Effect of Colostomy on the Circadian Rhythm in DNA Synthesis in the Rat Colon

Synthesis of DNA and mitosis in gut epithelium are not constant or random events but rather are characterized by circadian rhythmicity, which we reported persists even in fasted rats. Others suggest that rhythms persist because rats anticipate food, causing nerve impulses to propagate caudally in the gut at usual meal times, or that digestive products from previous feedings cause rhythms in the lower tract. We studied colonic DNA synthesis in rats that had been given colostomies. In one study, the distal colon was isolated neurally from proximal gut by means of an end colostomy. In a second study, rats were subjected to loop colostomy; some intrinsic innervation of the gut wall remained intact. Sprague-Dawley male rats, 8 weeks old, were acclimated to a 12:12 light-dark cycle. Colostomies were performed after a 48-h fast. The rats were fed ad libitum for 4 weeks after surgery. Operated rats and an equal number (n = 30) of control rats (unoper-ated) were divided into four subgroups that were killed at 07:00,13:00,19:00, and 01:00 h. Each rat was injected with tritiated thymidine 30 min before it was killed. Proximal and distal colon were analyzed for incorporation of radioactivity (DNA synthesis). Results are reported as counts per minute per microgram of DNA and were analyzed using analysis of variance and the t test. Significant daily variation was found in proximal colon, both from control and operated rats. Rhythms were still present in colon distal to loop colostomy but were lost in the distal stump in rats that received an end colostomy. The mammalian “biological clock” regulates most circadian rhythms by neurohumoral mechanisms; however, our results suggest that the intrinsic enteric nervous system is important in the propagation of a signal that causes rhythmic cell proliferation in the gut in the intact animal.     

Ethanol-induced phospholipase C activation is inhibited by phorbol esters in isolated hepatocytes.

Ethanol causes a transient activation of the phosphoinositide-specific phospholipase C in intact hepatocytes and mimics the action of receptor-mediated agonists [Hoek, Thomas, Rubin & Rubin (1987) J. Biol. Chem. 262, 682-691]. Preincubation of the hepatocytes with phorbol esters which activate protein kinase C prevented this effect of ethanol: phorbol ester treatment inhibited the ethanol-induced phosphorylase activation, the increase in intracellular free Ca2+ concentrations measured in quin 2-loaded hepatocytes, and the changes in concentrations of inositol phosphates, phosphoinositides and phosphatidic acid. Several lines of evidence indicate that these effects were mediated by protein kinase C. Phorbol esters acted in a concentration range where they activate protein kinase C; phorbol esters that do not activate protein kinase C were not effective in inhibiting the effects of ethanol. The permeant diacylglycerol oleoyl-acetylglycerol also inhibited the effects of ethanol, but other diacylglycerols were not effective in the intact cells. The inhibition of ethanol-induced Ca2+ mobilization by phorbol esters was prevented by preincubating the cells with the protein kinase C inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) and sphingosine. H7 also enhanced the Ca2+ mobilization induced by ethanol in cells that were not pretreated with phorbol esters, indicating that the transient nature of the ethanol-induced Ca2+ mobilization may be due to an activation of protein kinase C caused by the accumulation of diacylglycerol. These data support a model whereby ethanol activates the phosphoinositide-specific phospholipase C, possibly by affecting receptor-G-protein-phospholipase C interactions in the membrane.     

Partial Revertants of the Transposable Element-Associated Suppressible Allele White-Apricot in Drosophila Melanogaster: Structures and Responsiveness to Genetic Modifiers

The eye color phenotype of white-apricot (wa), a mutant allele of the white locus caused by the insertion of the transposable element copia into a small intron, is suppressed by the extragenic suppressor suppressor-of-white-apricot (su(wa] and enhanced by the extragenic enhancers suppressor-of-forked su(f] and Enhancer-of-white-apricot (E(wa]. Derivatives of wa have been analyzed molecularly and genetically in order to correlate the structure of these derivatives with their response to modifiers. Derivatives in which the copia element is replaced precisely by a solo long terminal repeat (sLTR) were generated in vitro and returned to the germline by P-element mediated transformation; flies carrying this allele within a P transposon show a nearly wild-type phenotype and no response to either su(f) or su(wa). In addition, eleven partial phenotypic revertants of wa were analyzed. Of these, one appears to be a duplication of a large region which includes wa, three are new alleles of su(wa), two are sLTR derivatives whose properties confirm results obtained using transformation, and five are secondary insertions into the copia element within wa. One of these, waR84h, differs from wa by the insertion of the most 3' 83 nucleotides of the I factor. The five insertion derivatives show a variety of phenotypes and modes of interaction with su[f) and su(wa). The eye pigmentation of waR84h is affected by su(f) and E(wa), but not su(wa). These results demonstrate that copia (as opposed to the interruption of white sequences) is essential for the wa phenotype and its response to genetic modifiers, and that there are multiple mechanisms for the alteration of the wa phenotype by modifiers.     

Transient accumulation of inositol (1,3,4,5)-tetrakisphosphate in response to alpha 1-adrenergic stimulation in adult cardiac myocytes

To investigate the response to catecholamine stimulation of adult cardiac myocytes and the metabolism of inositol (1,4,5)-trisphosphate (1,4,5-IP3) and inositol (1,3,4,5)-tetrakisphosphate (IP4), we have employed a procedure developed in our laboratory to directly measure the mass of inositol phosphates after separation of individual isomers of inositol phosphates by high performance liquid chromatography. Control, unstimulated myocytes, contained low levels of inositol (1,4)-bisphosphate (1,4-IP2), inositol (1,3)-bisphosphate (1,3-IP2), inositol (3,4)-bisphosphate (3,4-IP2), inositol (1,3,4)-trisphosphate (1,3,4-IP3), 1,4,5-IP3 and IP4. Stimulation with norepinephrine for 30 seconds produced peak 1,4,5-IP3 and IP4 levels which rapidly returned to basal values by 60 seconds of norepinephrine stimulation. 1,4-IP2, 1,3-IP2 and 1,3,4-IP3 were increased markedly but only after stimulation with norepinephrine for 60 seconds. These results indicate a rapid yet transient increase in 1,4,5-IP3 and IP4 in response to norepinephrine stimulation and are the first quantitative measurements of the isomers of inositol phosphates in cardiac tissue.     

