Immunohistochemical localization of Na+,K+-ATPase in rodent and human salivary and lacrimal glands

The enzyme Na+,K+-ATPase was localized immunohistochemically in major salivary glands of mouse, rat, and human and in exorbital lacrimal glands of the rodents. Immunoreactive Na+,K+-ATPase was abundant in the basolateral membranes of all epithelial cells lining striated and intra- and interlobular ducts of all glands. Reactivity of intercalated ducts varied among gland type and species. Cells lining granular ducts in rodent submandibular gland showed a heterogeneous staining pattern in rat but stained homogeneously in mouse. Secretory cells varied greatly in their content of immunoreactive Na+,K+-ATPase. As with all duct cells, staining was present only at the basolateral surface and was never observed at the luminal surface of reactive secretory cells. Mucous cells failed to show any reactivity in any gland examined. Serous cells showed a gradient of immunostaining intensity ranging from strongly positive in demilunes of human sublingual gland to negative in rat submandibular gland and lacrimal glands of rats and mice. The presence of basolaterally localized Na+,K+-ATPase in most serous cells but not in mucous cells suggests that the enzyme contributes to the ion and water content of copious, low-protein serous secretions. The intense immunostaining of cells in most if not all segments of the duct system supports the idea that the ducts are involved with modification of the primary saliva, and extends this concept to include all segments of the duct system.     

Genome instability in interspecific cell hybrids I. Unstable expression of genes in divergent cell hybrids

Somatic cell hybrids between cells of widely divergent mammalian species display a range of chromosomal and genetic anomalies which may be the equivalent of the “genomic shock” phenomena observed in many plant and animal interspecific hybrids. Mouse-kangaroo hybrids show extreme segregation and fragmentation of the kangaroo chromosomes. Here 1 show that, in addition to the chromosomal instability, some hybrids display unstable expression of three genes borne on the kangaroo active maternal X chromosome. These genes (HPRT, G6PD andPGK) may be co-ordinately inactivated at high frequency, then reactivated once more. I suggest that this reversible inactivation in interspecific hybrids may be the result of an unstable change at an X inactivation centre located in the kangaroo X_q.     

The cytogenetic and hepatotoxic effects of dioxin on mouse liver cells

The cytogenetic and hepatotoxic effects of 2,3,7,8-tetrachlorodibenzo p-dioxin (TCDD) on mouse liver cells were investigated. Male C57BL/6J strain mice, which have TCDD receptors, were given single intraperitoneal injections of 25, 37.5, 75 and 150 g of TCDD/kg body weight or corn oil carrier alone. Two-thirds hepatectomies were carried out at 1 or 7 days after injection and chromosomal aberrations and mitotic indexes of the regenerating hepatocytes were scored 54 hr after hepatectomy. Liver sections from additional intact mice were studied for TCDD-hepatotoxicity at 1, 7 and 30 days after injection. The three high doses of TCDD caused hepatotoxicity with necrosis of liver cells and focal architectural collapse by 30 days after injection. No evidence was obtained of an increase in the frequency of chromosomal structural aberrations at doses that allowed sufficient mitotic activity for cytogenetic evaluation. We conclude that TCDD is not a clastogen for mouse hepatocytes, although high doses cause marked hepatocellular necrosis.Abbreviations CSD chromosome deletion - META metacentric chromosome - TCDD 2,3,7,8-tetrachlorobenzo-p-dioxin     

Seismic signal transmission between burrows of the Cape mole-rat,Georychus capensis

Summary Both seismic and auditory signals were tested for their propagation characteristics in a field study of the Cape mole-rat (Georychus capensis), a subterranean rodent in the family Bathyergidae. This solitary animal is entirely fossorial and apparently communicates with its conspecifics by alternately drumming its hind legs on the burrow floor. Signal production in this species is sexually dimorphic, and mate attraction is likely mediated primarily by seismic signalling between individuals in neighboring burrows. Measurements within, and at various distances away from, natural burrows suggest that seismic signals propagate at least an order of magnitude better than auditory signals. Moreover, using a mechanical thumper which could be triggered from a tape recording of the mole-rat's seismic signals, we established that the vertically-polarized surface wave (Rayleigh wave) propagates with less attenuation than either of the two horizontally-polarized waves. Thus, we tentatively hypothesize that Rayleigh waves subserve intraspecific communication in this species.Abbreviations PPM pulses per min - SB simulated burrow - SD standard deviation - SPL sound pressure level     

Rapid analysis of glycolipid anchors in amphiphilic dimers of acetylcholinesterases

1. We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs). 2. Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing a diacylglycerol, but not those with an alkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Roberts et al., J. Biol. Chem. 263:18766-18775, 1988; Biochem. Biophys. Res. Commun. 150:271-277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE from Drosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols). 3. Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutant et al., Eur. J. Biochem. 180:503-508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC. 4. In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.     

