Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

GC-1 mRHBDD1 knockdown spermatogonia cells lose their spermatogenic capacity in mouse seminiferous tubules

Background  

Apoptosis is important for regulating spermatogenesis. The protein mRHBDD1 (mouse homolog of human RHBDD1)/rRHBDD1 (rat homolog of human RHBDD1) is highly expressed in the testis and is involved in apoptosis of spermatogonia. GC-1, a spermatogonia cell line, has the capacity to differentiate into spermatids within the seminiferous tubules. We constructed mRHBDD1 knockdown GC-1 cells and evaluated their capacity to differentiate into spermatids in mouse seminiferous tubules.     

The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.     

The cDNA cloning and molecular evolution of reptile and pigeon lactate dehydrogenase isozymes

The cDNAs encoding lactate dehydrogenase isozymes LDH-A (muscle) and LDH-B (heart) from alligator and turtle and LDH-A, LDH-B, and LDH-C (testis) from pigeon were cloned and sequenced. The evolutionary relationships among vertebrate LDH isozymes were analyzed. Contrary to the traditional belief that the turtle lineage branched off before the divergence between the lizard/alligator and bird lineages, the turtle lineage was found to be clustered with either the alligator lineage or the alligator-bird clade, while the lizard lineage was found to have branched off before the divergence between the alligator/turtle and bird lineages. The pigeon testicular LDH-C isozyme was evidently duplicated from LDH-B (heart), so it is not orthologous to the mammalian testicular LDH-C isozymes.      

New insights into the pathological role of TNF-alpha in early cardiac dysfunction and subsequent heart failure after infarction in rats

A marked increase in plasma TNF-alpha has been described in patients with chronic heart failure (CHF). Nevertheless, little is known about the direct role of this cytokine early after myocardial infarction (MI) and its possible effects on the subsequent development of CHF. Wistar rats were subjected to permanent in vivo coronary artery ligation. At 5, 7, and 9 days after MI, cardiac function, passive compliance of the left ventricle (LV), and cardiac geometry were evaluated. The same model was used to perform pharmacological studies 7 days and 10 wk after MI in rats treated with monomeric recombinant human soluble TNF-alpha receptor type II (sTNF-RII, 40 microg/kg iv) or a placebo on day 3. Maximal alterations of cardiac function and geometry occurred 7 days after MI, which correlated chronologically with a peak of cardiac and serum TNF-alpha, as shown by immunohistochemistry and ELISA, respectively. sTNF-RII improved LV end-diastolic pressure under basal conditions and after volume overload 7 days and 10 wk after MI. Moreover, a significant leftward shift of the pressure-volume curve in the sTNF-RII-treated group 7 days after MI indicated a preservation of LV volume. Infarct expansion index was also significantly improved by sTNF-RII 7 days after MI (P < 0.01). Nevertheless, 10 wk after MI, geometric indexes and passive pressure-volume curves were not significantly improved by the treatment. In conclusion, TNF-alpha plays a major role in cardiac alterations 7 days after MI in rats and contributes to hemodynamic derangement, but not to cardiac remodeling, in subsequent CHF.     

Histamine receptor H1 in the nucleus tractus solitarii regulates arterial pressure and heart rate in rats

Axons of histamine (HA)-containing neurons are known to project from the posterior hypothalamus to many areas of the brain, including the nucleus tractus solitarii (NTS), a central brain structure that plays an important role in regulating arterial pressure. However, the functional significance of NTS HA is still not fully established. In this study, we microinjected HA or 2-pyridylethylamine, a HA-receptor H(1)-specific agonist, into the NTS of urethane-anesthetized Wister rats to identify the potential functions of NTS HA on cardiovascular regulation. When HA or H(1)-receptor-specific agonist was bilaterally microinjected into the NTS, mean arterial pressure (MAP) and heart rate (HR) were significantly increased, whereas pretreatment with the H(1)-receptor-specific antagonist cetirizine into the NTS significantly inhibited the cardiovascular responses. The maximal responses of MAP and HR changes induced by HA or H(1)-receptor-specific agonist were dose dependent. We also confirmed gene expression of HA receptors in the NTS and that the expression level of H(1) mRNA was higher than that of the other subtypes. In addition, we found that H(1) receptors are mainly expressed in neurons of the NTS. These findings suggested that HA within the NTS may play a role in regulating cardiovascular homeostasis via activation of H(1) receptors expressed in the NTS neurons.     

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.     

Impact of Anti-Peroxynitrite-Damaged-Thymidine- Monophosphate Antibodies on Disease Activity in Patients with Systemic Lupus Erythematosus

Present study probes the role of peroxynitrite (ONOO^-)-modified thymidine-5′-monophosphate (TMP) in SLE patients with different disease activity scores according to the SLE Disease Activity Index (SLEDAI). Serum analysis showed significant increased number of subjects positive for anti-ONOO^--TMP-protein antibodies in SLE patients with different SLEDAI scores. Interestingly, the levels of these antibodies were significantly higher among SLE patients, whose SLEDAI scores were ≥20. In addition, a significant correlation was observed between the levels of anti-ONOO^--TMP-protein antibodies and the SLEDAI score (r = 0.595, p < 0.0001). In short, this study shows a positive association between anti-ONOO^--TMP-protein antibodies and SLEDAI. The stronger response observed in patients with higher SLEDAI scores suggests that anti-ONOO^--TMP-protein antibodies may be useful in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.     

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [{"type":"entrez-nucleotide","attrs":{"text":"KF611679","term_id":"560187972","term_text":"KF611679"}}KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.