Pulmonary surfactant proteins insert cation-permeable channels in planar bilayers.

Pulmonary surfactant, a mixture of lipids and proteins, promotes lung ventilation by reduction of surface tension at air-fluid interfaces. Exogenous surfactants containing hydrophobic proteins induce biological effects in lungs that are not fully explained by reduction of surface tension and are not induced by surfactants lacking proteins. We hypothesized that hydrophobic proteins from surfactant insert in membranes to induce channel activities that contribute to the observed biological effects of surfactant. To test for channel insertion by surfactant, planar lipid bilayers were monitored electrophysiologically in the presence of either intact pulmonary surfactant or extracted surfactant proteins reconstituted with phospholipids or directly added to bilayer lipids prior to membrane casting. In this in vitro model, both intact surfactant and extracted surfactant proteins initiated gated channel activities with slope conductances averaging 40 pS. Observed reversal potentials confirmed monovalent cation conductance, and conductance of smaller monovalent cations was selective. Voltage dependence of channel openings and rectification of channel current were not observed. These results confirm that hydrophobic surfactant proteins induce channel-mediated transport in artificial membranes. We speculate that pulmonary surfactants, in addition to reducing surface tension at air-fluid interfaces, initiate physiological and therapeutic effects in lung by cation channel insertion at exposed epithelial membranes.

Surfactant modulates calcium response of neutrophils to physiologic stimulation via cell membrane depolarization.

Pulmonary surfactant (PS) reduces inflammation in the lung by poorly understood mechanisms. We have observed that surfactant-associated proteins (SAP) insert monovalent cation channels in artificial membranes. Neutrophils are primary mediators of acute pulmonary inflammation, and their functions are activated by increases in cytosolic ionized calcium concentration ([Ca2+]) and by changes in membrane potential. We hypothesize that PS inserts SAP-dependent cation channels in neutrophils, causing membrane depolarization, altered [Ca2+] response, and depressed activation. Human neutrophils were isolated, exposed to PS+SAP (1% Survanta), PS-SAP (1% Exosurf), or buffer, and washed before activating with selected stimulants. PS+SAP reduced phorbol ester- and formyl peptide-stimulated adherence and aggregation by 38% (p < 0.05) and 54% (p < 0.02), respectively. PS+SAP also inhibited the formyl peptide-induced [Ca2+] response of neutrophils (p < 0.01), but only in the presence of external Ca2+. Further characterization of this inhibition demonstrated that PS+SAP blocked formyl peptide-induced influx of both Ca2+ and Mn2+, and that this inhibition was present during activation by other neutrophil stimulants (IL-8, immune complexes). Prior depolarization of neutrophils with gramicidin-D similarly inhibited the [Ca2+] response of neutrophils to formyl peptide, and analysis of neutrophil membrane potential by 3,3'-dipentyloxaearbocyanine iodide (diOC5(3)) fluorescence revealed that PS+SAP induced rapid neutrophil depolarization. In contrast, PS-SAP exhibited little effect on neutrophil function, [Ca2+], or membrane potential. We conclude that PS+SAP decreases neutrophil adherence and aggregation responses, blocks Ca2+ influx after physiologic stimulation, and decreases membrane potential. We speculate that these effects are caused by membrane depolarization via SAP-dependent cation channel insertion, and that all of these effects contribute to the antiinflammatory properties of PS+SAP.

Detection of pathogen transmission in neonatal nurseries using DNA markers as surrogate indicators.

