An altered zinc-binding site confers resistance to a covalent inactivator of New Delhi metallo-beta-lactamase-1 (NDM-1) discovered by high-throughput screening

Due to the global threat of antibiotic resistance mediated by New Delhi metallo-beta-lactamase-1 (NDM-1) and the lack of structurally diverse inhibitors reported for this enzyme, we developed screening and counter-screening assays for manual and automated formats. The manual assay is a trans-well absorbance-based endpoint assay in 96-well plates and has a Z′ factor of 0.8. The automated assay is an epi-absorbance endpoint assay in 384-well plates, has a Z′ factor of ?0.8, good signal/baseline ratios (>3.8), and is likely scalable for high-throughput screening (HTS). A TEM-1-based counter-screen is also presented to eliminate false positives due to assay interference or off-target activities. A pilot screen of a pharmacologically characterized compound library identified two thiol-modifying compounds as authentic NDM-1 inhibitors: p-chloromecuribenzoate (p-CMB) and nitroprusside. Recombinant NDM-1 has one Cys residue that serves as a conserved active-site primary zinc ligand and is selectively modified by p-CMB as confirmed by LC–MS/MS. However a C208D mutation results in an enzyme that maintains almost full lactamase activity, yet is completely resistant to the inhibitor. These results predict that covalent targeting of the conserved active-site Cys residue may have drawbacks as a drug design strategy.     

Ultrastructural carbohydrate cytochemistry of gastric epithelium

Synopsis On examination with ultrastructural methods for visualizing thevicinal glycols and acid groups of complex carbohydrates, the most superficial surface epithelium of the rat gastric corpus displayed biphasic mucous droplets consisting of a cortex of hexose-rich (i.e. periodate-reactive) neutral mucosubstance and an uncharacterized denser core plus monophasic droplets with the neutral mucosubstance. In many surface epithelial cells of the foveolae, the biphasic and monophasic droplets with the neutral mucosubstance intermingled in varying proportions with monophasic droplets showing uniform periodate reactivity, a variable degree of dialyzed ironbinding—demonstrative of acidic glycoconjugate, and high iron—diamine affinity—demonstrative of sulphomucin. Deep foveolar epithelium displayed only monophasic droplets, most of which contained acidic periodate-reactive complex carbohydrate. Underiying cells, designated isthmus cells, exhibited monophasic or occasional biphasic granules containing sulphated, hexose-rich mucosubstance. Nascent droplets or granules near the Golgi zone differed from the mature organelles in the distribution of the glycoconjugate. Mucous neck cells occupied a deeper stratum and displayed a uniform population of monophasic mucous droplets with a loose meshwork of neutral mucosubstance.Techniques for demonstrating hexoses ultrastructurally stained all Golgi cisternae in the mucigenic epithelium, showing increasing reactivity toward the maturing face. Distinctive cistemae with moderate reactivity in the Golgi complex of isthmus cells were interpreted as GERL. Acidic mucosubstances were visualized only in the inner, mature cisternae of the Golgi complex of cells storing acidic glycoconjugates, and not in cisternae interpretable as GERL.The apical plasmalemma of isthmus cells uniquely exhibited abundant sulphated glycoconjugate and that of parietal cells revealed a less prominent, periodic neutral mucosubstance. Lateral and basal plasmalemmae varied from unstained to slightly reactive; basement membranes showed moderate reactivity with methods for visualizing complex carbohydrates. Abundance of glycogen further characterized surface epithelial cells of the corpus and of some parietal cells     

Drinking, Conduct Disorder, and Social Change: Navajo Experiences

Drinking, Conduct Disorder, and Social Change: Navajo Experiences. Stephen J. Kunitz and Jerrold E. Levy. Oxford: Oxford University Press, 2000. xiii. 262 pp.     

Insulin-like growth factor (IGF) 2 stimulates steroidogenesis and mitosis of bovine granulosa cells through the IGF1 receptor: role of follicle-stimulating hormone and IGF2 receptor

Little is known regarding the role of insulin-like growth factor 2 (IGF2) and the regulation of the IGF2 receptor (IGF2R) during follicular development. Granulosa cells were collected from small (1-5 mm) and large (8-22 mm) bovine follicles and were treated with IGF2 for 1-2 days in serum-free medium, and steroid production, cell proliferation, specific (125)I-IGF2 binding, and gene expression were quantified. IGF2 increased both estradiol and progesterone production by granulosa cells, and cells from large follicles were more responsive to the effects of IGF2 than those from small follicles. Abundance of aromatase (CYP19A1) mRNA was stimulated by IGF2 and IGF1. The effective dose (ED(50)) of IGF2 stimulating 50% of the maximal estradiol production was 63 ng/ml for small follicles and 12 ng/ml for large follicles, and these values were not affected by FSH. The ED(50) of IGF2 for progesterone production was 20 ng/ml for both small and large follicles. IGF2 also increased proliferation of granulosa cells by 2- to 3-fold, as determined by increased cell numbers and (3)H-thymidine incorporation into DNA. Treatment with IGF1R antibodies reduced the stimulatory effect of IGF2 and IGF1 on estradiol production and cell proliferation. Specific receptors for (125)I-IGF2 existed in granulosa cells, and 2-day treatment with estradiol, FSH, or cortisol had no significant effect on specific (125)I-IGF2 binding. Also, FSH treatment of small- and large-follicle granulosa cells had no effect on IGF2R mRNA levels, whereas IGF1 decreased IGF2R mRNA and specific (125)I-IGF2 binding. Granulosa cell IGF2R mRNA abundance was 3-fold greater in small than in large follicles. These findings support the hypothesis that both IGF2 and its receptor may play a role in granulosa cell function during follicular development. In particular, increased free IGF1 in developing follicles may decrease synthesis of IGF2R, thereby allowing for more IGF2 to be bioavailable (free) for induction of steroidogenesis and mitogenesis via the IGF1R.     

EVOLUTION OF MULTIPLE KINDS OF FEMALE SPERM-STORAGE ORGANS IN DROSOPHILA

Females of all species belonging to the family Drosophilidae have two kinds of sperm-storage organs: paired spherical spermathecae and a single elongate tubular seminal receptacle. We examined 113 species belonging to the genus Drosophila and closely allied genera and describe variation in female sperm-storage organ use and morphology. The macroevolutionary pattern of organ dysfunction and morphological divergence suggests that ancestrally both kinds of organs stored sperm. Loss of use of the spermathecae has evolved at least 13 times; evolutionary regain of spermathecal function has rarely if ever occurred. Loss of use of the seminal receptacle has likely occurred only once; in this case, all descendant species possess unusually elaborate spermathecae. Data further indicate that the seminal receptacle is the primary sperm-storage organ in Drosophila. This organ exhibits a pattern of strong correlated evolution with the length of sperm. The evolution of multiple kinds of female sperm-storage organs and the rapidly divergent and correlated evolution of sperm and female reproductive tract morphology are discussed.