Glycoprotein N of Human Cytomegalovirus Protects the Virus from Neutralizing Antibodies

Herpes viruses persist in the infected host and are transmitted between hosts in the presence of a fully functional humoral immune response, suggesting that they can evade neutralization by antiviral antibodies. Human cytomegalovirus (HCMV) encodes a number of polymorphic highly glycosylated virion glycoproteins (g), including the essential envelope glycoprotein, gN. We have tested the hypothesis that glycosylation of gN contributes to resistance of the virus to neutralizing antibodies. Recombinant viruses carrying deletions in serine/threonine rich sequences within the glycosylated surface domain of gN were constructed in the genetic background of HCMV strain AD169. The deletions had no influence on the formation of the gM/gN complex and in vitro replication of the respective viruses compared to the parent virus. The gN-truncated viruses were significantly more susceptible to neutralization by a gN-specific monoclonal antibody and in addition by a number of gB- and gH-specific monoclonal antibodies. Sera from individuals previously infected with HCMV also more efficiently neutralized gN-truncated viruses. Immunization of mice with viruses that expressed the truncated forms of gN resulted in significantly higher serum neutralizing antibody titers against the homologous strain that was accompanied by increased antibody titers against known neutralizing epitopes on gB and gH. Importantly, neutralization activity of sera from animals immunized with gN-truncated virus did not exhibit enhanced neutralizing activity against the parental wild type virus carrying the fully glycosylated wild type gN. Our results indicate that the extensive glycosylation of gN could represent a potentially important mechanism by which HCMV neutralization by a number of different antibody reactivities can be inhibited.     

Identification of Proteins from Interstitium of Trapezius Muscle in Women with Chronic Myalgia Using Microdialysis in Combination with Proteomics

Background

Microdialysis (MD) of the trapezius muscle has been an attractive technique to investigating small molecules and metabolites in chronic musculoskeletal pain in human. Large biomolecules such as proteins also cross the dialysis membrane of the catheters. In this study we have applied in vivo MD in combination with two dimensional gel electrophoresis (2-DE) and mass spectrometry to identify proteins in the extracellular fluid of the trapezius muscle.

Materials and Methods

Dialysate from women with chronic trapezius myalgia (TM; n = 37), women with chronic wide spread pain (CWP; n = 18) and healthy controls (CON; n = 22) was collected from the trapezius muscle using a catheter with a cut-off point of 100 kDa. Proteins were separated by two-dimensional gel electrophoresis and visualized by silver staining. Detected proteins were identified by nano liquid chromatography in combination with tandem mass spectrometry.

Results

Ninety-seven protein spots were identified from the interstitial fluid of the trapezius muscle; 48 proteins in TM and 30 proteins in CWP had concentrations at least two-fold higher or lower than in CON. The identified proteins pertain to several functional classes, e.g., proteins involved in inflammatory responses. Several of the identified proteins are known to be involved in processes of pain such as: creatine kinase, nerve growth factor, carbonic anhydrase, myoglobin, fatty acid binding protein and actin aortic smooth muscle.

Conclusions

In this study, by using in vivo microdialysis in combination with proteomics a large number of proteins in muscle interstitium have been identified. Several of the identified proteins were at least two-fold higher or lower in chronic pain patients. The applied techniques open up for the possibility of investigating protein changes associated with nociceptive processes of chronic myalgia.     