OBJECTIVE: Nosocomial infections are a major problem confronting neonatal intensive care units (NICUs). This study was conducted to determine if DNA markers designed from the cauliflower mosaic virus (CaMV 35S DNA) can serve as surrogate indicators of nosocomial pathogen transmission in NICUs. METHODS: Regions of cauliflower CaMV 35S promoter DNA were designed to serve as surrogate markers of microbial transmission pathways. Each of 6 pods within the NICU under study houses 8 newborn infants. DNA marker was placed on the telephone handle in only 1 of the 6 NICU pods (study pod). Bedside caregivers were blinded as to when placebo or marker were placed in the pod. Thirty-two samples were collected from predetermined sites within each pod at 0, 4, 8, 24, and 48 hours and 7 days after DNA placement. Similar sites were sampled in each of the 6 pods. Additional samples were collected concurrently from areas of the NICU segregated from direct patient care. Polymerase chain reactions were performed on collected samples, and products were analyzed by agarose gel electrophoresis. RESULTS: One thousand three hundred samples of the environment and hands of personnel were collected and analyzed. Within the study pod, 58% of sites tested positive for the DNA marker throughout all time points; positive sites peaked at 8 hours (78%) and declined to 23% positive at 7 days. The other 5 pods had a mean of 18% of sites positive throughout the 7 days and exhibited a similar decline throughout time. The most consistently positive sites within all pods were the blood gas analyzers, computer mice, telephone handles, medical charts, ventilator knobs, door handles, radiant warmer control buttons, patient monitors, and personnel hands. In areas outside the pods, the nurse's station, resident physician charting area, changing room, and staff break room had a mean of 50% positive sites throughout all time points. CONCLUSIONS: DNA markers proved useful as safe, surrogate indicators of microorganism transmission within and outside pods in the NICU. We speculate that utilization of these techniques in the hospital environment will provide important information about transmission of pathogens in the NICU, assist in developing and enforcing cleaning procedures, and permit testing of educational intervention programs targeting a decrease in nosocomial infections.nosocomial infection, neonatal intensive care, DNA marker, polymerase chain reaction, infection control.

Human milk effects on neutrophil calcium metabolism: blockade of calcium influx after agonist stimulation.

Neutrophils are the predominant cellular mediators of acute inflammation, and human milk suppresses multiple neutrophil functions. We sought to determine whether these effects were mediated through disruption of normal intracellular Ca2+ homeostasis. Exposure of human neutrophils to human milk, followed by washing, resulted in altered Ca2+ transient responses to formyl-peptide stimulation in which the peak cytosolic free Ca2+ concentration ([free Ca]) was the same as in unexposed cells, but the postpeak decline in [free Ca] was more rapid. This effect was observed after human milk exposures as brief as 10 s, persisted for up to 4 h after human milk removal, and was concentration dependent. On the basis of experiments examining Ca2+-free conditions followed by Ca2+ supplementation, and experiments examining spontaneous and stimulated manganese and barium influx into neutrophils, the human milk effect was due to blockade of Ca2+ influx. Decreased Ca2+ transient responses to other physiologic stimuli (IL-8, opsonized Staphylococcus aureus, and immune complexes) were observed after human milk exposures. Rat intestinal epithelial cells and HL-60 cells failed to show these effects, suggesting a selective effect on mature inflammatory cells. Characterization of the Ca2+-blocking activity showed it was heat and acid stable in human milk with a molecular mass between 30-100 kD. Commercial human milk lactoferrin exhibited Ca2+ influx blockade activity, but recombinant human lactoferrin showed none. Separation of the activity by heparin affinity chromatography showed that it was distinct from lactoferrin. Human milk-induced blockade of Ca2+ influx provides a potential mechanism for broad suppression of neutrophil functions that may contribute to the antiinflammatory properties of human milk.

Conductive choline transport by alveolar epithelial plasma membrane vesicles.

Choline is an important substrate in alveolar epithelia for both surfactant production and cellular maintenance. The underlying mechanisms of uptake and sites of membrane transport remain uncertain. To test the hypothesis that choline transport occurs at the basolateral side of alveolar epithelia by both Na+-independent and -dependent mechanisms, plasma membrane vesicles were prepared from the apical and basolateral membranes of mature porcine type II pneumocytes. Choline+ transport was assayed by uptake of [3H]choline+ by enriched apical or basolateral vesicles. In the presence of imposed, inside-negative charge gradients, basolateral vesicles exhibited early overshoot of [3H]choline+ uptake unaffected by the presence or absence of external Na+ (541 +/- 53 vs 564 +/- 79 pmol/mg protein (NS)). High sensitivity to hemicholinium-3 was observed in the presence or absence of Na+. In the absence of inside-negative charge gradients, uptake was reduced 12-fold in the presence or absence of Na+, and external choline+ induced internal alkalization of acidified basolateral vesicles. Accumulative [3H]choline+ uptakes by apical vesicles in the presence or absence of inside-negative charge gradients and Na+ were insignificant. We conclude that predominant choline+ uptake by type II pneumocytes occurs at the basolateral membrane by Na+-independent, electrogenic choline+ conductance. The presence of electroneutral choline+/H+ exchange is suggested.

