Broad-spectrum antimicrobial activity of hemoglobin

While hemoglobin is one of the most well characterized proteins due to its function in oxygen transport, few additional properties of hemoglobin have been described. While screening serum samples for novel antimicrobial factors, it was found that intact hemoglobin tetramers, including that from human, exhibited considerable activity against gram-positive and gram-negative bacteria, and fungi. To further characterize this surprising activity, the antimicrobial potency of sections of human hemoglobin was tested against a panel of microorganisms. In all cases separate testing of the alpha and beta subunits provided activity at least as potent as the intact tetramer. This activity is derived from the protein portion of hemoglobin since removal of the heme prosthetic group did not lead to decreases in potency. In addition, cyanogen bromide cleavage of both subunits provided fragments that still contained substantial antimicrobial activity. It has been possible to map specific regions of the human hemoglobin molecule that are responsible for significant antimicrobial activity. The carboxyl terminal thirty amino acids of the beta subunit, which form a cationic alpha-helix based on the crystal structure of the intact tetramer, were active against Escherichia coli, Staphylococcus aureus and Candida albicans. In view of the fact that different hemoglobin-derived peptide fragments exhibit diverse antibiotic activities, it is conceivable that, in addition to its role in oxygen transport. hemoglobin functions as an important multi-defense agent against a wide range of microorganisms.     

Evidence that the kinesin light chain domain contains tetratricopeptide repeat units

Homology models were built for various length sequences of the kinesin-1 light chain (KLC) domain of Drosophila melanogaster and subjected to 200 ns of all-atom molecular dynamics. We also cloned, expressed and characterized these regions spectroscopically. Results confirm that KLC contains tetratricopeptide repeat units; a regular array of repeating 34-residue helix-loop-helix monomers. Experimental and computational evidence is provided confirming the stability and overall helicity of individual TPR repeats as well as individual TPR units incorporated into a multi-TPR structure.     

Instability of the acetamide-inducible expression vector pJAM2 in Mycobacterium tuberculosis

The Escherichia coli-mycobacterium shuttle vector pJAM2 has been used to inducibly express genes in mycobacteria. The vector carries the promoter region from the highly inducible acetamidase gene of Mycobacterium smegmatis which is used to drive expression of heterologous genes. We used pJAM2 to over-express the Mycobacterium tuberculosis gene Rv2868c, a homologue of gcpE. In M. smegmatis the plasmid was stable, but the promoter region was readily deleted when the parental vector or recombinant plasmids were transformed into M. tuberculosis. We mapped the deletion by sequencing and found that it encompassed the entire acetamidase promoter and adjacent sequence totalling approximately 7.3 kb and occurred very soon after introduction into M. tuberculosis. This is the first report of instability of a vector carrying the acetamidase promoter in M. tuberculosis.     

Too dangerous to ignore: self-tolerance and the control of ignorant autoreactive T cells

Self-reactive T cells often escape thymic negative selection and are released into the periphery. While many of these T cells are tolerized by peripheral deletion or anergy, a proportion persists in a na?ve (or ignorant) state. Self-ignorant T cells are probably one of the greatest threats to the maintenance of self-tolerance, as inadvertent activation of these cells may provoke autoimmune pathology. Nevertheless, despite the presence of self-ignorant T cells within most individuals, the majority of people fail to develop autoimmunity. The means by which self-ignorant T cells are silenced by the immune system remains a major issue within the tolerance field and it has received surprisingly little attention within the literature. In this review, we first summarize the factors that allow such cells to persist in a self-ignorant state, with a particular focus on the role of self-antigen dose. We next consider the conditions under which such self-reactive cells may become activated and speculate on how the immune system is able to prevent such cells from precipitating autoimmune disease.     

Automatically extracting functionally equivalent proteins from SwissProt

Background  

There is a frequent need to obtain sets of functionally equivalent homologous proteins (FEPs) from different species. While it is usually the case that orthology implies functional equivalence, this is not always true; therefore datasets of orthologous proteins are not appropriate. The information relevant to extracting FEPs is contained in databanks such as UniProtKB/Swiss-Prot and a manual analysis of these data allow FEPs to be extracted on a one-off basis. However there has been no resource allowing the easy, automatic extraction of groups of FEPs – for example, all instances of protein C.     

Protein chaperones Q8ZP25_SALTY from Salmonella typhimurium and HYAE_ECOLI from Escherichia coli exhibit thioredoxin-like structures despite lack of canonical thioredoxin active site sequence motif

The structure of the 142-residue protein Q8ZP25_SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE_ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247–D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the ‘thioredoxin-like clan’. However, protein HYAE_ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE_ECOLI was previously classified as a [NiFe] hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.     

