CGRP-immunoreactive sensory nerve fibers in the submandibular gland of the rat

Summary Indirect immunofluorescence technique was used to study the occurrence and distribution of CGRP immunoreactivity in the submandibular gland of normal rats and after unilateral sensory and sympathetic denervations. In normal rats, CGRP-immunoreactive nerve fibers and nerve trunks were seen around or in close contact with interlobular salivary ducts as well as around small blood vessels of the gland. Occasionally, CGRP-immunoreactive nerve fibers were also detected between or around the acini of the gland.The submandibular ganglia contained CGRP-immunoreactive nerve fibers, but the ganglion cells were not immunoreactive for CGRP. The trigeminal ganglion contained a population of CGRP-immunoreactive, mainly small sized ganglion cells and nerve fibers distributed throughout the ganglion. Unilateral electrocoagulation of the trigeminal nerve caused a significant reduction in the number of immunoreactive nerve fibers in the gland, although some fibers still were present in the ipsilateral glandular tissue. Unilateral superior cervical ganglionectomy caused no detectable effect on the number of CGRP-immunoreactive nerve fibers in the gland.The present results suggest that the rat submandibular gland contains CGRP-immunoreactive nerve fibers both around blood vessels and in glandular secretory elements. Denervation experiments support the view that the majority, but perhaps not all of them originate from the trigeminal ganglion.     

Effect of a Single Dose of β,β''-Iminodipropionitrile In Vivo on the Properties of Neurofilaments In Vitro: Comparison with the Effect of Iminodipropionitrile Added Directly to Neurofilaments In Vitro

The biochemical properties of neurofilaments isolated from control and iminodipropionitrile-treated rats were compared with regard to autophosphorylation capacity, hydrolysis of ATP, and the formation of a viscous gel between filaments. Both preparations exhibited a similar polypeptide composition, and no covalent cross-linking between neurofilament subunits was induced by iminodipropionitrile in vivo. An ATPase activity, systematically present in all preparations, was unaffected by the administration of iminodipropionitrile to the rats. Conversely, the autophosphorylation of neurofilament subunits in vitro was significantly higher in preparations from iminodipropionitrile-treated rats than from control animals, with a marked increase of the phosphorylation of a high molecular weight neurofilament-associated protein. Iminodipropionitrile provoked a higher gelation capacity of neurofilaments as measured in vitro, with a lower critical concentration for the preparation from treated animals. A similar increased interaction was obtained with millimolar concentrations of iminodipropionitrile added to bovine neurofilaments in vitro, involving likely neurofilament-associated molecules, because the effect of the drug was lost after their extraction by 0.8 M KCl. These results support the hypothesis that iminodipropionitrile interferes with the neurofilament networks through a preferential interaction with the neurofilament-associated proteins, resulting in a change in their properties and consequently in an increased capacity of interaction between the polymers.     

Diet and urinary estrogen profile in premenopausal omnivorous and vegetarian women and in premenopausal women with breast cancer

The urinary estrogen profile was studied in the midfollicular phase twice, and diet four times during 1 yr in 10 premenopausal breast cancer (BC) patients consuming an omnivorous normal Finnish diet and in two control groups, one consuming an omnivorous (n = 12) and the other a lactovegetarian (n = 11) diet. Total fat intake in relation to caloric intake was almost identical in all three groups. Only with regard to grain fiber intake did the BC patients differ significantly from both other groups. No differences were found between the groups with regard to urinary excretion of 13 individual estrogens and total estrogens, with the exception of 4-hydroxyestrone (4-OH-E1), which was significantly lower (P less than 0.05) in the BC group than in the vegetarians. A high carbohydrate to protein ratio in the diet had a negative correlation with the excretion of 2-hydroxyestrogens and 2-hydroxyesterone (2-OH-E1) to 4-OH-E1 ratio. The BC group had significantly higher urinary 2-OH-E1 to E1 ratio (P less than 0.05) compared to the vegetarians. The 2-OH-E1 to 4-OH-E1 ratio was highest in the BC group (= 7.1) and differed significantly from that of the omnivores (= 4.3; P less than 0.02) and vegetarians (= 3.6; P less than 0.005). This ratio showed a negative correlation with intake of carbohydrates, starch, total and grain fiber. Urinary excretion of 4-OH-E1 correlated positively with total and grain fiber intake and plasma SHBG. Protein intake correlated positively with urinary 2-methoxy-E1 excretion, and retinol intake positively with catechol estrogen, E1 and E2 excretion. It is concluded that estrogen production and urinary estrogen profile in premenopausal breast cancer patients is normal with the exception of a low 4-OH-E1 excretion and high urinary 2-OH-E1 to 4-OH-E1 ratio. This ratio, which seems to depend on diet, is the only urinary estrogen parameter separating premenopausal BC patients from the control omnivorous and lactovegetarian women.     