New Insights into [FeFe] Hydrogenase Activation and Maturase Function

[FeFe] hydrogenases catalyze H₂ production using the H-cluster, an iron-sulfur cofactor that contains carbon monoxide (CO), cyanide (CN^–), and a dithiolate bridging ligand. The HydE, HydF, and HydG maturases assist in assembling the H-cluster and maturing hydrogenases into their catalytically active form. Characterization of these maturases and in vitro hydrogenase activation methods have helped elucidate steps in the H-cluster biosynthetic pathway such as the HydG-catalyzed generation of the CO and CN^– ligands from free tyrosine. We have refined our cell-free approach for H-cluster synthesis and hydrogenase maturation by using separately expressed and purified HydE, HydF, and HydG. In this report, we illustrate how substrates and protein constituents influence hydrogenase activation, and for the first time, we show that each maturase can function catalytically during the maturation process. With precise control over the biomolecular components, we also provide evidence for H-cluster synthesis in the absence of either HydE or HydF, and we further show that hydrogenase activation can occur without exogenous tyrosine. Given these findings, we suggest a new reaction sequence for the [FeFe] hydrogenase maturation pathway. In our model, HydG independently synthesizes an iron-based compound with CO and CN^– ligands that is a precursor to the H-cluster [2Fe]_H subunit, and which we have termed HydG-co. We further propose that HydF is a transferase that stabilizes HydG-co and also shuttles the complete [2Fe]_H subcluster to the hydrogenase, a translocation process that may be catalyzed by HydE. In summary, this report describes the first example of reconstructing the [FeFe] hydrogenase maturation pathway using purified maturases and subsequently utilizing this in vitro system to better understand the roles of HydE, HydF, and HydG.     

Multimerization of tegument protein pp28 within the assembly compartment is required for cytoplasmic envelopment of human cytomegalovirus

Human cytomegalovirus (HCMV) UL99-encoded pp28 is an essential tegument protein required for envelopment and production of infectious virus. Nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with the UL99 gene deleted. Previous results have suggested that a key function of pp28 in the envelopment of infectious HCMV is expressed after the protein localizes in the assembly compartment (AC). In this study, we investigated the potential role of pp28 multimerization in the envelopment of the infectious virion. Our results indicated that pp28 multimerized during viral infection and that interacting domains responsible for self-interaction were localized in the amino terminus of the protein (amino acids [aa] 1 to 43). The results from transient-expression and/or infection assays indicated that the self-interaction took place in the AC. A mutant pp28 molecule containing only the first 35 aa failed to accumulate in the AC, did not interact with pp28 in the AC, and could not support virus replication. In contrast, the first 50 aa of pp28 was sufficient for the self-interaction within the AC and the assembly of infectious virus. Recombinant viruses encoding an in-frame deletion of aa 26 to 33 of pp28 were replication competent, whereas infectious virus was not recovered from HCMV BACs lacking aa 26 to 43. These findings suggested that the accumulation of pp28 was a prerequisite for multimerization of pp28 within the AC and that pp28 multimerization in the AC represented an essential step in the envelopment and production of infectious virions.     

Influences of the N700S thrombospondin-1 polymorphism on protein structure and stability

Thrombospondins (THBSs) are multimodular, secreted proteins characterized by a signature domain comprising a unique set of 13 calcium-binding repeats flanked by epidermal growth factor (EGF)-like and lectin-like modules. A polymorphism that changes a conserved Asn to Ser at residue 700 in the most N-terminal calcium-binding repeat of THBS-1 (repeat 1C) is found in 8-10% of European populations and has been linked to increased risk of premature coronary artery disease. The Ser substitution leads to altered stability in the EGF-like and wire modules of the THBS-1 signature domain as assessed by differential scanning calorimetry carried out in 2 mm or 200 mum calcium. Studies of the melting profiles of the THBS-2 signature domain proteins with Asn or Ser at position 702 (homologous to 700 in THBS-1) revealed that the impact of the Ser allele is similar in both THBS-1 and THBS-2. Structure determination of the Ser(702) THBS-2 variant in 2 mm calcium showed that repeat 1C contains two bound calcium ions as in the crystal of the Asn(702) protein, including the ion that is coordinated by Asn(702), and is associated with changes in conformation of repeat 1C and the adjacent EGF-like modules. The Ser substitution leads to the decreased ability of soluble THBS-2 signature domain protein to bind 4B6.13, a conformation-sensitive monoclonal antibody that recognizes an epitope in repeat 1C. These results indicate that although THBS harboring the Ser allele binds a full complement of calcium ions, repeat 1C is altered, leading to destabilization of surrounding structures.     

