The protein import pore Tom40 in the microsporidian Nosema bombycis

Microsporidia, an unusual group of unicellular parasites related to fungi, possess a highly reduced mitochondrion known as the mitosome. Since mitosomes lack an organellar genome, their proteins must be translated in the cytosol before being imported into the mitosome via translocases. We have identified a Tom40 gene (NbTom40), the main component of the translocase of the outer mitochondrial membrane, in the genome of the microsporidian Nosema bombycis. NbTom40 is reduced in size, but it is predicted to form a β-barrel structure composed of 19 β-strands. Phylogenetic analysis confirms that NbTom40 forms a clade with Tom40 sequences from other species, distinct from a related clade of voltage-dependent anion channels (VDACs). The NbTom40 contains a β-signal motif that the polar residue is substituted by glycine. Furthermore, we show that expression of NbTom40, as a GFP fusion protein within yeast cells, directs GFP to mitochondria of yeast. These findings suggest that NbTom40 may serve as an import channel of the microsporidian mitosome and facilitate protein translocation into this organelle.     

Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line

Background

The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions.

Methodology

Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot.

Principal Findings

The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence.

Conclusions

We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.     

Excitability decreasing central motor plasticity is retained in multiple sclerosis patients

ABSTRACT: BACKGROUND: Compensation of brain injury in multiple sclerosis (MS) may in part work through mechanisms involving neuronal plasticity on local and interregional scales. Mechanisms limiting excessive neuronal activity may have special significance for retention and (re-)acquisition of lost motor skills in brain injury. However, previous neurophysiological studies of plasticity in MS have investigated only excitability enhancing plasticity and results from neuroimaging are ambiguous. Thus, the aim of this study was to probe long-term depression-like central motor plasticity utilizing continuous theta-burst stimulation (cTBS), a non-invasive brain stimulation protocol. Because cTBS also may trigger behavioral effects through local interference with neuronal circuits, this approach also permitted investigating the functional role of the primary motor cortex (M1) in force control in patients with MS. METHODS: We used cTBS and force recordings to examine long-term depression-like central motor plasticity and behavioral consequences of a M1 lesion in 14 patients with stable mild-to-moderate MS (median EDSS 1.5, range 0 to 3.5) and 14 age-matched healthy controls. cTBS consisted of bursts (50 Hz) of three subthreshold biphasic magnetic stimuli repeated at 5 Hz for 40 s over the hand area of the left M1. Corticospinal excitability was probed via motor-evoked potentials (MEP) in the abductor pollicis brevis muscle over M1 before and after cTBS. Force production performance was assessed in an isometric right thumb abduction task by recording the number of hits into a predefined force window. RESULTS: cTBS reduced MEP amplitudes in the contralateral abductor pollicis brevis muscle to a comparable extent in control subjects (69 +/- 22 % of baseline amplitude, p < 0.001) and in MS patients (69 +/- 18 %, p < 0.001). In contrast, post-cTBS force production performance was only impaired in controls (2.2 +/- 2.8, p = 0.011), but not in MS patients (2.0 +/- 4.4, p = 0.108). The decline in force production performance following cTBS correlated with corticomuscular latencies (CML) in MS patients, but did not correlate with MEP amplitude reduction in patients or controls. CONCLUSIONS: Long-term depression-like plasticity remains largely intact in mild-to-moderate MS. Increasing brain injury may render the neuronal networks less responsive toward lesion-induction by cTBS.     

Early Priming Minimizes the Age-Related Immune Compromise of CD8+ T Cell Diversity and Function

The elderly are particularly susceptible to influenza A virus infections, with increased occurrence, disease severity and reduced vaccine efficacy attributed to declining immunity. Experimentally, the age-dependent decline in influenza-specific CD8⁺ T cell responsiveness reflects both functional compromise and the emergence of ‘repertoire holes’ arising from the loss of low frequency clonotypes. In this study, we asked whether early priming limits the time-related attrition of immune competence. Though primary responses in aged mice were compromised, animals vaccinated at 6 weeks then challenged >20 months later had T-cell responses that were normal in magnitude. Both functional quality and the persistence of ‘preferred’ TCR clonotypes that expand in a characteristic immunodominance hierarchy were maintained following early priming. Similar to the early priming, vaccination at 22 months followed by challenge retained a response magnitude equivalent to young mice. However, late priming resulted in reduced TCRβ diversity in comparison with vaccination earlier in life. Thus, early priming was critical to maintaining individual and population-wide TCRβ diversity. In summary, early exposure leads to the long-term maintenance of memory T cells and thus preserves optimal, influenza-specific CD8⁺ T-cell responsiveness and protects against the age-related attrition of naïve T-cell precursors. Our study supports development of vaccines that prime CD8⁺ T-cells early in life to elicit the broadest possible spectrum of CD8⁺ T-cell memory and preserve the magnitude, functionality and TCR usage of responding populations. In addition, our study provides the most comprehensive analysis of the aged (primary, secondary primed-early and secondary primed-late) TCR repertoires published to date.     

