Functional Analysis of Glycoprotein L (gL) from Rhesus Lymphocryptovirus in Epstein-Barr Virus-Mediated Cell Fusion Indicates a Direct Role of gL in gB-Induced Membrane Fusion

Glycoprotein L (gL), which complexes with gH, is a conserved herpesvirus protein that is essential for Epstein-Barr virus (EBV) entry into host cells. The gH/gL complex has a conserved role in entry among herpesviruses, yet the mechanism is not clear. To gain a better understanding of the role of gL in EBV-mediated fusion, chimeric proteins were made using rhesus lymphocryptovirus (Rh-LCV) gL (Rh gL), which shares a high sequence homology with EBV gL but does not complement EBV gL in mediating fusion with B cells. A reduction in fusion activity was observed with chimeric gL proteins that contained the amino terminus of Rh gL, although they retained their ability to process and transport gH/gL to the cell surface. Amino acids not conserved within this region in EBV gL when compared to Rh gL were further analyzed, with the results mapping residues 54 and 94 as being functionally important for EBV-mediated fusion. All chimeras and mutants displayed levels of cell surface expression similar to that of wild-type gL and interacted with gH and gp42. Our data also suggest that the role of gL involves the activation or recruitment of gB with the gH/gL complex, as we found that reduced fusion of Rh gL, EBV/Rh-LCV chimeras, and gL point mutants could be restored by replacing EBV gB with Rh gB. These observations demonstrate a distinction between the role of gL in the processing and trafficking of gH to the cell surface and a posttrafficking role in cell-cell fusion.Epstein-Barr virus (EBV) is a member of the lymphocryptovirus (LCV) subgroup of gammaherpesviruses and primarily infects epithelial and B lymphocytes, where it establishes productive and latent infections, respectively (8, 40). EBV is extremely prevalent in humans, with more than 90% of the population being infected. Following primary infection, EBV persists in a latent state in memory B lymphocytes, where it can remain indefinitely (3, 43). The expansion and proliferation of these cells can lead to the development of a number of EBV-related malignancies, including tumors of lymphoid tissues, such as Hodgkin''s lymphoma, Burkitt''s lymphoma, and some T-cell lymphomas, as well as tumors associated with epithelial tissues, such as nasopharyngeal carcinoma and gastric carcinoma (5, 32, 36, 48). EBV is also associated with lymphoproliferative disorders in immunocompromised patients, such as oral hairy leukoplakia and posttransplant lymphoproliferative disorders (12, 15, 43, 45).Herpesvirus entry is a multistep process that includes the initial binding of the virus to the cell surface, interaction with a cellular entry receptor, membrane-virion fusion, and internalization of the virion (40). The enveloped membrane of EBV contains numerous glycoproteins that are necessary for mediating attachment and the subsequent fusion between viral and cell membranes, although the required glycoproteins differ for epithelial and B-cell infection (13, 22). Infection of B cells is initiated upon the binding of EBV glycoprotein gp350/220 to the CD21/CR2 cellular receptor expressed on target cells (9, 27, 41). Following the initial binding, viral glycoprotein gp42 binds to human leukocyte antigen class II (HLA class II) and triggers fusion, which is mediated by the concerted actions of glycoproteins gB, gH, and gL (21, 41). Glycoproteins gH and gL form a heteromeric complex that is conserved among the herpesvirus family despite a low degree of sequence homology in gH and gL across herpesviruses (33, 40). The gH/gL complex has an essential role in entry of herpesviruses, yet the exact role of each protein is not well understood. The role of gH is thought to be in cell fusion, while gL mediates processing and transport of gH to the cell surface (34, 35, 47, 51). Since gL is generally essential for the proper processing and transport of gH in all herpesviruses, it has been difficult to examine the role of gL separate from gH.While little is known about the role of gL in EBV-mediated fusion, data from studies of other herpesviruses have suggested a more direct role of gL in fusion. Antibodies specific for the C terminus of herpes simplex virus (HSV) gL, mapped to residues 168 to 224, were found to inhibit fusion with some strains of HSV type 1 (HSV-1), suggesting a role of this region in fusion (28). Deletion analysis of HSV-1 gL determined that the first 161 amino acids of gL are sufficient for binding gH, and further deletion identified the region between amino acids 155 and 161 of HSV-1 gL as critical for gH transport and cell fusion (19, 20, 33). Together, the results of these studies suggest that the inhibition of fusion observed with gL antibodies (Abs) is not due to an inhibition of gH/gL complex formation. Another study found that mutations to the amino-terminal region of HSV-2 gH permitted transport of the glycoprotein to the cell surface independent of gL but that gL was still required for fusion, suggesting a role of gL in fusion after the complex has trafficked to the plasma membrane (6). Most recently, the results of an investigation of the functional homology of the primate rhesus LCV (Rh-LCV) gL (Rh gL) suggest that gL may have a functional role in EBV fusion with B cells. Rh gL shares a high degree of sequence similarity with EBV (81.6%), and yet, the glycoprotein was unable to mediate fusion with human B cells when expressed with either EBV gH or Rh gH, even though the gH/gL complex was detected on the cell surface and Rh gL could associate with gH and gp42 (31). The LCV that infects rhesus primates is biologically similar to EBV in that it shares a similar genome organization, repertoire of lytic and latent genes, and pathogenesis (7, 25, 37, 46).To gain a better understanding of the role of gL in EBV-mediated fusion with B cells, we constructed chimeric gL proteins that included portions of Rh gL inserted into the sequence of EBV gL. We identified the amino terminus of EBV gL as being functionally necessary in mediating fusion with B cells, and this role is distinguishable from the role of gL in the processing and transport of gH to the cell surface. Single and double point mutations were constructed in both EBV and Rh gL, and our results indicated that residues 54 and 94 are functionally critical in mediating fusion with B cells. Interestingly, our studies also identified a species-specific reliance between gL and gB, suggesting a possible association between these two glycoproteins that is necessary in mediating fusion. Thus, our studies demonstrated that EBV gL has a more substantial role in mediating fusion with B cells that is distinct from its role in the processing and trafficking of gH.     