Inhibition of hepatic sterol synthesis and reduction of serum cholesterol in rats by 5α-cholest-8(14)-en-3β-ol-15-one

The preparation of 5α-cholest-8(14)-en-3β-ol-15-one from 3β-benzoyloxy-5α-cholest-8(14)-en-15-one is described herein. Subcutaneous administration of the former compound (2 mg per day for 15 days) resulted in a significant depression of the incorporation of the label of [2-¹⁴C]-acetate, but not of [2-¹⁴C]-3RS-mevalonate, into digitonin-precipitable sterols in rat liver homogenate preparations. Subcutaneous administration of the inhibitor, 2 mg per day or 5 mg per day, for 3 days resulted in a 12% and 22% reduction of serum cholesterol levels, respectively.     

Development and characterization of highly polymorphic long TC repeat microsatellite markers for genetic analysis of peanut

Background

The immune system has paradoxical roles during cancer development and the prognostic significance of immune modulating factors is controversial. The aim of this study was to determine the expression of cyclooxygenase 2 (COX-2), transforming growth factor-beta (TGF- beta), interleukin-10 (IL-10) and their prognostic significance in breast cancers. Ki67 was included as a measure of growth fraction of tumor cells.

Methods

On immunohistochemical stained slides from 38 breast cancer patients, we performed digital video analysis of tumor cell areas and adjacent tumor stromal areas from the primary tumors and their corresponding lymph node metastases. COX-2 was recorded as graded staining intensity.

Results

The expression of TGF-beta, IL-10 and Ki67 were recorded in tumor cell areas and adjacent tumor stromal areas. In both primary tumors and metastases, the expression of COX-2 was higher in the tumor stromal areas than in the tumor cell areas (both P < 0.001). High stromal staining intensity in the primary tumors was associated with a 3.9 (95% CI 1.1-14.2) times higher risk of death compared to the low staining group (P = 0.036). The expression of TGF-beta was highest in the tumor cell areas of both primary tumors and metastases (both P < 0.001). High stromal expression of TGF-beta was associated with increased mortality. For IL-10, the stromal expression was highest in the primary tumors (P < 0.001), whereas in the metastases the expression was highest in tumor cell areas (P < 0.001). High IL-10 expression in tumor- and stromal cell areas of primary tumors predicted mortality. Ki67 was higher expressed in tumor stromal areas of the metastases, and in tumor cell areas of the primary tumors (P < 0.001). Ki67 expression in tumor cell areas and stromal areas of the metastases was independently associated with breast cancer mortality.

Conclusions

Stromal expression of COX-2, TGF-beta and Ki67 may facilitate tumor progression in breast cancer.     

Methidiumpropyl-EDTA-iron(II) cleavage of ribosomal DNA chromatin from Dictyostelium discoideum.

We have used methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)] in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dictyostelium. Confirming our earlier results with micrococcal nuclease (1,2), MPE.Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced. In the central non-transcribed spacer region of the palindrome, MPE.Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern. The difference is attributed to the lower sequence specificity of MPE.Fe(II) compared to micrococcal nuclease. In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern. In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence.     

Direct Imaging of Cl‐ and Cu‐Induced Short‐Circuit Efficiency Changes in CdTe Solar Cells

To achieve high‐efficiency polycrystalline CdTe‐based thin‐film solar cells, the CdTe absorbers must go through a post‐deposition CdCl₂ heat treatment followed by a Cu diffusion step. To better understand the roles of each treatment with regard to improving grains, grain boundaries, and interfaces, CdTe solar cells with and without Cu diffusion and CdCl₂ heat treatments are investigated using cross‐sectional electron beam induced current, electron backscatter diffraction, and scanning transmission electron microscope techniques. The evolution of the cross‐sectional carrier collection profile due to these treatments that cause an increase in short‐circuit current and higher open‐circuit voltage are identified. Additionally, an increased carrier collection in grain boundaries after either/both of these treatments is revealed. The increased current at the grain boundaries is shown to be due to the presence of a space charge region with an intrinsic carrier collection profile width of ≈350 nm. Scanning transmission electron microscope electron‐energy loss spectroscopy shows a decreased Te and increased Cl concentration in grain boundaries after treatment, which causes the inversion. Each treatment improves the overall carrier collection efficiency of the cell separately, and, therefore, the benefits realized by each treatment are shown to be independent of each other.