Cystic mammary adenocarcinoma associated with a prolactin-secreting pituitary adenoma in a New Zealand white rabbit (Oryctolagus cuniculus)

Pituitary adenoma in a rabbitA 44-mo-old, female, nulliparous New Zealand White Rabbit (Oryctolagus cuniculus) presented with bilaterally diffusely enlarged mammary glands with enlarged, discolored teats that exuded brown, mucoid discharge. The complete blood count and serum chemistry panels were within normal limits, bacteria were not isolated from a culture of the discharge, and the clinical signs did not resolve with antibiotic treatment. Computed tomography and serum prolactin levels supported the diagnosis of mammary gland dysplasia, possibly due to a prolactin-secreting pituitary adenoma. Histologic evaluation confirmed the presence of a pituitary adenoma, mammary hyperplasia, dysplasia, and cystic mammary adenocarcinoma. Immunohistochemical staining confirmed the presence of abundant prolactin secreting cells in the pituitary adenoma. This is the second report of hyperprolactinemia with mammary dysplasia in rabbits, and the first report of cystic mammary adenocarcinoma associated with a prolactin-secreting pituitary adenoma in a rabbit.     

Stimulation of osteoblast activity by homocysteine

Homocysteine (HCY) has recently been linked to fragility fractures. Moreover, HCY activates osteoclasts. Little is known about the effect of HCY on activity of human osteoblasts (OBs). We hypothesized that HCY decreases the activity of OBs. Osteoblasts obtained from tra-becular human bone specimens of eight donors were cultured with conditioned medium. Culture medium was adjusted to 0, 100, 500, 1000 and 2000 muM HCY. After 14 days alkaline phosphatase (AP) activity, pro-collagen type I N-terminal peptide (PINP) and osteocalcin (OC) secretion in the supernatant were measured. After 20 days the formation of mineralized matrix was analyzed. HCY-stimulated AP activity gradually (100 muM HCY: 118%, P= 0.006; 500 muM HCY: 125%, P < 0.001). At 1000 and 2000 muM HCY the increase of AP activity was reversible (1000 muM HCY: 106%, P= 0.317; 2000 muM HCY: 102%, P < 0.737). The PINP secretion was also stimulated by HCY reaching a maximum of 260 +/- 154 mug/l at 500 mumol/l versus 205 +/- 94 mu,g/l in controls. After 20 days of culture the formation of bone matrix was increased at 100 and 500 muM HCY. OC secretion was not significantly changed. The results of the present study consistently demonstrate a moderate stimulation of primary human OB activity by increasing concentrations of HCY. However, the magnitude of this effect seems to be less pronounced than recent observations on primary human osteoclasts, suggesting a dysbalance between OBs and osteoclasts in favour of osteoclasts.     

Distinct ecological niches of marine symbiotic N2‐fixing cyanobacterium Candidatus Atelocyanobacterium thalassa sublineages

A recently described symbiosis between the metabolically streamlined nitrogen‐fixing cyanobacterium UCYN‐A and a single‐celled eukaryote prymnesiophyte alga is widely distributed throughout tropical and subtropical marine waters, and is thought to contribute significantly to nitrogen fixation in these regions. Several UCYN‐A sublineages have been defined based on UCYN‐A nitrogenase (nifH) sequences. Due to the low abundances of UCYN‐A in the global oceans, currently existing molecular techniques are limited for detecting and quantifying these organisms. A targeted approach is needed to adequately characterize the diversity of this important marine cyanobacterium, and to advance understanding of its ecological importance. We present findings on the distribution of UCYN‐A sublineages based on high throughput sequencing of UCYN‐A nifH PCR amplicons from 78 samples distributed throughout many major oceanic provinces. These UCYN‐A nifH fragments were used to define oligotypes, alternative taxonomic units defined by nucleotide positions with high variability. The data set was dominated by a single oligotype associated with the UCYN‐A1 sublineage, consistent with previous observations of relatively high abundances in tropical and subtropical regions. However, this analysis also revealed for the first time the widespread distribution of the UCYN‐A3 sublineage in oligotrophic waters. Furthermore, distinct assemblages of UCYN‐A oligotypes were found in oligotrophic and coastally influenced waters. This unique data set provides a framework for determining the environmental controls on UCYN‐A distributions and the ecological importance of the different sublineages.     

