Drosophila protein kinase CK2 is rendered temperature-sensitive by mutations of highly conserved residues flanking the activation segment

CK2 is a Ser/Thr protein kinase essential for animal development. Although null alleles for CK2 are available in the mouse and Drosophila models, they are lethal when homozygous, thus necessitating conditional alleles for analysis of its developmental roles. We describe the isolation of temperature-sensitive (ts) alleles of Drosophila CK2α (dCK2α). These alleles efficiently rescue lethality of yeast lacking endogenous CK2 at 29°C, but this ability is lost at higher temperatures in an allele-specific manner. These ts-variants exhibit properties akin to the wild type protein, and interact robustly with dCK2β. Modeling of these ts-variants using the crystal structure of human CK2α indicates that the affected residues are in close proximity to the active site. We find that substitution of Asp²¹² elicits potent ts-behavior, an important finding because this residue contributes to stability of the activation segment and is invariant in other Ser/Thr protein kinases.     

The effect of oxygenated mycolic acid composition on cell wall function and macrophage growth in Mycobacterium tuberculosis

There are three major structural classes of mycolic acids in the cell envelope of Mycobacterium tuberculosis (MTB): alpha-, methoxy- and ketomycolate. The two oxygen-containing classes are biosynthetically related through a common α-methyl hydroxymycolate intermediate. BCG strains that fail to produce methoxymycolate and instead produce only keto- and alpha-mycolic acids show apparent defects in the O -methyltransferase MMAS-3. Overproduction of MMAS-3 from MTB resulted in a complete replacement of ketomycolate by methoxymycolate in both BCG and MTB. In vitro growth of these recombinant strains lacking ketomycolate was impaired at reduced temperatures but appeared to be normal at 37°C. Glucose uptake was significantly decreased in such strains, but uptake of chenodeoxycholate and glycine was unaffected. Although sensitivity to INH remained unchanged, these cells were found to be hypersensitive to ampicillin and rifampicin. Infectivity of BCG and H37Rv wild type or MMAS-3 overproducers in THP-1 cells was somewhat affected, but the ability of the strains lacking ketomycolate to grow within this macrophage-like cell line was severely compromised. In vivo labelling of mycolic acids during growth of H37Rv within THP-1 cells revealed a substantial increase in ketomycolate and alphamycolate synthesized by intracellularly grown mycobacteria. These results establish a critical role for mycolate composition in proper cell wall function during the growth of MTB in vivo .     

HISTOLOGICAL EFFECTS OF AQUEOUS ACIDS AND GASEOUS HYDROGEN CHLORIDE ON BEAN LEAVES

Primary leaves of Phaseolus vulgaris L. (pinto bean), 9 or 12 days from sowing, were exposed to aqueous acids, chloride salts, or hydrogen chloride gas. Leaves were examined for the presence and severity of resultant visible injury and sampled for light and scanning electron microscopy. Exposure to 0.06 n HCl, HNO₃, H₂SO₄ or 14.5–19.0 mg m^-3 gaseous HCl for 20 min evoked similar foliar injury including glazing and necrosis of the laminas. This injury appeared to result initially from plasmolysis and collapse of the epidermis and subsequently of the underlying mesophyll. Cellular injury was accompanied by various cytoplasmic alterations. Microscopic symptoms observed in leaves exposed to gaseous HCl or aqueous acids included vesicles and particulates within the larger vacuoles. Similar symptoms were present in leaves exposed to polyethylene glycol 6000. Differential effects included formation of necrotic “pits” and preferential injury to paravascular tissues in leaves treated with aqueous acids and crystalline chloroplast inclusions in gaseous HCl-treated and water-stressed leaves. The visible and microscopic appearances of leaves exposed to aqueous acids or gaseous HCl were compared and related to injury stemming from acid precipitation and a possible mechanism of action for gaseous HCl phytotoxicity.     

Modified Methodology to Improve Flow Cytometric Dna Histograms from Paraffin-Embedded Material

Flow Cytometric (FCM) DNA content analysis of paraffin-embedded tissues has become a widely accepted procedure in assessing the biologic course in some tumors from archival material. Difficulty in interpreting histograms is frequently due to high levels of debris, and wide coefficients of variation (CV). These may lead to underestimating near-diploid abnormality. Although the clinical significance of low-degree aneuploidy has yet to be established, the procedure reported here improved our endeavor to detect DNA Indices (DI) of at least 1.1%. Experience has shown that careful technique can result in overall improvement of DNA histograms by lowering levels of debris and % CV, dramatically so in some cases. Archival histograms can be generated that rival in quality fresh tissue results. Archival material is currently employed for diagnostic and research purposes.