial to the enhanced biological activity of the MybEnVP22 fusion. The "intrinsic" bystander activity of TK was 4-fold greater than was "engineered" by VP22 fusion, probably reflecting the abundance of gap junctions between VSMC. VP22 fusion may enhance the efficiency of non-viral gene delivery when combined with the appropriate therapeutic transgene, target tissue and transfection method.     

Differences between the trypanosomal and human GlcNAc-PI de-N-acetylases of glycosylphosphatidylinositol membrane anchor biosynthesis

De-N-acetylation of N-acetylglucosaminyl-phosphatidylino-sitol (GlcNAc-PI) is the second step of glycosylphosphatidylino-sitol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent processing of glucosaminyl-phosphatidylinositol (GlcN-PI) that leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. In this article, we used a direct de-N-acetylase assay, based on the release of [14C]acetate from synthetic GlcN[14C]Ac-PI and analogues thereof, and an indirect assay, based on the mannosylation of GlcNAc-PI analogues, to study the substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells. The HeLa enzyme was found to be more fastidious than the trypanosomal enzyme such that, unlike the trypanosomal enzyme, it was unable to act on a GlcNAc-PI analogue containing 2-O-octyl-d- myo -inositol or on the GlcNAc-PI diastereoisomer containing l- myo -inositol (GlcNAc-P(l)I). These results suggest thatselective inhibition of the trypanosomal de-N-acetylase may be possible and that this enzyme should be considered as a possible therapeutic target. The lack of strict stereospecificity of the trypanosomal de-N-acetylase for the d- myo -inositol component was also seen for the trypanosomal GPI alpha-manno-syltransferases when GlcNAc-P(l)I was added to the trypanosome cell-free system, but not when GlcN-P(l)I was used. In an attempt to rationalize these data, we modeled the structure and dynamics of d-GlcNAcalpha1-6d- myo -inositol-1-HPO4-( sn )-3-glycerol and its diastereoisomer d-GlcNAcalpha1-6l- myo -inositol-1-HPO4-( sn )-3-glycerol. These studies indicate that the latter compound visits two energy minima, one of which resembles the low-energy conformer of former compound. Thus, it is conceivable that the trypanosomal de-N-acetylase acts on GlcNAc-P(l)I when it occupies a GlcNAc-PI-likeconformation and that GlcN-P(l)I emerging from the de-N-acetylase may be channeled to the alpha-mannosyltransferases in this conformation.     

Nine months in space: effects on human autonomic cardiovascular regulation.

We studied three Russian cosmonauts to better understand how long-term exposure to microgravity affects autonomic cardiovascular control. We recorded the electrocardiogram, finger photoplethysmographic pressure, and respiratory flow before, during, and after two 9-mo missions to the Russian space station Mir. Measurements were made during four modes of breathing: 1) uncontrolled spontaneous breathing; 2) stepwise breathing at six different frequencies; 3) fixed-frequency breathing; and 4) random-frequency breathing. R wave-to-R wave (R-R) interval standard deviations decreased in all and respiratory frequency R-R interval spectral power decreased in two cosmonauts in space. Two weeks after the cosmonauts returned to Earth, R-R interval spectral power was decreased, and systolic pressure spectral power was increased in all. The transfer function between systolic pressures and R-R intervals was reduced in-flight, was reduced further the day after landing, and had not returned to preflight levels by 14 days after landing. Our results suggest that long-duration spaceflight reduces vagal-cardiac nerve traffic and decreases vagal baroreflex gain and that these changes may persist as long as 2 wk after return to Earth.     

Human tribbles-1 controls proliferation and chemotaxis of smooth muscle cells via MAPK signaling pathways

Migration and proliferation of smooth muscle cells are key to a number of physiological and pathological processes, including wound healing and the narrowing of the vessel wall. Previous work has shown links between inflammatory stimuli and vascular smooth muscle cell proliferation and migration through mitogen-activated protein kinase (MAPK) activation, although the molecular mechanisms of this process are poorly understood. Here we report that tribbles-1, a recently described modulator of MAPK activation, controls vascular smooth muscle cell proliferation and chemotaxis via the Jun kinase pathway. Our findings demonstrate that this regulation takes place via direct interactions between tribbles-1 and MKK4/SEK1, a Jun activator kinase. The activity of this kinase is dependent on tribbles-1 levels, whereas the activation and the expression of MKK4/SEK1 are not. In addition, tribbles-1 expression is elevated in human atherosclerotic arteries when compared with non-atherosclerotic controls, suggesting that this protein may play a role in disease in vivo. In summary, the data presented here suggest an important regulatory role for trb-1 in vascular smooth muscle cell biology.     

Injection of excitatory amino acid antagonists into the medial pallidal segment of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated primate reverses motor symptoms of parkinsonism

Intracerebral injections of the broad spectrum excitatory amino acid antagonist kynurenic acid (50 ug) alleviated the symptoms of akinesia, tremor and rigidity in a severely parkinsonian monkey. Unilateral injection of kynurenic acid within the medial pallidal segment produced rotational behaviour away from the side of the injection, and the limbs on the contralateral side showed relief of the MPTP-induced parkinsonian symptoms. The subsequent bilateral injection of the excitatory amino acid antagonist allowed the monkey to move freely, unhindered by tremor or rigidity. In addition unilateral injections of the NMDA antagonist MK-801 (5, 25 and 50 ug) within the medial pallidum also produced dose-related rotational behaviour, with alleviation of parkinsonian symptoms in the contralateral limbs. Systemic administration of MK-801 (1 ng/kg - 1 ug/kg i.m.) was without effect.     

A comparative study between two different artificial substrate samplers and regular sampling techniques

One of the major problems confronting aquatic biologists in locating and sampling ecologically comparable habitats both exposed and not exposed to the variable one wishes to study (e.g. pollutional stress). This not only entails finding areas with comparable physico-chemical characteristics, but should also include samplibility and proximity of sites to each other to provide valid comparative data for proper evaluation of the similarities and differences. One solution to this problem is the use of artificial substrate devices which insure habitat similarity and may be placed where one wishes.     

Dispersal and social organization of the northern hairy-nosed wombat Lasiorhinus krefftii

Burrows of the northern hairy-nosed wombat were arranged in loose clusters, each of which was used in common by a group of up to 10 wombats. However, individual wombats rarely used the same burrows on the same days and feeding ranges tended to be separate within each sex. The incidence of breeding dispersal among females was quite high (at least 50%); dispersal distances ranged up to almost the full extent of the population's range. Dispersal by adult males was rare, and no juvenile males were observed to disperse.