Membrane depolarization and depletion of intracellular calcium stores are associated with delay of apoptosis in human neutrophils.

Apoptosis occurs rapidly in human polymorphonuclear leukocytes (PMN) after exposure to 1 mM cycloheximide (CHX). We examined whether this form of stimulated apoptosis altered either resting cytosolic free Ca2+ concentrations ([free Ca]) or membrane potential (psi) in PMN and found no significant effects. However, manipulation of either PMN intracellular Ca2+ stores or psi was found to delay CHX-induced apoptosis. Depletion of PMN intracellular Ca2+ stores with thapsigargin caused membrane depolarization and significantly delayed CHX-induced apoptosis based on both morphological and annexin-V-fluorescein isothiocyanate binding criteria. Short-term suspension (4 h) of PMN in Ca2+-free buffer depleted internal Ca2+ stores, induced membrane depolarization at 2.5 h, and delayed spontaneous (24 h) apoptosis but had no effect on CHX-induced apoptosis. Rapid membrane depolarization with 150 mM KCl buffer significantly delayed CHX-induced apoptosis, suggesting that depolarization rather than Ca2+ stores depletion was the crucial event. Timing experiments revealed that depolarization within 12 min of CHX exposure significantly delayed apoptosis. Collectively, these observations suggest an early psi-sensitive step in the apoptosis pathway initiated by CHX. CHX exposure alone does not alter either resting PMN [free Ca] or psi; accompanying depolarization of plasma membrane (either electrochemically or via depletion of internal Ca2+ stores) delays CHX-induced apoptosis in a time-dependent manner.

Sodium-coupled transport of glucose by plasma membranes of type II pneumocytes.

Sodium-dependent absorption of alveolar fluid promotes efficient gas exchange. In animal models, alveolar glucose stimulates phlorizin-sensitive, Na(+)-dependent fluid absorption. It is hypothesized that Na+/glucose cotransporters are localized to apical membranes of type II pneumocytes. Enriched apical and basolateral plasma membrane vesicles were isolated from adult bovine type II pneumocytes. Uptakes of 22Na+ and [3H]glucose by enriched apical and basolateral vesicles were monitored over time. Following addition of external glucose (75 mM), 22Na+ uptake by mannitol-loaded, apically-enriched vesicles was significantly increased over controls. Substitution of interior-negative charge gradients for internally directed Na+ gradients increased glucose-dependent Na+ uptakes even greater. By contrast, external glucose did not significantly promote 22Na+ uptake by enriched basolateral vesicles. External Na+ (75 mM) significantly increased [3H]glucose uptakes by enriched apical vesicles with evidence of overshoot. Phlorizin (100 microM) inhibited both glucose-coupled 22Na+ uptakes and Na(+)-coupled [3H]glucose uptakes. These observations support localization of electrogenic, Na+/glucose cotransporters to enriched apical membranes of mature type II pneumocytes.

Sodium chloride cotransport at the basolateral membrane of type II pneumocytes.

Current evidence indirectly supports the hypothesis that Na+/Cl- cotransport occurs at basolateral membranes of type II pneumocytes. To test this hypothesis, enriched apical and basolateral plasma membrane vesicles were prepared from adult bovine type II pneumocytes. Uptake of 22Na+ or 36Cl- by these vesicle populations was monitored over time. Using enriched basolateral vesicles, substitution of formate- for Cl- nearly eliminated 22Na+ uptake, and substitution of Tris+ for Na+ significantly reduced 36Cl- uptake. These observations of Cl(-)-dependent 22Na+ uptake and Na(+)-dependent 36Cl- uptake demonstrated coupling between Na+ and Cl- absorption at the basolateral membrane. Na+/Cl- cotransport inhibitors, furosemide (1 mM) and bumetanide (0.1 mM), also reduced 22Na+ and 36Cl- uptakes by enriched basolateral vesicles. By contrast, furosemide and bumetanide did not affect 22Na+ and 36Cl- uptakes by enriched apical vesicles. Collectively, these observations support localization of Na+/Cl- cotransport to the basolateral membranes of mature type II pneumocytes.