Beclin 1 knockdown inhibits autophagic activation and prevents the secondary neurodegenerative damage in the ipsilateral thalamus following focal cerebral infarction

Cerebral infarction can cause secondary degeneration of thalamus and delay functional recovery. However, the mechanisms underlying secondary degeneration are unclear. The present study aimed to determine the occurrence and contribution of autophagy to the thalamic degeneration after cerebral infarction. Focal cerebral infarction was induced by distal middle cerebral artery occlusion (MCAO). Autophagic activation, Beclin 1 expression and amyloid β (Aβ) deposits were determined by immunofluorescence, immunoblot and electron microscopy. Secondary damage to thalamus was assessed with Nissl staining and immunofluorescence analysis. Apoptosis was determined using TUNEL staining. The contribution of autophagy to the secondary damage was evaluated by shRNA-mediated downregulation of Beclin 1 and the autophagic inhibitor, 3-methyladenine (3-MA). The potential role of Aβ in autophagic activation was determined with N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). The results showed that the conversion of LC3-II, the formation of autophagosomes, and the levels of activated cathepsin B and Beclin 1 were significantly increased in the ipsilateral thalamus at 7 and 14 days after MCAO (p < 0.05 or 0.01). Both Beclin 1 knockdown and 3-MA treatment significantly reduced LC3-II conversion and autophagosome formation, which were accompanied by obvious decreases in neuronal loss, gliosis and apoptosis in the ipsilateral thalamus (p < 0.05 or 0.01). Additionally, DAPT treatment markedly reduced Aβ deposits, which coincided with decreases in LC3-II conversion and autophagosome formation (p < 0.01). These results suggest that inhibition of autophagy by Beclin 1 knockdown can attenuate the secondary thalamic damage after focal cerebral infarction. Furthermore, Aβ deposits may be involved in the activation of autophagy.     

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

Background

Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.

Methodology/Principal Findings

Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.

Conclusions/Significance

The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.     

The antiapoptotic herpes simplex virus glycoprotein J localizes to multiple cellular organelles and induces reactive oxygen species formation

The Us5 gene of herpes simplex virus (HSV) encodes glycoprotein J (gJ). The only previously reported function of gJ was its ability to inhibit apoptosis. However, the mechanism by which gJ prevents apoptosis is not understood, and it is not known whether gJ mediates additional cellular effects. In this study, we evaluated the expression, localization, and cellular effects of Us5/gJ. Us5 was first expressed 4 h after infection. gJ was detectable at 6 h and was expressed in glycosylated and unglycosylated forms. Us5 was regulated as a late gene, with partial dependency on DNA replication for expression. Us5 expression was delayed in the absence of ICP22; furthermore, expression of Us5 in trans protected cells from apoptosis induced by an HSV mutant with deletion of ICP27, suggesting that the antiapoptotic effects of ICP22 and ICP27 are mediated in part through effects on gJ expression. Within HSV-infected or Us5-transfected cells, gJ was distributed widely, especially to the endoplasmic reticulum, trans-Golgi network, and early endosomes. gJ interacted with F_oF₁ ATP synthase subunit 6 by a yeast two-hybrid screen and had strong antiapoptotic effects, which were mediated by protein rather than mRNA. Antiapoptotic activity required the extracellular and transmembrane domains of gJ, but not the intracellular domain. Consistent with inhibition of F_oF₁ ATP synthase function, Us5 was required for HSV-induced reactive oxygen species (ROS) formation, and gJ was sufficient to induce ROS in Us5-transfected cells. Thus, HSV gJ is a multifunctional protein, modulating other cellular processes in addition to inhibition of apoptosis.     

Diverse Tetracycline Resistant Bacteria and Resistance Genes from Coastal Waters of Jiaozhou Bay

Environmental microbiology investigation was carried out in Jiaozhou Bay to determine the source and distribution of tetracycline-resistant bacteria and their resistance mechanisms. At least 25 species or the equivalent molecular phylogenetic taxa in 16 genera of resistant bacteria could be identified based on 16S ribosomal deoxyribonucleic acid sequence analysis. Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae constituted the majority of the typical resistant isolates. Indigenous estuarine and marine Halomonadaceae, Pseudoalteromonadaceae, Rhodobacteraceae, and Shewanellaceae bacteria also harbored tetracycline resistance. All the six resistance determinants screened, tet(A)–(E) and tet(G), could be detected, and the predominant genes were tet(A), tet(B), and tet(G). Both anthropogenic activity-related and indigenous estuarine or coastal bacteria might contribute to the tet gene reservoir, and resistant bacteria and their molecular determinants may serve as bioindicators of coastal environmental quality. Our work probably is the first identification of tet(E) in Proteus, tet(G) in Acinetobacter, tet(C) and tet(D) in Halomonas, tet(D) and tet(G) in Shewanella, and tet(B), tet(C), tet(E), and tet(G) in Roseobacter. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ndel1 promotes axon regeneration via intermediate filaments

Failure of axons to regenerate following acute or chronic neuronal injury is attributed to both the inhibitory glial environment and deficient intrinsic ability to re-grow. However, the underlying mechanisms of the latter remain unclear. In this study, we have investigated the role of the mammalian homologue of aspergillus nidulans NudE, Ndel1, emergently viewed as an integrator of the cytoskeleton, in axon regeneration. Ndel1 was synthesized de novo and upregulated in crushed and transected sciatic nerve axons, and, upon injury, was strongly associated with neuronal form of the intermediate filament (IF) Vimentin while dissociating from the mature neuronal IF (Neurofilament) light chain NF-L. Consistent with a role for Ndel1 in the conditioning lesion-induced neurite outgrowth of Dorsal Root Ganglion (DRG) neurons, the long lasting in vivo formation of the neuronal Ndel1/Vimentin complex was associated with robust axon regeneration. Furthermore, local silencing of Ndel1 in transected axons by siRNA severely reduced the extent of regeneration in vivo. Thus, Ndel1 promotes axonal regeneration; activating this endogenous repair mechanism may enhance neuroregeneration during acute and chronic axonal degeneration.