Mitochondrial DNA divergence in the critically imperilled pygmy madtom,Noturus stanauli (Siluriformes: Ictaluridae)

The mitochondrial cytochrome b gene was sequenced for six individuals of the pygmy madtom, Noturus stanauli, a globally imperilled catfish, from both known localities in Tennessee (U.S.A.) separated by over 1055 river km. Phylogenetic and population genetic analyses revealed little divergence between these populations for this locus.     

Periplasmic Domains of Pseudomonas aeruginosa PilN and PilO Form a Stable Heterodimeric Complex

Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 Å resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.     

NDUFS4: Creation of a mouse model mimicking a Complex I disorder

The Complex I NADH dehydrogenase–ubiquinone–FeS 4 (NDUFS4) subunit gene is involved in proper Complex I function such that the loss of NDUFS4 decreases Complex I activity resulting in mitochondrial disease. Therefore, a mouse model harboring a point mutation in the NDUFS4 gene was created. An embryonic lethal phenotype was observed in homozygous (NDUFS4^?/?) mutant fetuses. Mitochondrial function was impaired in heterozygous animals based on oxygen consumption, and Complex I activity in NDUFS4 mouse mitochondria. Decreased Complex I activity with unaltered Complex II activity, along with an accumulation of lactate, were consistent with Complex I disorders in this mouse model.     

Characterization of a clinical polymer‐drug conjugate using multiscale modeling

The molecular conformation of certain therapeutic agents has been shown to affect the ability to gain access to target cells, suggesting potential value in defining conformation of candidate molecules. This study explores how the shape and size of poly‐γ‐glutamyl‐glutamate paclitaxel (PGG‐PTX), an amphiphilic polymer‐drug with potential chemotherapeutic applications, can be systematically controlled by varying hydrophobic and hydrophilic entities. Eighteen different formulations of PGG‐PTX varying in three PTX loading fractions (f_PTX) of 0.18, 0.24, and 0.37 and six spatial arrangements of PTX (‘clusters’, ‘ends’, ‘even’, ‘middle’, ‘random’, and ‘side’) were explored. Molecular dynamics (MD) simulations of all‐atom (AA) models of PGG‐PTX were run until a statistical equilibrium was reached at 100 ns and then continued as coarse‐grained (CG) models until a statistical equilibrium was reached at an effective time of 800 ns. Circular dichroism spectroscopy was used to suggest initial modeling configurations. Results show that a PGG‐PTX molecule has a strong tendency to form coil shapes, regardless of the PTX loading fraction and spatial PTX arrangement, although globular shapes exist at f_PTX = 0.24. Also, less uniform PTX arrangements such as ‘ends’, ‘middle’, and ‘side’ produce coil geometries with more curvature. The prominence of coil shapes over globules suggests that PGG‐PTX may confer a long circulation half‐life and high propensity for accumulation to tumor endothelia. This multiscale modeling approach may be advantageous for the design of cancer therapeutic delivery systems. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 936–951, 2010.     

Constitutive Endocytic Recycling and Protein Kinase C-mediated Lysosomal Degradation Control KATP Channel Surface Density

Pancreatic ATP-sensitive potassium (K_ATP) channels control insulin secretion by coupling the excitability of the pancreatic β-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed K_ATP channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that K_ATP channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases K_ATP channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic β-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native K_ATP channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic β-cells, we propose that PKC-regulated K_ATP channel trafficking may play a role in the regulation of insulin secretion.     

DmsD,a Tat system specific chaperone,interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein–protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a “node” in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.     

Epidemiologic Investigation of Immune-Mediated Polyradiculoneuropathy among Abattoir Workers Exposed to Porcine Brain

Background

In October 2007, a cluster of patients experiencing a novel polyradiculoneuropathy was identified at a pork abattoir (Plant A). Patients worked in the primary carcass processing area (warm room); the majority processed severed heads (head-table). An investigation was initiated to determine risk factors for illness.

Methods and Results

Symptoms of the reported patients were unlike previously described occupational associated illnesses. A case-control study was conducted at Plant A. A case was defined as evidence of symptoms of peripheral neuropathy and compatible electrodiagnostic testing in a pork abattoir worker. Two control groups were used - randomly selected non-ill warm-room workers (n = 49), and all non-ill head-table workers (n = 56). Consenting cases and controls were interviewed and blood and throat swabs were collected. The 26 largest U.S. pork abattoirs were surveyed to identify additional cases. Fifteen cases were identified at Plant A; illness onsets occurred during May 2004–November 2007. Median age was 32 years (range, 21–55 years). Cases were more likely than warm-room controls to have ever worked at the head-table (adjusted odds ratio [AOR], 6.6; 95% confidence interval [CI], 1.6–26.7), removed brains or removed muscle from the backs of heads (AOR, 10.3; 95% CI, 1.5–68.5), and worked within 0–10 feet of the brain removal operation (AOR, 9.9; 95% CI, 1.2–80.0). Associations remained when comparing head-table cases and head-table controls. Workers removed brains by using compressed air that liquefied brain and generated aerosolized droplets, exposing themselves and nearby workers. Eight additional cases were identified in the only two other abattoirs using this technique. The three abattoirs that used this technique have stopped brain removal, and no new cases have been reported after 24 months of follow up. Cases compared to controls had higher median interferon-gamma (IFNγ) levels (21.7 pg/ml; vs 14.8 pg/ml, P<0.001).

Discussion

This novel polyradiculoneuropathy was associated with removing porcine brains with compressed air. An autoimmune mechanism is supported by higher levels of IFNγ in cases than in controls consistent with other immune mediated illnesses occurring in association with neural tissue exposure. Abattoirs should not use compressed air to remove brains and should avoid procedures that aerosolize CNS tissue. This outbreak highlights the potential for respiratory or mucosal exposure to cause an immune-mediated illness in an occupational setting.     

Α-galactosylceramide analogs with weak agonist activity for human iNKT cells define new candidate anti-inflammatory agents

CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.