Morphological and Molecular Characterization of Punctodera Stonei Brzeski, 1998 (Nematoda: Heteroderidae) from Virginia,USA

In August of 2021, several cysts with juveniles and eggs were discovered during a vegetation survey conducted at the Arlington National Cemetery, Virginia. Eight soil samples were collected from the rhizosphere region of the common grass (Festuca arundinacea L.) and processed at the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL). Cysts were light to dark brown in color, and oval to pear-shaped without bullae in young cysts but present in older cysts and with prominent vulval cone. The juveniles had slightly concave stylet knobs projecting sometimes anteriorly, tail tapering gradually to a narrowly rounded terminus, and hyaline tail terminus conspicuous at least twice the length of stylet. The molecular analysis included the analysis of three gene sequence fragments: D2–D3 of 28S rRNA, ITS rRNA, and COI. The nematode species was identified by both morphological and molecular means as Stone''s cyst nematode, Punctodera stonei. Detection of P. stonei in Virginia represents a new record of this species in the United States, and a second report after Canada in North America.     

Excitation-contraction coupling in resistance mesenteric arteries: Evidence for NKCC1-mediated pathway

Bumetanide and other high-ceiling diuretics (HCD) attenuate myogenic tone and contractions of vascular smooth muscle cells (VSMC) triggered by diverse stimuli. HCD outcome may be mediated by their interaction with NKCC1, the only isoform of Na⁺, K⁺, 2Cl⁻ cotransporter expressed in VSMC as well as with targets distinct from this carrier. To examine these hypotheses, we compared the effect of bumetanide on contractions of mesenteric arteries from wild-type and NKCC1 knockout mice. In mesenteric arteries from wild-type controls, 100 μM bumetanide evoked a decrease of up to 4-fold in myogenic tone and contractions triggered by modest [K⁺]_o-induced depolarization, phenylephrine and UTP. These actions of bumetanide were preserved after inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester, but were absent in mesenteric arteries from NKCC1^-/- mice. The data show that bumetanide inhibits VSMC contractile responses via its interaction with NKCC1 and independently of nitric oxide production by endothelial cells.     

Distribution of Tetrahydromethanopterin-Dependent Enzymes in Methylotrophic Bacteria and Phylogeny of Methenyl Tetrahydromethanopterin Cyclohydrolases

The methylotrophic proteobacterium Methylobacterium extorquens AM1 possesses tetrahydromethanopterin (H(4)MPT)-dependent enzymes, which are otherwise specific to methanogenic and sulfate-reducing archaea and which have been suggested to be involved in formaldehyde oxidation to CO(2) in M. extorquens AM1. The distribution of H(4)MPT-dependent enzyme activities in cell extracts of methylotrophic bacteria from 13 different genera are reported. H(4)MPT-dependent activities were detected in all of the methylotrophic and methanotrophic proteobacteria tested that assimilate formaldehyde by the serine or ribulose monophosphate pathway. H(4)MPT-dependent activities were also found in autotrophic Xanthobacter strains. However, no H(4)MPT-dependent enzyme activities could be detected in other autotrophic alpha-proteobacteria or in gram-positive methylotrophic bacteria. Genes encoding methenyl H(4)MPT cyclohydrolase (mch genes) were cloned and sequenced from several proteobacteria. Bacterial and archaeal Mch sequences have roughly 35% amino acid identity and form distinct groups in phylogenetic analysis.     

Low efficiency of functional translation of ouabain-resistant α2-Na(+),K(+)-ATPase mRNA in C7-MDCK epithelial cells

Na(+),K(+)-ATPase is a heterodimer consisting of catalytic α1-α4 and regulatory β1-β3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na(+),K(+)-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K(+) ((86)Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na(+),K(+)-ATPase. α2R mRNA in transfected cells was ～8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10?μmol/L ouabain led to complete inhibition of (86)Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of (86)Rb influx in α1R-transfectd cells was observed at 1000?μmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3?μmol/L ouabain , α1R-cells survived after 24?h incubation with 1000?μmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-Na(+),K(+)-ATPase subunit mRNA and immunoreactive protein does not contribute to Na(+)/K(+) pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2-Na(+),K(+)-ATPase on Na(+)/K(+) pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells.     

