Does progressive nuclear staining with hemalum (alum hematoxylin) involve DNA,and what is the nature of the dye-chromatin complex?

Previous investigators have disagreed about whether hemalum stains DNA or its associated nucleoproteins. I review here the literature and describe new experiments in an attempt to resolve the controversy. Hemalum solutions, which contain aluminum ions and hematein, are routinely used to stain nuclei. A solution containing 16 Al³⁺ ions for each hematein molecule, at pH 2.0–2.5, provides selective progressive staining of chromatin without cytoplasmic or extracellular “background color.” Such solutions contain a red cationic dye-metal complex and an excess of Al³⁺ ions. The red complex is converted to an insoluble blue compound, assumed to be polymeric, but of undetermined composition, when stained sections are blued in water at pH 5.5–8.5. Staining experiments with DNA, histone and DNA + histone mixtures support the theory that DNA, not histone, is progressively colored by hemalum. Extraction of nucleic acids, by either a strong acid or nucleases at near neutral pH, prevented chromatin staining by a simple cationic dye, thionine, pH 4, and by hemalum, with pH adjustments in the range, 2.0–3.5. Staining by hemalum at pH 2.0–3.5 was not inhibited by methylation, which completely prevented staining by thionine at pH 4. Staining by hemalum and other dye-metal complexes at pH ≤ 2 may be due to the high acidity of DNA-phosphodiester (pK_a ~ 1). This argument does not explain the requirement for a much higher pH to stain DNA with those dyes and fluorochromes not used as dye-metal complexes. Sequential treatment of sections with Al₂(SO₄)₃ followed by hematein provides nuclear staining that is weaker than that attainable with hemalum. Stronger staining is seen if the pH is raised to 3.0–3.5, but there is also coloration of cytoplasm and other materials. These observations do not support the theory that Al³⁺ forms bridges between chromatin and hematein. When staining with hematein is followed by an Al₂(SO₄)₃ solution, there is no significant staining. Taken together, the results of my study indicate that the red hemalum cation is electrostatically attracted to the phosphate anion of DNA. The bulky complex cation is too large to intercalate between base pairs of DNA and is unlikely to fit into the minor groove. The short range van der Waals forces that bind planar dye cations to DNA probably do not contribute to the stability of progressive hemalum staining. The red cation is precipitated in situ as a blue compound, insoluble in water, ethanol and water-ethanol mixtures, when a stained preparation is blued at pH > 5.5.     

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network.     

Biologically relevant effects of mRNA amplification on gene expression profiles

Background  

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results.     

Comparative Genome Structure,Secondary Metabolite,and Effector Coding Capacity across Cochliobolus Pathogens

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP–encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.     

Description of Euroleptochromus gen.n. (Coleoptera,Staphylinidae, Scydmaeninae) from Baltic amber,with discussion of biogeography and mouthpart evolution within Clidicini

A new extinct species of the ant‐like stone beetle supertribe Mastigitae, Euroleptochromus sabathi gen. & sp.n. is described from Eocene Baltic amber. A phylogenetic analysis of Clidicini, with representatives of Leptomastacini and Mastigini as out‐group taxa, provided strong support for a sister‐group relationship between the Neotropical Leptochromus and the new genus. The monophyly of Clidicini is questioned because of an alternative placement of Nearctic Papusus as a sister taxon to Leptomastacini + [Clidicus + (Palaeoleptochromus + (Euroleptochromus + Leptochromus))]. A dispersal‐vicariance analysis provided three alternative scenarios for the evolution of Mastigitae; with Laurasia as the ancestral area of the supertribe, major branching events occurring within either Eurasia or Laurentia and two trans‐Beringia dispersals in Late Cretaceous and Eocene. Euroleptochromus, Palaeoleptochromus and Leptochromus share highly derived structures on postgenae and maxillary palps, probably as part of a specialised feeding or prey capture mechanism. The formation of these modifications in Clidicini is demonstrated to involve a process (traced back to the Campanian, 79 Ma) of elongation and narrowing of maxillary palps and forming a cuticular setal projection from a broadened insertion site of sensory setae.     

Effects of plant height and row spacing on kenaf forage potential with multiple harvests

Kenaf (Hibiscus cannabinus L.) forage potential can be enhanced through its regrowth capacity and higher production in narrow rows. A field experiment was conducted in Matamoros, Coahuila, Mexico, during 2 growing seasons (2004 and 2005) to study the effects of plant height and row spacing on kenaf forage potential with multiple harvests. This study evaluated the effects of (1) 2 plant heights at cutting (1.0-1.2 m and 1.8-2.0 m) and (2) 4 inter row spacings (0.19, 0.38, 0.57 and 0.76 m) using a 2 x 4 factorial arrangement of treatments in a completely randomized block design with 4 replications. Dry matter (DM) and crude protein (CP) yields, DM partitioning, neutral detergent fiber (NDF) and CP concentrations were determined. Heights at cutting × row spacing interactions were not significant for the monitored variables (p>0.05). Kenaf response to treatments was only relevant for main effects (p≤0.05). Row spacing and plant height affected DM and CP yields (p≤0.05), whereas only plant height affected chemical composition and DM partitioning (p≤0.05). Dry matter (17.0%-26.0%), and CP (12.4%-15.6%) yields were higher (p≤0.05) when plant heights had reached 1.8 to 2.0 m. Row spacing reduction from 0.76 m to 0.38 and 0.19 m increased DM yield (20.4-33.4%) and CP yield (24.2-38.5%) (p≤0.05). Kenaf forage potential increases when planted in narrow rows and harvested 2 or 3 times during the growing season.     

Statin-derived 1,3-oxazinan-2-ones as submicromolar inhibitors of LFA-1/ICAM-1 interaction: stabilization of the metabolically labile vanillyl side chain

Modification of the vanillyl substituent on a potent, semisynthetic lymphocyte function-associated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1 binding inhibitor of the statin family resulted in metabolically more stable analogues that displayed submicromolar inhibitory activity in vitro and considerable anti-inflammatory activity in vivo. The benzodioxole derivative 2b emerged with the best overall profile.