Incorporation of three endocytosed varicella-zoster virus glycoproteins, gE, gH, and gB, into the virion envelope

The cytoplasmic tails of all three major varicella-zoster virus (VZV) glycoproteins, gE, gH, and gB, harbor functional tyrosine-based endocytosis motifs that mediate internalization. The aim of the present study was to examine whether endocytosis from the plasma membrane is a cellular route by which VZV glycoproteins are delivered to the final envelopment compartment. In this study, we demonstrated that internalization of the glycoproteins occurred in the first 24 h postinfection but was reduced later in infection. Using surface biotinylation of VZV-infected cells followed by a glutathione cleavage assay, we showed that endocytosis was independent of antibody binding to gE, gH, and gB. Subsequently, with this assay, we demonstrated that biotinylated gE, gH, and gB retrieved from the cell surface were incorporated into nascent virus particles isolated after density gradient sedimentation. To confirm and extend this finding, we repeated the above sedimentation step and specifically detected envelopes decorated with Streptavidin-conjugated gold beads on a majority of complete virions through examination by transmission electron microscopy. In addition, a gE-gI complex and a gE-gH complex were found on the virions. Therefore, the above studies established that VZV subsumed a postendocytosis trafficking pathway as one mechanism by which to deliver viral glycoproteins to the site of virion assembly in the cytoplasm. Furthermore, since a recombinant VZV genome lacking only endocytosis-competent gE cannot replicate, these results supported the conclusion that the endocytosis-envelopment pathway is an essential component of the VZV life cycle.     

Calcium channels, transporters and exchangers in placenta: a review

Calcium (Ca2+) entry in cells is crucial for development and physiology of virtually all cell types. It acts as an intracellular (second) messenger to regulate a diverse array of cellular functions, from cell division and differentiation to cell death. Among candidates for Ca2+ entry in cells are-voltage-dependant Ca2+ channels (VDCCs), transient receptor potential (TRP)-related Ca2+ channels and store-operated Ca2+ (SOC) channels. Plasma membrane Ca2+-ATPases (PMCA) and Na+/Ca2+ exchanger (NCX) are mainly responsible for Ca2+ extrusion. These different Ca2+channels/transporters and exchangers exhibit specific distribution and physiological properties. During pregnancy, the syncytiotrophoblast layer of the human placenta transfers as much as 30 g of Ca2+ from the mother to the fetus, especially in late gestation where Ca2+ transport through different channels must increase in response to the demands of accelerating bone mineralization of the fetus. The identification and characterization of the different Ca2+ channels/transporters and exchangers on the brush-border membrane (BBM) facing the maternal circulation, and the basal plasma membrane (BPM) facing the fetal circulation; placental membrane of the syncytiotrophoblasts have been the focus of numerous studies. This review discusses current views in this field regarding localization and functions during transcellular Ca2+ entry and extrusion from cells particularly in the placenta.     

Galantamine antiacetylcholinesterase activity in rat brain influenced by L-carnitine

Galantamine (GAL) is a selective, competitive and reversible acetylcholinesterase (AChE) inhibitor, which increases the activity of the cholinergic system and hence gives rise to an improvement of cognitive functions in patients suffering from dementia of Alzheimer type. L-Carnitine (CAR) is a natural component of the mammalian tissue and is known to increase penetration of some chemical compounds/groups across biological membranes. The aim of this study was to evaluate the influence of pretreatment with CAR on AChE inhibition caused by GAL in selected brain parts in rat (basal ganglia, septum, frontal cortex, hippocampus) and in hypophysis, which does not lay beyond the blood-brain-barrier. During the first stage of the study, GAL was administered i.m. in different doses ranging from 2.5 to 10 mg/kg. The highest degree of AChE dose dependent inhibition was observed in hypophysis, while that in CNS was lower and became apparent in frontal cortex and hippocampus only after the administration of the dose of 10 mg/kg i.m. In the second stage, CAR was administered daily during 3 consecutive days at a dose of 250 mg/kg p.o. prior to the administration of GAL (10 mg/kg i.m.). Pretreatment with CAR enhanced trend of AChE inhibition in all selected brain parts comparing with single GAL administration, however, significant difference was not observed. Comparing these results with control group, statistical significance was found in frontal cortex, hippocampus and hypophysis.     