In-stent restenosis after revascularization of myocardium with drug-eluting stents is accompanied by elevated level of blood plasma eosinophil cationic protein

The aim of this study was to assess the involvement of eosinophil cationic protein, a marker of eosinophil activation, in the development of in-stent restenosis after drug-eluting stent implantation. Follow-up angiography at 6 to 12?months was performed in 32 patients who were treated with percutaneous coronary intervention and implantation of sirolimus-eluting stents. Blood plasma levels of eosinophil cationic protein (ECP) and total immunoglobulin E (IgE) were measured by enzyme-linked immunosorbent assay and the level of C-reactive protein (hs-CRP) by high-sensitivity nephelometry. According to angiography data, in-stent restenosis occurred in 13 patients, while 19 patients did not develop it. There were no differences between the hs-CRP and IgE levels in patients with or without restenosis. In contrast, ECP level was higher in patients with restenosis compared with that in patients without restenosis [17.7?ng/mL (11.2-24.0) vs. 9.0?ng/mL (6.4-12.9), p?= 0.017]. The incidence of in-stent restenoses was 63% in patients with ECP level higher than or equal to 11?ng/mL, and 19% in patients with an ECP level lower than 11?ng/mL (p?= 0.019). These findings suggest that elevated eosinophil activation may play an important role in the pathogenesis of in-stent restenosis after implantation of drug-eluting stents.     

A cis-acting element that directs circular adeno-associated virus replication and packaging

A novel pathway of adeno-associated virus (AAV) replication marked by the assembly of circular monomer duplex intermediates (cAAV) has been recently discovered. In the present report we identify a single AD domain of the inverted terminal repeat as a minimal origin of cAAV replication. A small internal palindrome (BB'), necessary for optimal Rep-inverted terminal repeat interaction, does not contribute to the efficiency of cAAV replication, while the terminal resolution site is an essential cis-acting element. Furthermore, recombinant cAAV vectors that encompass only the AD domain replicate exclusively in a circular form and no detectable linear duplex replicative intermediates are generated, suggesting that both pathways of AAV replication are independent and can be separated. In addition, we show that cAAVs are efficient templates for encapsidation of single-stranded DNA genomes, an observation that assigns a biological role for these novel replication species. Together, these findings shed new light on the current model of AAV replication and packaging.     

Identification of Nedd4 E3 ubiquitin ligase as a binding partner and regulator of MAK-V protein kinase

MAK-V/Hunk is a scantily characterized AMPK-like protein kinase. Recent findings identified MAK-V as a pro-survival and anti-apoptotic protein and revealed its role in embryonic development as well as in tumorigenesis and metastasis. However molecular mechanisms of MAK-V action and regulation of its activity remain largely unknown. We identified Nedd4 as an interaction partner for MAK-V protein kinase. However, this HECT-type E3 ubiquitin ligase is not involved in the control of MAK-V degradation by the ubiquitin-proteasome system that regulates MAK-V abundance in cells. However, Nedd4 in an ubiquitin ligase-independent manner rescued developmental defects in Xenopus embryos induced by MAK-V overexpression, suggesting physiological relevance of interaction between MAK-V and Nedd4. This identifies Nedd4 as the first known regulator of MAK-V function.     

An evolutionary-network model reveals stratified interactions in the V3 loop of the HIV-1 envelope

The third variable loop (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope is a principal determinant of antibody neutralization and progression to AIDS. Although it is undoubtedly an important target for vaccine research, extensive genetic variation in V3 remains an obstacle to the development of an effective vaccine. Comparative methods that exploit the abundance of sequence data can detect interactions between residues of rapidly evolving proteins such as the HIV-1 envelope, revealing biological constraints on their variability. However, previous studies have relied implicitly on two biologically unrealistic assumptions: (1) that founder effects in the evolutionary history of the sequences can be ignored, and; (2) that statistical associations between residues occur exclusively in pairs. We show that comparative methods that neglect the evolutionary history of extant sequences are susceptible to a high rate of false positives (20%–40%). Therefore, we propose a new method to detect interactions that relaxes both of these assumptions. First, we reconstruct the evolutionary history of extant sequences by maximum likelihood, shifting focus from extant sequence variation to the underlying substitution events. Second, we analyze the joint distribution of substitution events among positions in the sequence as a Bayesian graphical model, in which each branch in the phylogeny is a unit of observation. We perform extensive validation of our models using both simulations and a control case of known interactions in HIV-1 protease, and apply this method to detect interactions within V3 from a sample of 1,154 HIV-1 envelope sequences. Our method greatly reduces the number of false positives due to founder effects, while capturing several higher-order interactions among V3 residues. By mapping these interactions to a structural model of the V3 loop, we find that the loop is stratified into distinct evolutionary clusters. We extend our model to detect interactions between the V3 and C4 domains of the HIV-1 envelope, and account for the uncertainty in mapping substitutions to the tree with a parametric bootstrap.