Sodium/proton transport by apical membranes of type-II pneumocytes.

Recent studies fail to confirm the coexistence of Na+ channels and Na+/H+ exchange at the apical membranes of lower airway epithelia. Availability of plasma membrane vesicles simplifies the investigation of membrane transport processes. Apical and basolateral plasma membrane vesicles of disrupted type-II pneumocytes were fractionated upon nonlinear, continuous sucrose gradients. To investigate sodium transport, 22Na+ uptake by apical membrane vesicles was assayed in the presence and absence of transmembrane sodium diffusion potentials. Interior-negative sodium diffusion potentials promoted 22Na+ uptake 1.5-fold. Internally-directed H+ gradients or NH+4 gradients inhibited 22Na+ uptake 40-50%. Amiloride (1-1000 microM) inhibited uptake 10-79%. To investigate H+ transport, decay of transmembrane pH gradients was monitored with pH probe acridine orange. In the presence or absence of externally-directed H+ gradients, external sodium promoted internal alkalinization, except in the presence of external amiloride. These observations of amiloride-sensitive, electrogenic Na+ uptake and amiloride-sensitive, electroneutral, Na+/H+ coupling indicate coexistence of Na+ channels and Na+/H+ exchange at the apical membrane of type-II pneumocytes.

Gestational age correlates with skin reflectance in newborn infants of 24-42 weeks gestation.

Accurate gestational age determination is limited in very low birthweight infants using neurological and physical assessments. As one of the markers of intrauterine development, skin maturation is assessed qualitatively by pediatricians. Based on this observation, we hypothesize that skin reflectance relates directly to gestational age. Light was delivered and collected from the skin through a topically placed optical patch. Reflected light was detected by the spectrophotometer and corrected by an adjacent laptop computer to yield the true total diffuse reflectance as a function of wavelength between 380-820 nm. The calculated reflectance at 837 nm (R837) where it is independent of melanin, was determined by extrapolation from the reflectance at 650 and 750 nm. Sixty-four neonates of different races with gestational ages of 24-42 weeks were studied at 2-151 h of age. R837 was related exponentially to gestational age (GA) by the equation R837 = Rmax(1-exp[-(GA-G0)/tau]), where Rmax is the maximal value of R837, G0 is an apparent delay time before dermal scattering increases rapidly, and tau is a time constant, r = 0.88, p < 0.001. In summary, the extrapolated skin reflectance offers a quantitative and objective assessment of gestational age which is independent of melanin and sex.

Alpha 1-adrenergic and muscarinic receptors in adult and neonatal rat type II pneumocytes.

Binding characteristics of the alpha 1-adrenergic radioloigand [3H]prazosin, and the muscarinic cholinergic radioligand, [3H]quinuclidinyl benzilate, were determined both in intact cell preparations of rat alveolar type II pneumocytes (TIIPs) and in membrane preparations of rat lung tissue. Binding in adult and neonatal (< 24 h postnatal age) rats was also compared. Binding affinities for both receptor classes on TIIPs and whole lung membrane preparations alike did not vary significantly with age. In lung membrane preparations, the concentrations of both receptor classes were higher in neonates than adults. In TIIPs, the alpha 1-adrenergic receptor concentration was higher in neonates, but muscarinic receptor concentration was higher in adults. To begin investigation of the functional significance of these receptors, the effects of alpha 1-adrenergic and muscarinic agonists on intracellular calcium ion concentration ([Ca2+]i) were also measured. Both agonists induced consistent increases in [Ca2+]i, which were blocked by respective antagonists. These data indicate the presence of receptors on TIIPs for alpha 1-adrenergic and muscarinic agonists that may influence cellular function via modulation of [Ca2+]i.

Taurine-conjugated bile acids act as Ca2+ ionophores.