Effect of chronic ethanol ingestion on pancreatic protein synthesis

The effect of chronic ethanol feeding on pancreatic protein synthesis was assessed by studying the rate of incorporation of [3H]leucine into proteins in isolated rat pancreatic acini in vitro. Chronic ethanol feeding increased the rate of protein synthesis (2-3-fold) compared to controls fed an isocaloric diet. The onset of the increase in protein synthesis was detectable 2 days after the beginning of ethanol feeding, reached a maximum after 7 days and remained constant for up to 4 months. The increased incorporation of [3H]leucine was not due to an increased turnover of proteins as measured in pulse-chase experiments. After separation of individual digestive enzymes by SDS-polyacrylamide gel electrophoresis and determination of the distribution of radioactivity in different proteins, a general increase in the rate of incorporation of the label into all of the proteins was observed. In contrast to the observations made with isolated acini, there was no significant difference between the control and ethanol-fed groups when the rate of pancreatic protein synthesis was measured in vivo. However, overnight withdrawal of ethanol led to an increase of approx. 70% in protein synthesis in the ethanol-fed group. These results suggest that chronic ethanol ingestion modifies the control of pancreatic protein synthesis; the enhanced protein synthesis is expressed in isolated acini, i.e., in the absence of physiological factors present during chronic ethanol ingestion and in vivo after ethanol withdrawal.     

Spectral, Photophysical, and Stability Properties of Isolated Photosystem II Reaction Center

Photosystem II reaction center (RC) preparations isolated from spinach (Spinacea oleracea) by the Nanba-Satoh procedure (O Nanba, K Satoh 1987 Proc Natl Acad Sci USA 84: 109-112) are quite labile, even at 4°C in the dark. Simple spectroscopic criteria were developed to characterize the native state of the material. Degradation of the RC results in (a) blue-shifting of the red-most absorption maximum, (b) a shift of the 77 K fluorescence maximum from ~682 nm to ~670 nm, and (c) a shift of fluorescence lifetime components from 1.3-4 nanoseconds and >25 nanoseconds to ~6-7 nanoseconds. Fluorescence properties at 77 K seem to be a more sensitive spectral indicator of the integrity of the material. The >25 nanosecond lifetime component is assigned to P680⁺ Pheophytin⁻ recombination luminescence, which suggests a correlation between the observed spectral shifts and the photochemical competence of the preparation. Substitution of lauryl maltoside for Triton X-100 immediately after RC isolation stabilizes the RCs and suggests that Triton may be responsible for the instability.     

Mitogenic properties of neuraminidase and galactose oxidase-treated lymphoblastoid cells and plasma membranes for peripheral blood lymphocytes

Generation of aldehydes on terminal D-galactose or N-acetyl-D-galactosamine residues of cell surface glycoproteins by treatment with neuraminidase and galactose oxidase (NAGO) renders some types of cells mitogenic for T lymphocytes. The cell surface molecules required for the presentation of mitogenic signals by NAGO-treated cells are unknown. We tested the mitogenic properties of NAGO-treated lymphoblastoid cell lines (LCL) and subcellular fractions as an initial step in the isolation and characterization of cell surface molecules required for stimulation. We report here that the NAGO-LCL of B cell lineage were potent stimulators, whereas the NAGO-LCL of T cell lineage were weaker and more variable stimulators of lymphocyte proliferation. T-LCL that were stimulatory in indirect stimulation did not induce a mixed lymphocyte response, whereas the B-LCL were positive in both assays. Aldehyde-bearing plasma membrane-enriched subcellular fractions, depleted of nuclear, cytosolic, and mitochondrial components, were mitogenic, and the stimulatory activity was dose dependent. The ability to induce mitogenesis was abrogated by reduction of cell surface aldehyde groups. The results indicate that lymphocyte activation, induced by NAGO-treated stimulatory cells, is a plasma membrane-associated event and does not require the metabolic activity of intact cells. Furthermore, the aldehyde moiety is required but not sufficient for presentation of mitogenic signals. The LCL provide a suitable and reproducible source for isolation and characterization of stimulatory cell surface structures.     

A viscoplastic model for the active component in cardiac muscle

The HMK model (Hunter et al. in Prog Biophys Mol Biol 69:289–331, 1998) proposes mechanobiological equations for the influence of intracellular calcium concentration \(\hbox {Ca}_\mathrm{i}\) on the evolution of bound calcium concentration \(\hbox {Ca}_\mathrm{b}\) and the tropomyosin kinetics parameter z, which model processes in the active component of the tension in cardiac muscle. The inelastic response due to actin-myosin crossbridge kinetics is modeled in the HMK model with a function Q that depends on the history of the rate of total stretch of the muscle fiber. Here, an alternative model is proposed which models the active component of the muscle fiber as a viscoplastic material. In particular, an evolution equation is proposed for the elastic stretch \(\lambda _\mathrm{a} \) in the active component. Specific forms of the constitutive equations are proposed and used to match experimental data. The proposed viscoplastic formulation allows for separate modeling of three processes: the high rate deactivation of crossbridges causing rapid reduction in active tension; the high but lower rate reactivation of crossbridges causing recovery of active tension; and the low rate relaxation effects characterizing the Hill model of muscles.