The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells.

Vascular endothelial cell adhesion molecule 1 (VCAM-1) is an adherence molecule that is induced on endothelial cells by cytokine stimulation and can mediate binding of lymphocytes or tumor cells to endothelium. Because these interactions often occur at the level of the microvasculature, we have examined the regulation of expression of VCAM-1 in human dermal microvascular endothelial cells (HDMEC) and compared it to the regulation of VCAM-1 in large vessel human umbilical vein endothelial cells (HUVEC). Both cell populations were judged pure as assessed by expression of von Willebrand factor and uptake of acetylated low density lipoprotein. Expression of VCAM-1 was not detectable on either unstimulated HDMEC or HUVEC when assessed by ELISA or flow cytometry. Stimulation of either HDMEC or HUVEC with TNF-alpha resulted in a time- and dose-dependent induction of VCAM-1. However, although TNF-alpha-induced cell surface and mRNA expression of VCAM-1 in HDMEC was transient, peaking after 16 h of stimulation, TNF stimulation led to persistently elevated cell surface expression of VCAM-1 on HUVEC. IL-1 alpha also induced cell surface expression of VCAM-1 on HUVEC in a time- and dose-dependent manner, but stimulation of HDMEC with IL-1 alpha at doses up to 1000 U/ml failed to induce significant cell surface expression. However, IL-1 alpha induced time- and dose-dependent increases in ICAM-1 on HDMEC. Similarly, IL-4 induced VCAM-1 expression and augmented TNF-alpha-induced expression on HUVEC but did not affect VCAM-1 expression on HDMEC. Binding of Ramos cells to cytokine-stimulated endothelial cell monolayers correlated with VCAM-1 induction. Increased binding was seen after stimulation of HDMEC with TNF-alpha, which was blocked by anti-VCAM-1 mAb, but no increases in binding were noted after stimulation of HDMEC monolayers with IL-1 alpha. These data provide additional evidence for the existence of endothelial cell heterogeneity and differences in cell adhesion molecule regulation on endothelial cells derived from different vascular beds.     

Transforming growth factor beta 1 dysregulation in a human oral carcinoma tumour progression model

A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.     

Signaling Dependent and Independent Mechanisms in Pemphigus Vulgaris Blister Formation

Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). Significant advances in our understanding of pemphigus pathomechanisms have been derived from the generation of pathogenic monoclonal Dsg3 antibodies. However, conflicting models for pemphigus pathogenicity have arisen from studies using either polyclonal PV patient IgG or monoclonal Dsg3 antibodies. In the present study, the pathogenic mechanisms of polyclonal PV IgG and monoclonal Dsg3 antibodies were directly compared. Polyclonal PV IgG cause extensive clustering and endocytosis of keratinocyte cell surface Dsg3, whereas pathogenic mouse monoclonal antibodies compromise cell-cell adhesion strength without causing these alterations in Dsg3 trafficking. Furthermore, tyrosine kinase or p38 MAPK inhibition prevents loss of keratinocyte adhesion in response to polyclonal PV IgG. In contrast, disruption of adhesion by pathogenic monoclonal antibodies is not prevented by these inhibitors either in vitro or in human skin explants. Our results reveal that the pathogenic activity of polyclonal PV IgG can be attributed to p38 MAPK-dependent clustering and endocytosis of Dsg3, whereas pathogenic monoclonal Dsg3 antibodies can function independently of this pathway. These findings have important implications for understanding pemphigus pathophysiology, and for the design of pemphigus model systems and therapeutic interventions.     

Structure and genetic complexity of the genomes of herpesvirus defective-interfering particles associated with oncogenic transformation and persistent infection.

The complexity and structural organization of defective-interfering (DI) particle DNA of equine herpesvirus type 1 (EHV-1) have been elucidated by using restriction enzyme and Southern blot hybridization analyses. DI particles were generated by serial high-multiplicity passage of EHV-1 in L-M cells, and total viral DNA was extracted from virus purified from supernatants of these serial passages. EHV-1 DI particle DNA was quantitatively separated from standard (STD) DNA by several cycles of CsCl isopycnic banding in a vertical rotor. Restriction endonuclease digestion profiles of pure DI DNA were completely different from the mapped patterns observed for EHV-1 STD DNA. Digestion of pure defective DNA with restriction enzymes (Bg/II, EcoRI, and XbaI), for which there are few or no cleavage sites within the S (short) region of the EHV-1 STD genome, yielded high-molecular-weight supermolar DNA bands, suggesting that a large subgenomic repeat unit was present in defective DNA. DNA blot hybridization analysis with the Bg/II supermolar fragment of defective DNA, intact DI particle genomic DNA, and EHV-1 STD DNA restriction enzyme fragments as 32P-labeled probes indicated that the EHV-1 DI particle genome originates predominately from the STD DNA S region (0.77 to 1.00 map units) and to a lesser extent from the left terminus of the unique long (UL) region (0.00 to 0.05 map units). None of the EHV-1 DNA sequences associated to date with EHV-1 oncogenesis (0.32 to 0.38 map units; O'Callaghan et al. in B. Roizman [ed.], Herpesviruses, in press; Robinson et al., Cell 32:204-219, 1983, and Proc. Natl. Acad. Sci., U.S.A., 78:6684-6688, 1981) were detected in the DI particle DNA. The importance of these data with regard to DNA replication of DI particles and the role of DI particles in one model system of EHV-1 oncogenic transformation are discussed.     