The ionophoretic properties of several taurine-conjugated bile acids have been investigated in two experimental systems: in a two-phase bulk partitioning system and in proteoliposomes. In the former, a bile acid/Ca2+ complex was extracted into the bulk organic phase and had an experimental stoichiometry of 1.75. Extraction was specific for Ca2+ over Mg2+; Na+ and K+ did not compete with the extraction of Ca2+. In the second system, bile acids at concentrations as low as 5-100 molecules/vesicle lowered the steady-state Ca2+ gradient maintained by a reconstituted sarcoplasmic reticulum Ca(2+)-ATPase. The effect was not due to nonspecific membrane perturbation. In addition to releasing intravesicular Ca2+ in a transmembraneous process, bile acids caused partition of Ca2+/bile acid complexes into the hydrophobic core of the bilayer. In both experimental systems, the Ca2+ ionophoretic activity correlated well with the concentration and the hydrophobicity of the bile acid. Taurolithocholate was most active, with a significant effect measurable at 10 microM in either system. Since bile acid concentrations equal to those used in our experiments can occur in the blood in certain liver diseases, the results support the notion that bile acids can increase the intracellular Ca2+ concentration bypassing the regulatory systems that maintain cellular Ca2+ homeostasis.

Neurocristopathy syndrome: review of four cases.

Aberrant embryologic development of the neural crest may clinically result in one of several patterns of malformation. Four examples of the neurocristopathy syndrome are described that collectively feature a very broad range of expression in these infants. Because of the many expressions, the syndrome may not be recognized. We believe that greater familiarity with the disorder will aid in the discovery of unsuspected anomalies and provide clues about predisposing etiologies.

Bile salt-induced intracellular Ca++ accumulation in type II pneumocytes.

The pathogenesis of pneumonitis associated with meconium aspiration is poorly understood. To explore the possibility of pulmonary cytotoxicity in association with bile salt exposure and calcium accumulation, we compared cell viability, radiolabeled calcium accumulation, and intracellular [calcium] in the presence and absence of bile salts, chenodeoxycholate, and 3 beta-OH-5-cholenoate. We assessed viability of type II pneumocytes in culture by cell permeability to trypan blue dye, incorporation of leucine into cellular proteins, and cellular morphology. Intracellular calcium concentrations were monitored with fluorescent dye methodology. At micromolar concentrations, the above bile salts increased cell permeability by as much as 9-fold and decreased leucine incorporation by as much as 5-fold. Radiolabeled calcium accumulation increased by as much as 2.5-fold and intracellular [calcium] transiently increased by as much as 6-fold. Studies using bile salts extracted from meconium yielded similar results. Correlation of calcium accumulation to viability studies yielded a direct relationship with cell permeability and an inverse relationship with leucine incorporation. We speculate that bile salt-induced accumulation of intracellular calcium in lung cells may contribute to the pathogenesis of meconium aspiration pneumonitis.

Maternal-neonatal serum vitamin A concentrations.

Prevention of neonatal vitamin A deficiency is related to the adequacy of maternal vitamin A stores. In this study we investigated maternal and cord serum vitamin A and retinol-binding protein (RBP) values in an Indian population including, for the first time, clinically vitamin A-deficient mothers. Twenty-eight maternal-neonatal pairs were selected from maternal cohorts of high socioeconomic status without clinical evidence of vitamin A deficiency (group I) and low socioeconomic status with conjunctival xerosis and Bitot's spots (group II). Maternal education, caloric and vitamin A intakes, weight, height, hemoglobin, and birth weight were significantly lower in group II. Serum vitamin A levels were significantly higher in group I mothers and newborns as were RBP levels in group I mothers. However, a significant difference between groups I and II in cord blood RBP was not observed. Upon correlation of maternal vitamin A levels with cord blood vitamin A levels, a logarithmic relationship was revealed, suggesting saturable transplacental transport of vitamin A.

Effect of bile acids on calcium efflux from isolated rat hepatocytes and perfused rat livers.