Binding of a distamycin-ellipticine hybrid molecule to DNA and chromatin: spectroscopic, biochemical, and molecular modeling investigations.

A bifunctional molecule in which an ellipticine chromophore is attached to a distamycin residue via a diaminopropyl tether has been designed and synthesized in the expectation of creating a hybrid molecule capable of bidentate binding to DNA by both intercalation and minor-groove interactions. The strength and mode of binding to DNA of this conjugate have been studied by means of circular and linear dichroism as well as by stopped-flow kinetics and measurements of reactivity toward a chemical probe. The results converge to reveal that the ellipticine moiety of the hybrid largely dominates the binding reaction with DNA. In the presence of chromatin, the hybrid molecule binds preferentially to the internucleosomal DNA, a preference dictated by its intercalating chromophore. Theoretical computations were performed on the comparative complexation energies of distamycin, the ellipticine derivative, and the hybrid ligand with a B-representative octanucleotide, d(GCATATGC)2. The best binding configuration of the ellipticine derivative locates its aminoalkyl side chain in the minor groove where distamycin is also present. The molecular modeling analysis fully supports the involvement of a bimodal binding process for the hybrid and reveals that the binding of the conjugate to DNA favors a pronounced bending toward the minor groove. This effect is attributed to intercalation of the ellipticine chromophore. An interesting link is established between the DEPC reactivity experiments and the theoretical computations, suggesting that DEPC can be used as a probe for drug-induced DNA bending. On the basis of these results, we propose the design of a new hybrid ligand bearing an additional positively-charged amidine side chain to confer higher DNA-binding affinity.     

Characteristics of ouabain binding to isolated trout hepatocytes

Summary Na⁺, K⁺ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10^–4 M produced maximal inhibition (95%) of K⁺ uptake and enhanced intracellular Na⁺ accumulation, showing that active fluxes account for a very large proportion of Na⁺ and K⁺ exchanges. Inhibition of the Na–K pump by ouabain was significant at low concentrations (10^–8 M). When external K⁺ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC₅₀) of K⁺ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [³H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K⁺ concentration in the medium, the other linear and independent of external K⁺. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5·10⁵ per cell) and independent of the external K⁺ concentration (7, 0.5 or 0 mM), while the dissociation constant (K_D) decreased from 4.2 M to 7.3 nM when K⁺ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min^–1), as estimated from ouabain-sensitive K⁺ flux, in comparison to those described in other cell types of higher vertebrates. At each external K⁺ concentration studied, the inhibition of K⁺ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside.     

Influence of the Molecular Structure of Steroids on Their Ability to Interrupt Gap Junctional Communication

17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism. Received: 10 April 1995/Revised: 27 October 1995     

Preference for plants damaged by conspecific larvae in ovipositing cabbage root flies: influence of stimuli from leaf surface and roots