Neuropeptide regulation of human dermal microvascular endothelial cell ICAM-1 expression and function

There isincreasing evidence that sensory nerves may participate in cutaneousinflammatory responses by the release of neuropeptides such assubstance P (SP). We examined the direct effect of SP on human dermalmicrovascular endothelial cell (HDMEC) intercellular adhesion molecule1 (ICAM-1) expression and function. Our results indicated that,although cultured HDMEC expressed mRNA for neurokinin receptors 1, 2, and 3 (NK-1R, NK-2R, and NK-3R), SP initiated a rapid increase in HDMECintracellular Ca²⁺ levels,primarily by the activation of NK-1R. Immunohistochemistry studieslikewise demonstrated that HDMEC predominantly expressed NK-1R. Theaddition of SP to HDMEC resulted in a rapid increase in cellular ICAM-1mRNA levels, followed by a fivefold increase in ICAM-1 cell surfaceexpression. This functionally resulted in a threefold increase in⁵¹Cr-labeled binding of J-Ylymphoblastoid cells to HDMEC. In vivo studies demonstrated a markedincrease in microvascular ICAM-1 immunostaining 24 and 48 h afterapplication of capsaicin to the skin. These results indicate thatneuropeptides such as SP are capable of directly activating HDMEC toexpress increased levels of functional ICAM-1 and further support therole of the cutaneous neurological system in modulating inflammatoryprocesses in the skin.

     

Combined monte carlo and molecular dynamics simulation of fully hydrated dioleyl and palmitoyl-oleyl phosphatidylcholine lipid bilayers

We have applied a new equilibration procedure for the atomic level simulation of a hydrated lipid bilayer to hydrated bilayers of dioleyl-phosphatidylcholine (DOPC) and palmitoyl-oleyl phosphatidylcholine (POPC). The procedure consists of alternating molecular dynamics trajectory calculations in a constant surface tension and temperature ensemble with configurational bias Monte Carlo moves to different regions of the configuration space of the bilayer in a constant volume and temperature ensemble. The procedure is applied to bilayers of 128 molecules of POPC with 4628 water molecules, and 128 molecules of DOPC with 4825 water molecules. Progress toward equilibration is almost three times as fast in central processing unit (CPU) time compared with a purely molecular dynamics (MD) simulation. Equilibration is complete, as judged by the lack of energy drift in 200-ps runs of continuous MD. After the equilibrium state was reached, as determined by agreement between the simulation volume per lipid molecule with experiment, continuous MD was run in an ensemble in which the lateral area was restrained to fluctuate about a mean value and a pressure of 1 atm applied normal to the bilayer surface. Three separate continuous MD runs, 200 ps in duration each, separated by 10,000 CBMC steps, were carried out for each system. Properties of the systems were calculated and averaged over the three separate runs. Results of the simulations are presented and compared with experimental data and with other recent simulations of POPC and DOPC. Analysis of the hydration environment in the headgroups supports a mechanism by which unsaturation contributes to reduced transition temperatures. In this view, the relatively horizontal orientation of the unsaturated bond increases the area per lipid, resulting in increased water penetration between the headgroups. As a result the headgroup-headgroup interactions are attenuated and shielded, and this contributes to the lowered transition temperature.     

Duplicate <Emphasis Type="Italic">dmbx1</Emphasis>genes regulate progenitor cell cycle and differentiation during zebrafish midbrain and retinal development

Background  

The Dmbx1 gene is important for the development of the midbrain and hindbrain, and mouse gene targeting experiments reveal that this gene is required for mediating postnatal and adult feeding behaviours. A single Dmbx1 gene exists in terrestrial vertebrate genomes, while teleost genomes have at least two paralogs. We compared the loss of function of the zebrafish dmbx1a and dmbx1b genes in order to gain insight into the molecular mechanism by which dmbx1 regulates neurogenesis, and to begin to understand why these duplicate genes have been retained in the zebrafish genome.