An intronic duplication in the alanine: glyoxylate aminotransferase gene facilitates identification of mutations in compound heterozygote patients with primary hyperoxaluria type 1

Summary We report here the identification of a duplication within the first intron of the gene encoding human alanine:glyoxylate aminotransferase (AGT); this duplication is closely linked to two point mutations associated with peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1 (PH1) patients. Polymerase chain reaction amplification of regions of the AGT gene including the insertion site from individuals heterozygous for this duplication, produces allele-specific fragments of different sizes. We have taken advantage of this to identify a nonsense mutation within a non-expressed allele of a compound heterozygote PH1 patient with mitochondrial AGT.     

Heterogeneity of apical glycoconjugates in kidney collecting ducts: Further studies using simultaneous detection of lectin binding sites and immunocytochemical detection of key transport enzymes

Summary In the search for a functional role for the polarized glycoconjugates of rat collecting duct epithelial cells, the relation between binding of various lectins and expression of cellular transport enzyme profile of the cells was studied. For this purpose, principal and intercalated cells of rat kidney collecting duct were identified by morphological criteria and by their immunocytochemically determined content of (Na⁺+K⁺)-ATPase and carbonic anhydrase (CA II), respectively. VariousN-acetylgalactosamine-specific lectins such as those fromHelix pomatia andMaclura pomifera revealed heterogeneity among both principal and intercalated cells, whereas -N-acetylgalactosa nine-specific lectin fromDolichos biflorus andVicia villosa bound preferentially to principal cells. Still another lectin fromArachis hypogaea reacted with most collecting duct cells in the cortex and outer medulla, but only with a subpopulation of cells in the inner medulla. Interestingly, some lectins reacted exclusively with the apical aspect of the collecting duct epithelial cells, whereas others revealed both an apical and basolateral distribution of lectin reactive glycoconjugates. The results thus show subtle differences in the glycocalyx structure of principal and intercalated cells and differences in the intracellular polarization of glycoconjugates of these cells. Thus, lectins may be useful tools in the study of the molecular mechanisms which establish and maintain the polarized functions of principal and intercalated cells.     

Immunocytochemistry of ion transport mediators in the genital tract of female rodents

With immunocytochemistry, we have determined distribution of sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) and of three isoenzymes of carbonic anhydrase (CA) and have shown absence of the chloride channel, Band 3 protein, in the genital tract of female rodents. Staining for Na+,K+-ATPase was strongest in the ampullary oviduct and uterine glands in the mouse. In the mouse and rat ovary, immunostaining evidenced CA I, II, and III in theca interna cells where the enzyme could affect the pH of follicular fluid. The zona pellucida of the ovary and cytoplasmic foci in follicular granulosa cells stained for content of only CA I in mouse ovary, suggesting synthesis of a zona pellucida component by granulosa cells. CA II in mouse oviductal epithelium increased from the negative infundibulum to the variably positive ampulla and isthmus to the uniformly positive interstitial segment. The content of CA III varied inversely to that of CA II. The prevalence of CA II-positive cells apparently corresponded with that of nonciliated cells, whereas abundance of CA III-positive cells concurred with that of ciliated cells in regions of the mouse oviduct. The rat oviduct lacked CA II but, like that of the mouse, showed CA III in the proximal region. The staining for CA II in surface epithelium exceeded the reactivity in glandular epithelium in the mouse uterus, except during estrus. In contrast, rat uterus evidenced CA II in glandular but not surface epithelium. These results testify to possible significance of various ion transport mechanisms for biologic activities of diverse cells in the female genital tract.     

Relationship between concentrations of immunoreactive insulin-like growth factor-I in follicular fluid and various biochemical markers of differentiation in bovine antral follicles

Three experiments were conducted to determine the relationship between concentrations of insulin-like growth factor-I (IGF-I) in ovarian follicular fluid and various biochemical markers of follicular differentiation in bovine follicles. In Experiment I, ovaries were removed on Days 7, 14, 28, 42, or 56 after parturition from a total of 21 cows. In Experiment II, ovaries of 31 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH, 500 ng/5 ml saline) injections given every 2 h via jugular cannulae. In Experiment III, ovaries of six cows were removed 48-50 h after a 35-mg injection of prostaglandin F2 alpha during the midluteal phase of an estrous cycle. In Experiments I and II, all follicles greater than or equal to 8.0 mm in diameter were removed from each ovary (n = 33 and 46, respectively). In Experiment III, fluid from all follicles greater than 4 mm in diameter were removed individually (n = 10), and fluid from follicles 1-4 mm in diameter were pooled for each cow. Follicles for each experiment were further categorized as either estrogen-active (E-A, concentration of estradiol greater than progesterone in follicular fluid) or estrogen-inactive (E-I, concentration of progesterone greater than estradiol in follicular fluid). Measurements of immunoreactive IGF-I (i-IGF-I) were made after separating IGFs from their binding proteins with an acid-ethanol extraction.(ABSTRACT TRUNCATED AT 250 WORDS)