In laboratory dual-choice assays females of the cabbage root fly, Delia radicum, prefer for oviposition plants with roots damaged by conspecific larvae to undamaged controls. Cauliflower and kale plants were inoculated with root fly eggs (25 per plant) and the hatching larvae were allowed to feed on the roots for various periods of time (1–17 days). After 4 (cauliflower) or 5 (kale) days of larval feeding the oviposition preference was most pronounced and flies laid between 64% and 68% of their eggs near plants with damaged roots. Later, with increasing damage but fewer surviving, and thus actively feeding, larvae, the magnitude of the preference declined. The preference for plants already damaged by conspecific larvae may contribute to the previously observed aggregated distribution of D. radicum eggs in Brassica crop fields.Further experiments revealed that the sensory cues inducing this oviposition preference originate from the complex consisting of the damaged roots, the surrounding substrate (soil) and associated microbes, rather than from the aerial plant parts. In choice assays using the root-substrate complex of damaged and control plants (aerial parts removed), the observed preference for damaged roots was similar to that found for the entire plant but was more pronounced. The damaged roots alone, compared to control roots, received up to 72% (cauliflower) and 75% (kale) of the eggs. By contrast, surrogate leaves sprayed with methanolic leaf surface extracts from the most preferred plants which had been damaged were not discriminated from surrogate leaved sprayed with extracts of the respective control plants. Analysis of glucosinolate levels in methanolic leaf surface extracts revealed that root damage resulted in enhanced concentrations of indole-glucosinolates on the leaf surface in kale but not in cauliflower. Although indole-glucosinolates are oviposition stimulants for the cabbage root fly, the induced changes were apparently too small to influence oviposition behaviour.     

Cloning of Choriocarcinoma Cells Shows That Invasion Correlates with Expression and Activation of Gelatinase A

Implantation and placental development are dependent upon trophoblast invasion of the endometrium. While the villous trophoblast does not display invasive behavior, the extravillous cytotrophoblast is highly invasive. By cloning BeWo choriocarcinoma cells, we have isolated two distinct clones that share similarities with villous and extravillous cytotrophoblasts. When cultured at the surface of a type I collagen gel, BeWo MC-1 cells were not invasive, whereas BeWo MC-2 cells rapidly invaded this matrix. When injected subcutaneously in nude mice, BeWo MC-1 cells developed a localized tumor and BeWo MC-2 cells developed larger tumors with micrometastases. Gelatinase A expression and minute amounts of gelatinase B were detected in the parental cell line and in both clones. However, the parental and the BeWo MC-2 cells secreted 5- to 10-fold more gelatinase A than the BeWo MC-1 cells. Laminin and matrigel stimulated the production of gelatinase A in BeWo MC-2 cells. Type I collagen promoted the conversion of the 72-kDa progelatinase A in an active enzyme only in parental BeWo and in BeWo MC-2 cells. These clones provide an interesting model for studying the complex mechanisms regulating implantation as well as the controlled invasiveness during implantation compared to tumor invasion.     

Life-history variations of the Lesser Sheathbill Chionis minor in contrasting habitats

From 1987 until 1995, life-history traits of the Lesser Sheathbill Chionis minor in the Kerguelen Archipelago were compared with those at other localities, where this species breeds in seabird colonies. At Kerguelen Island only, some pairs breed on shores free of penguin and cormorant colonies. Moreover, pairs, including nonbreeders, and solitary individuals maintain territories all the year round. Site and mate changes were not linked, and all divorces were permanent. Fidelity rates were similar in all localities and so were the annual survival rates of adults. The survival rate of immature individuals was highest at Kerguelen Island, where sheathbills laid smaller clutches, produced fewer fledglings and had lower breeding success than in the other islands. At Kerguelen Island, pairs breeding on shores had similar clutch sizes but fledged fewer offspring than those breeding in seabird colonies. Further, the proportion of vacant territories and nonbreeders was higher on shores. Kerguelen sheathbills devoted less time to food acquisition than those on Marion Island, being exceptionally kleptoparasitic, and spent a smaller part of their foraging time exploiting seabird colonies. Their diet was mainly algae. The differences on Kerguelen Island as compared with other localities were caused by the presence of an extensive intertidal zone on the former, which reduced competition, making many more sites suitable and the environment more predictable. These life-history and behavioural traits are discussed in relation to life-history theory. As differences were also found, although less extensive, within the Kerguelen Archipelago, we suggest that some traits represent an adaptive response to external constraints and that the life-history strategy of the Lesser Sheathbill is particularly opportunistic.