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     

Histochemical Identification of Basic Proteins with Biebrich Scarlet at Alkaline pH

Staining at graded alkaline pH levels with the sulfonated dye, Biebrich scarlet, shows basic proteins in various histologic sites, and differentiates the sites according to their relative basicity. Certain structures stain at pH 6.0 but not at 8.0 or above. Others stain maximally up to pH 93 and a few stain strongly as high as pH 103. The most strongly basic sites resist more than the others the destruction of acidophilia by nitrosation, acetylation or exposure to formaldehyde.     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

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.     

Identification of the RNA binding domain of T4 RegA protein by structure-based mutagenesis.

The T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H. , Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170-1173). Domain I of the RegA protein contains a four-stranded beta-sheet and two alpha-helices. Domain II contains a four-stranded beta-sheet and an unusual 3/10 helix. Since beta-sheet residues play a role in a number of protein-RNA interactions, one or both of the beta-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both beta-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of beta-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an alpha-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties.     

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

Trastuzumab (Herceptin), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.