A novel SINE family occurs frequently in both genomic DNA and transcribed sequences in ixodid ticks of the arthropod sub-phylum Chelicerata

Reassociation kinetics and flow cytometry data indicate that ixodid tick genomes are large, relative to most arthropods, containing>or=10(9) base pairs. The molecular basis for this is unknown. We have identified a novel small interspersed element with features of a tRNA-derived SINE, designated Ruka, in genomic sequences of Rhipicephalus appendiculatus and Boophilus (Rhipicephalus) microplus ticks. The SINE was also identified in expressed sequence tag (EST) databases derived from several tissues in four species of ixodid ticks, namely R. appendiculatus, B. (R.) microplus, Amblyomma variegatum and also the more distantly related Ixodes scapularis. Secondary structure predictions indicated that Ruka could adopt a tRNA structure that was, atypically, most similar to a serine tRNA. By extrapolation the frequency of occurrence in the randomly selected BAC clone sequences is consistent with approximately 65,000 copies of Ruka in the R. appendiculatus genome. Real time PCR analyses on genomic DNA indicate copy numbers for specific Ruka subsets between 5800 and 38,000. Several putative conserved Ruka insertion sites were identified in EST sequences of three ixodid tick species based on the flanking sequences associated with the SINEs, indicating that some Ruka transpositions probably occurred prior to speciation within the metastriate division of the Ixodidae. The data strongly suggest that Class I transposable elements form a significant component of tick genomes and may partially account for the large genome sizes observed.     

Myosin Vb Mediated Plasma Membrane Homeostasis Regulates Peridermal Cell Size and Maintains Tissue Homeostasis in the Zebrafish Epidermis

The epidermis is a stratified epithelium, which forms a barrier to maintain the internal milieu in metazoans. Being the outermost tissue, growth of the epidermis has to be strictly coordinated with the growth of the embryo. The key parameters that determine tissue growth are cell number and cell size. So far, it has remained unclear how the size of epidermal cells is maintained and whether it contributes towards epidermal homeostasis. We have used genetic analysis in combination with cellular imaging to show that zebrafish goosepimples/myosin Vb regulates plasma membrane homeostasis and is involved in maintenance of cell size in the periderm, the outermost epidermal layer. The decrease in peridermal cell size in Myosin Vb deficient embryos is compensated by an increase in cell number whereas decrease in cell number results in the expansion of peridermal cells, which requires myosin Vb (myoVb) function. Inhibition of cell proliferation as well as cell size expansion results in increased lethality in larval stages suggesting that this two-way compensatory mechanism is essential for growing larvae. Our analyses unravel the importance of Myosin Vb dependent cell size regulation in epidermal homeostasis and demonstrate that the epidermis has the ability to maintain a dynamic balance between cell size and cell number.     

The hTH-GFP Reporter Rat Model for the Study of Parkinson's Disease

Parkinson disease (PD) is the second leading neurodegenerative disease in the US. As there is no known cause or cure for PD, researchers continue to investigate disease mechanisms and potential new therapies in cell culture and in animal models of PD. In PD, one of the most profoundly affected neuronal populations is the tyrosine hydroxylase (TH)-expressing dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). These DA-producing neurons undergo degeneration while neighboring DA-producing cells of the ventral tegmental area (VTA) are largely spared. To aid in these studies, The Michael J. Fox Foundation (MJFF) partnered with Thomas Jefferson University and Taconic Inc. to generate new transgenic rat lines carrying the human TH gene promoter driving EGFP using a 11 kb construct used previously to create a hTH-GFP mouse reporter line. Of the five rat founder lines that were generated, three exhibited high level specific GFP fluorescence in DA brain structures (ie. SN, VTA, striatum, olfactory bulb, hypothalamus). As with the hTH-GFP mouse, none of the rat lines exhibit reporter expression in adrenergic structures like the adrenal gland. Line 12141, with its high levels of GFP in adult DA brain structures and minimal ectopic GFP expression in non-DA structures, was characterized in detail. We show here that this line allows for anatomical visualization and microdissection of the rat midbrain into SNpc and/or VTA, enabling detailed analysis of midbrain DA neurons and axonal projections after toxin treatment in vivo. Moreover, we further show that embryonic SNpc and/or VTA neurons, enriched by microdissection or FACS, can be used in culture or transplant studies of PD. Thus, the hTH-GFP reporter rat should be a valuable tool for Parkinson''s disease research.     

Biochemical and immunological characterization of pea nuclear intermediate filament proteins

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6–12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.Abbreviations IF Intermediate filament - L Lamin fraction - LM Lamina-matrix fraction - MAb JLA20 Anti-chicken actin monoclonal antibody - MAb LN43 Anti-human lamin B2 monoclonal antibody - MAb PL19 Anti-pea lamin #19 monoclonal antibody - MAb TIB 131 Anti-intermediate filament monoclonal antibody - N Nuclei fraction - NEM Nuclear envelope-matrix fraction - NIF Nuclear intermediate filament - PAb PL3 Anti-pea lamin #3 polyclonal antibody     

Expression of carboxylesterase and lipase genes in rat liver cell-types

Approximately 80% of the body vitamin A is stored in liver stellate cells with in the lipid droplets as retinyl esters. In low vitamin A status or after liver injury, stellate cells are depleted of the stored retinyl esters by their hydrolysis to retinol. However, the identity of retinyl ester hydrolase(s) expressed in stellate cells is unknown. The expression of carboxylesterase and lipase genes in purified liver cell-types was investigated by real-time PCR. We found that six carboxylesterase and hepatic lipase genes were expressed in hepatocytes. Adipose triglyceride lipase was expressed in Kupffer cells, stellate cells and endothelial cells. Lipoprotein lipase expression was detected in Kupffer cells and stellate cells. As a function of stellate cell activation, expression of adipose triglyceride lipase decreased by twofold and lipoprotein lipase increased by 32-fold suggesting that it may play a role in retinol ester hydrolysis during stellate cell activation.     

Appraisal of Transdermal Water-in-Oil Nanoemulgel of Selegiline HCl for the Effective Management of Parkinson’s Disease: Pharmacodynamic,Pharmacokinetic, and Biochemical Investigations

In the present study, the potential of transdermal nanoemulsion gel of selegiline hydrochloride for the treatment of Parkinson’s disease was investigated. Water-in-oil nanoemulsions were developed by comparing low- and high-energy methods and were subjected to thermodynamic stability tests, in vitro permeation, and characterization studies. In vitro studies indicated that components of nanoemulsion acted as permeation enhancers with highest flux of 3.531 ± 1.94 μg/cm²/h from nanoemulsion SB6 containing 0.5 mg selegiline hydrochloride, 3% distilled water, 21% S _mix (Span 85, Tween 80, PEG 400), and 76% isopropyl myristate by weight. SB6 with the least droplet size of 183.4 ± 0.35 nm, polydispersity index of 0.42 ± 0.06 with pH of 5.9 ± 0.32 and viscosity of 22.42 ± 0.14 cps was converted to nanoemulsion gel NEGS4 (viscosity = 22,200 ± 400 cps) by addition of Viscup160® for ease of application and evaluated for permeation, safety, and pharmacokinetic profile in Wistar rats. It provided enhancement ratio 3.69 times greater than conventional gel. NEGS4 showed 6.56 and 5.53 times increase in bioavailability in comparison to tablet and conventional gel, respectively, along with sustained effect. Therefore, the developed water-in-oil nanoemulsion gel promises to be an effective vehicle for transdermal delivery of selegiline hydrochloride.