p53--prognostic factor of malignant transformation of Barrett's esophagus

The most significant precancerosis in the esophageal cancer is Barrett's esophagus. The risk of malignant transformation is determined primarily in accordance with the degree of dysplastic alterations of the mucosa. Indication of "preventive" extirpation of the esophagus should be supported by other factors, for example by detection of p53 mutation or expression. The study reports on the evaluation of a group of 20 patients with Barrett's esophagus treated at the 1st Department of Surgery, the p53 level and its correlation with histological findings evaluated in these patients. A good correlation was found between the grade of Barrett's esophagus dysplasia and high p53 positivity. This correlation was also confirmed by detection of early carcinoma in patients with "preventive" extirpation of the esophagus due to a high-grade dysplasia. Preliminary results show that examination of p53 level in specimens taken from the esophageal mucosa may be helpful for the estimation of malignant potential of the dysplastic mucosa.     

Dominant portion of thyrotropin-releasing hormone receptor is excluded from lipid domains. Detergent-resistant and detergent-sensitive pools of TRH receptor and Gqalpha/G11alpha protein

Some G protein-coupled receptors might be spacially targetted to discrete domains within the plasma membrane. Here we assessed the localization in membrane domains of the epitope-tagged, fluorescent version of thyrotropin-releasing hormone receptor (VSV-TRH-R-GFP) expressed in HEK293 cells. Our comparison of three different methods of cell fractionation (detergent extraction, alkaline treatment/sonication and mechanical homogenization) indicated that the dominant portion of plasma membrane pool of the receptor was totally solubilized by Triton X-100 and its distribution was similar to that of transmembrane plasma membrane proteins (glycosylated and non-glycosylated forms of CD147, MHCI, CD29, CD44, transmembrane form of CD58, Tapa1 and Na,K-ATPase). As expected, caveolin and GPI-bound proteins CD55, CD59 and GPI-bound form of CD58 were preferentially localized in detergent-resistant membrane domains (DRMs). Trimeric G proteins G(q)alpha/G(11)alpha, G(i)alpha1/G(i)alpha2, G(s)alphaL/G(s)alphaS and Gbeta were distributed almost equally between detergent-resistant and detergent-solubilized pools. In contrast, VSV-TRH-R-GFP, Galpha, Gbeta and caveolin were localized massively only in low-density membrane fragments of plasma membranes, which were generated by alkaline treatment/sonication or by mechanical homogenization of cells. These data indicate that VSV-TRH-R-GFP as well as other transmembrane markers of plasma membranes are excluded from TX-100-resistant, caveolin-enriched membrane domains. Trimeric G protein G(q)alpha/G(11)alpha occurs in both DRMs and in the bulk of plasma membranes, which is totally solubilized by TX-100.     

A new type of peroxisomal acyl-coenzyme A synthetase from Arabidopsis thaliana has the catalytic capacity to activate biosynthetic precursors of jasmonic acid

Arabidopsis thaliana contains a large number of genes that encode carboxylic acid-activating enzymes, including nine long-chain fatty acyl-CoA synthetases, four 4-coumarate:CoA ligases (4CL), and 25 4CL-like proteins of unknown biochemical function. Because of their high structural and sequence similarity with bona fide 4CLs and their highly hydrophobic putative substrate-binding pockets, the 4CL-like proteins At4g05160 and At5g63380 were selected for detailed analysis. Following heterologous expression, the purified proteins were subjected to a large scale screen to identify their preferred in vitro substrates. This study uncovered a significant activity of At4g05160 with medium-chain fatty acids, medium-chain fatty acids carrying a phenyl substitution, long-chain fatty acids, as well as the jasmonic acid precursors 12-oxo-phytodienoic acid and 3-oxo-2-(2'-pentenyl)-cyclopentane-1-hexanoic acid. The closest homolog of At4g05160, namely At5g63380, showed high activity with long-chain fatty acids and 12-oxo-phytodienoic acid, the latter representing the most efficiently converted substrate. By using fluorescent-tagged variants, we demonstrated that both 4CL-like proteins are targeted to leaf peroxisomes. Collectively, these data demonstrate that At4g05160 and At5g63380 have the capacity to contribute to jasmonic acid biosynthesis by initiating the beta-oxidative chain shortening of its precursors.     