Radiation therapy in treatment of fibrodysplasia ossificans progressiva: a case report and review of the literature

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare genetic disorder with diffuse extra-skeletal bone formation. The genetic mutation responsible for FOP has recently been discovered and is connected with excessive activation of bone morphogenetic protein receptor. This disease usually begins with typical ossification pattern in early childhood, causing increasing disability and making patients totally disabled by the age of 30. Ectopic ossification develops spontaneously and can be triggered by any trauma and even intramuscular injections. The symptoms of FOP are often misdiagnosed as cancer, causing unnecessary biopsies, which can precipitate further progressive heterotopic ossification. There is no effective treatment for this severe condition. Radiotherapy can be helpful in impeding ossification, although the strict evidence for that is lacking. There are only two reports in the literature referring to the use of radiotherapy in treatment of FOP. Herein, we present a 35-year-old patient successfully treated with small doses of fractionated radiotherapy in several courses. This case indicates that radiotherapy can be useful in treating patients with FOP.     

Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase

Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and 4.8 mM (5.13 μmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed to 4-halocatechols at 2 mM within a 1–2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5–20-fold decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol products followed by column chromatography resulted in the production of purified products with a final yield of between 33% and 38%.     

The retinal specific CD147 Ig0 domain: from molecular structure to biological activity

CD147 is a type I transmembrane protein that is involved in inflammatory diseases, cancer progression, and multiple human pathogens utilize CD147 for efficient infection. CD147 expression is so high in several cancers that it is now used as a prognostic marker. The two primary isoforms of CD147 that are related to cancer progression have been identified, differing in their number of immunoglobulin (Ig)-like domains. These include CD147 Ig1-Ig2, which is ubiquitously expressed in most tissues, and CD147 Ig0-Ig1-Ig2, which is retinal specific and implicated in retinoblastoma. However, little is known in regard to the retinal specific CD147 Ig0 domain despite its potential role in retinoblastoma. We present the first crystal structure of the human CD147 Ig0 domain and show that the CD147 Ig0 domain is a crystallographic dimer with an I-type domain structure, which maintained in solution. Furthermore, we have utilized our structural data together with mutagenesis to probe the biological activity of CD147-containing proteins, both with and without the CD147 Ig0 domain, within several model cell lines. Our findings reveal that the CD147 Ig0 domain is a potent stimulator of interleukin-6 and suggest that the CD147 Ig0 domain has its own receptor distinct from that of the other CD147 Ig-like domains, CD147 Ig1-Ig2. Finally, we show that the CD147 Ig0 dimer is the functional unit required for activity and can be disrupted by a single point mutation.     