High Expression of Complement Components in Omental Adipose Tissue in Obese Men

Objective: Accumulation of visceral fat is recognized as a predictor of obesity‐related metabolic disturbances. Factors that are predominantly expressed in this depot could mediate the link between visceral obesity and associated diseases. Research Methods and Procedures: Paired subcutaneous and omental adipose tissue biopsies were obtained from 10 obese men. Gene expression was analyzed by DNA microarrays in triplicate and by real‐time polymerase chain reaction. Serum C3 and C4 were analyzed by radial immunodiffusion assays in 91 subjects representing a cross section of the general population. Body composition was measured by computerized tomography. Results: Complement components C2, C3, C4, C7, and Factor B had higher expression in omental compared with subcutaneous adipose tissue (～2‐, 4‐, 17‐, 10‐, and 7‐fold, respectively). In addition, adipsin, which belongs to the alternative pathway, and the classical pathway components C1QB, C1R, and C1S were expressed in both depots. Analysis of tissue distribution showed high expression of C2, C3, and C4 in omental adipose tissue, and only liver had higher expression of these genes. Serum C3 levels correlated with both visceral and subcutaneous adipose tissue in both men (r = 0.65 and p < 0.001 and r = 0.52 and p < 0.001, respectively) and women (r = 0.34 and p = 0.023 and r = 0.49 and p < 0.001, respectively), whereas C4 levels correlated with only visceral fat in men (r = 0.36, p = 0.015) and with both depots in women (visceral: r = 0.58, p < 0.001; and subcutaneous: r = 0.51, p < 0.001). Discussion: Recent studies show that the metabolic syndrome is associated with chronically elevated levels of several immune markers, some of which may have metabolic effects. The high expression of complement genes in intra‐abdominal adipose tissue might suggest that the complement system is involved in the development of visceral adiposity and/or contributes to the metabolic complications associated with increased visceral fat mass.     

HCMV-Encoded Glycoprotein M (UL100) Interacts with Rab11 Effector Protein FIP4

The envelope of human cytomegalovirus (HCMV) consists of a large number of glycoproteins. The most abundant glycoprotein in the HCMV envelope is the glycoprotein M (UL100), which together with glycoprotein N (UL73) form the gM/gN protein complex. Using yeast two-hybrid screening, we found that the gM carboxy-terminal cytoplasmic tail (gM-CT) interacts with FIP4, a Rab11-GTPase effector protein. Depletion of FIP4 expression in HCMV-infected cells resulted in a decrease in infectious virus production that was also associated with an alteration of the HCMV assembly compartment (AC) phenotype. A similar phenotype was also observed in HCMV-infected cells that expressed dominant negative Rab11(S25N). Recently, it has been shown that FIP4 interactions with Rab11 and additionally with Arf6/Arf5 are important for the vesicular transport of proteins in the endosomal recycling compartment (ERC) and during cytokinesis. Surprisingly, FIP4 interaction with gM-CT limited binding of FIP4 with Arf5/Arf6; however, FIP4 interaction with gM-CT did not prevent recruitment of Rab11 into the ternary complex. These data argued for a contribution of the ERC during cytoplasmic envelopment of HCMV and showed a novel FIP4 function independent of Arf5 or Arf6 activity.