Constitutive dimerization of human serotonin 5-HT4 receptors in living cells

Serotonin 5-HT4 receptor isoforms are G protein-coupled receptors (GPCRs) with distinct pharmacological properties and may represent a valuable target for the treatment of many human disorders. Here, we have explored the process of dimerization of human 5-HT4 receptor (h5-HT4R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT4(d)R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells. 5-HT4R ligands did not influence the constitutive energy transfer of the h5-HT4(d)R splice variant in intact cells and isolated plasma membranes. In addition, we found that h5-HT4(d)R and h5-HT4(g)R which structurally differ in the length of their C-terminal tails were able to form constitutive heterodimers independently of their activation state. Finally, we found that coexpression of h5-HT4R and beta2-adrenergic receptor (beta2AR) led to their heterodimerization. Given the large number of h5-HT4R isoforms which are coexpressed in a same tissue, our results points out the complexity by which this 5-HTR sub-type mediates its biological effects.     

Increased megakaryopoiesis in cultures of CD34-enriched cord blood cells maintained at 39 degrees C

Based on previous evidence suggesting positive effects of fever on in vivo hematopoiesis, we tested the effect of hyperthermia on megakaryopoiesis (MK) in ex vivo cultures of CD34-enriched cord blood (CB) cells. The cells were cultured at 37 degrees C or 39 degrees C for 14 days in cytokine conditions optimized for megakaryocyte development and analyzed periodically. Compared to 37 degrees C, the cultures maintained at 39 degrees C produced significantly more (up to 10-fold) total cells, myeloid and MK progenitors, and total MKs, and showed accelerated and enhanced MK maturation with increased yields of proplatelets and platelets. This observation could facilitate clinical applications requiring ex vivo expansion of hematopoietic cells.     

Identification of bilirubin reduction products formed by Clostridium perfringens isolated from human neonatal fecal flora

Urobilinoids belong to the heterogenous group of degradation products of bilirubin formed in the gastrointestinal tract by intestinal microflora. Among them urobilinogen and stercobilinogen with their respective oxidation products, urobilin and stercobilin, are the most important compounds. The aim of present study was to analyze the products of bacterial reduction of bilirubin in more detail. The strain of Clostridium perfringens isolated from neonatal stools, capable of reducing bilirubin, was used in the study. Bacteria were incubated under anaerobic conditions with various native as well as synthetic bile pigments, including radiolabeled unconjugated bilirubin (UCB). Their reduction products were extracted from media and separated following thin layer chromatography. Pigments isolated were analyzed by spectrophotometry, spectrofluorometry and mass spectrometry. In a special set of experiments, bilirubin diglucuronide was incubated with either bacterial lysate or partially purified bilirubin reductase and beta-glucuronidase to reveal whether bilirubin glucuronides may be directly reduced onto conjugated urobilinoids. A broad substrate activity was detected in the investigated strain of C. perfringens and a series of bilirubin reduction products was identified. These products were separated in the form of their respective chromogens and further oxidized. Based on their physical-chemical properties, as well as mass spectra, end-catabolic bilirubin products were identified to belong to urobilinogen species. The reduction process, catalyzed enzymatically by the studied bacterial strain, does not proceed to stercobilinogen. Bilirubin diglucuronide is not reduced onto urobilinoid conjugates, glucuronide hydrolysis must precede double bond reduction and thus UCB is reduced much faster.