Mechanism of GABAB receptor-induced BDNF secretion and promotion of GABAA receptor membrane expression

Recent studies have shown that GABA(B) receptors play more than a classical inhibitory role and can function as an important synaptic maturation signal early in life. In a previous study, we reported that GABA(B) receptor activation triggers secretion of brain-derived neurotrophic factor (BDNF) and promotes the functional maturation of GABAergic synapses in the developing rat hippocampus. To identify the signalling pathway linking GABA(B) receptor activation to BDNF secretion in these cells, we have now used the phosphorylated form of the cAMP response element-binding protein as a biological sensor for endogenous BDNF release. In the present study, we show that GABA(B) receptor-induced secretion of BDNF relies on the activation of phospholipase C, followed by the formation of diacylglycerol, activation of protein kinase C, and the opening of L-type voltage-dependent Ca(2+) channels. We further show that once released by GABA(B) receptor activation, BDNF increases the membrane expression of β(2/3) -containing GABA(A) receptors in neuronal cultures. These results reveal a novel function of GABA(B) receptors in regulating the expression of GABA(A) receptor through BDNF-tropomyosin-related kinase B receptor dependent signalling pathway.     

Conversion of post consumer polyethylene to the biodegradable polymer polyhydroxyalkanoate

A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer.     

Disease intensity of some tomato viroses in Serbia

In this paper we present the data on the disease intensity of the tomato plants grown in glass and plastic-houses, and in the open field. The infection was caused by the following viruses: Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Alfalfa mosaic virus (AMV), Potato virus X (PVX), Potato virus Y (PVY), Tomato black ring virus (TBRV), Tomato ringspot virus (ToRSV), Tomato aspermy virus (TAV), and Cucumber mosaic virus (CMV). These viruses represented most frequent tomato pathogens in Serbia. According to the obtained results, it could be concluded that 92.94% of the tested tomato plants grown in glass and plastic-houses, and 89.82% grown in the open field were infected by one of the above viruses. Most of the plant samples were infected by two or more viruses. The most frequent viruses — tomato pathogens in Serbia were ToMV, PVY and TMV.     

In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis

In vitro studies using isolated cells, mitochondria and submitochondrial fractions demonstrated that in steroid synthesizing cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein, preferentially located in the outer/inner membrane contact sites, involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Mitochondrial PBR ligand binding characteristics and topography are sensitive to hormone treatment suggesting a role of PBR in the regulation of hormone-mediated steroidogenesis. Targeted disruption of the PBR gene in Leydig cells in vitro resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the mutant cells with a PBR cDNA rescued steroidogenesis demonstrating an obligatory role for PBR in cholesterol transport. Molecular modeling of PBR suggested that it might function as a channel for cholesterol. This hypothesis was tested in a bacterial system devoid of PBR and cholesterol. Cholesterol uptake and transport by these cells was induced upon PBR expression. Amino acid deletion followed by site-directed mutagenesis studies and expression of mutant PBRs demonstrated the presence in the cytoplasmic carboxy-terminus of the receptor of a cholesterol recognition/interaction amino acid consensus sequence. This amino acid sequence may help for recruiting the cholesterol coming from intracellular sites to the mitochondria.     

The role of 26S proteasome-dependent proteolysis in the formation and restructuring of microtubule networks

In this review, we summarize the evidence pointing at the important role of 26S proteasome-dependent proteolysis in the regulation of microtubule synthesis and microtubule dynamics. Because most of the advances in this relatively unexplored research field originate from yeast and animal studies, we have considered those studies that describe the role of proteolysis in processes that are evolutionarily conserved and known to exist in plants. In addition, we place particular emphasis on the proteasome-dependent degradation of plant-specific microtubule-associated protein SPIRAL1 and its function in MT rearrangements associated with salt stress.     

Neuroprotective Potential and Chemical Profile of Alternatively Cultivated Ganoderma lucidum Basidiocarps

Various neurodegenerative diseases are the main challenges to the modern medicine and there is a great need for novel, natural, neuroprotective agents. Ganoderma lucidum is a well‐known medicinal mushroom, which health benefits have been confirmed by numerous studies. As demand for its basidiocarps is increased and traditional cultivation on hardwoods is not environmentally friendly and economically justified, finding of alternative substrates is necessary. The aim of the study was to assess the effect of alternative cultivation substrates on the chemical profile of G. lucidum basidiocarps and their capacity to inhibit acetylcholinesterase and tyrosinase, which higher activity is directly associated with neurodegenerative processes. Extracts of basidiocarps cultivated on alternative substrates, especially on clear wheat straw, showed significantly higher inhibition capacities than extracts of commercially‐grown ones. These extracts were considerably different chemically from commercial basidiocarps extracts and even nine new compounds were isolated from them. Our results suggest that cultivation substrate greatly affect the chemical profile and neuroprotective capacity of obtained basidiocarps and wheat straw is a promising cultivation substrate.