The changes in intracellular Ca2+ concentration [( Ca2+]i) of hepatocytes induced by certain bile acids are biphasic: an initial increase is followed by a more gradual decrease. This latter decline in [Ca2+]i may be due to an efflux of Ca2+ across the plasma membrane. This hypothesis was tested by studying the effect of different bile acids on the efflux of 45Ca from preloaded rat hepatocytes and isolated perfused rat livers. The following bile acids were studied: cholic (C), ursodeoxycholic (UDC), chenodeoxycholic (CDC), and deoxycholic (DC) acids; their taurine (T) conjugates (TC, TUDC, TCDC, and TDC); and the taurine, sulfate (S), and glucuronide (Glu) derivatives of lithocholic acid (TLC, LS, TLS, and LGlu, respectively). At 0.3 mM, all bile acids except C, TC, TCDC, UDC, and TUDC significantly increased 45Ca efflux from preloaded hepatocytes without affecting cell viability. Dose-response studies revealed that the minimum effective concentration needed to induce 45Ca efflux was 0.06 mM for LS, 0.8 mM for TCDC, and 10 mM for TC. Efflux of 86Rb from preloaded hepatocytes was not significantly altered by 0.1 mM LS, indicating relative specificity for calcium. TDC and DC, but not TC, increased 45Ca efflux from preloaded perfused rat livers. These results showed that bile acids known to increase [Ca2+]i (CDC, DC, TDC, and TLC) also increased 45Ca efflux from hepatocytes and perfused livers and that efflux was also stimulated by LS, TLS, and LGlu. The extent of this efflux was related to the hydrophobicity of the steroid nucleus of the bile acid. It is speculated that bile acid-induced increases in [Ca2+]i activate the plasma membrane Ca2+ pump resulting in increased Ca2+ efflux.

Vitamin concentrations in very low birth weight infants given vitamins intravenously in a lipid emulsion: measurement of vitamins A, D, and E and riboflavin.

Because total parenteral nutrition with vitamins added to the glucose-amino acid mixture is often associated with a reduction in blood levels of vitamin A (retinol) during the routine treatment of many very low birth weight (VLBW) infants (less than 1500 gm), and because retinol losses in the plastic delivery system can be prevented by adding the vitamins to an intravenous lipid emulsion, seven VLBW infants with a mean birth weight of 900 gm (range 450 to 1360 gm) were given 40% of a unit dose vial, per kilogram of body weight, of a multivitamin preparation (M.V.I. Pediatric) (280 micrograms retinol; 160 IU vitamin D; 2.8 mg tocopherol; 0.68 mg riboflavin) in a lipid emulsion, Intralipid. After treatment with the intralipid-vitamin mixture for 19 to 28 days, plasma vitamin A (retinol) concentrations increased significantly from 11.0 +/- 0.76 (mean +/- SEM) before intralipid to 19.2 +/- 0.97 micrograms/dl after the intralipid-vitamin mixture (p less than 0.01); 25-hydroxyvitamin D concentrations increased from an initial value of 12.6 +/- 2.6 to 20.2 +/- 1.9 mg/dl (p less than 0.01); alpha-tocopherol concentrations increased from an initial value of 0.31 +/- 0.06 to 2.44 +/- 0.13 mg/dl (p less than 0.01); and riboflavin levels increased from 64.1 +/- 7.8 ng/ml to concentrations between 20 and 100 times the initial level. Erythrocyte riboflavin levels increased from 71.8 +/- 14 initially to 166 +/- 41 ng/gm hemoglobin, and erythrocyte flavin-adenine dinucleotide levels increased similarly from 972 +/- 112 initially to 2005 +/- 294 ng/gm hemoglobin. These results show that the addition of M.V.I. Pediatric to Intralipid decreases the extensive in vivo loss of retinol and is associated with an increase in plasma retinol concentrations in VLBW infants. The daily doses of vitamins D (160 IU/kg) and E (2.8 mg/kg) appear sufficient, but the dose of vitamin A (280 micrograms/kg) is insufficient to raise blood levels of all infants into the normal range. The current dose of riboflavin is excessive and may be harmful.

Intestinal absorption of bile acid glucuronides in rats.

While the intestinal absorption of taurine, glycine, and sulfate conjugates of bile acids has been studied extensively, nothing is known about the absorption of bile acid glucuronides. In the present study, the intestinal phase of the enterohepatic circulation of two bile acid glucuronides was examined. [3 beta-3H]cholic acid 3-O-beta-D-glucuronide or [3 beta-3H]lithocholic acid 3-O-beta-D-glucuronide was perfused through isolated segments of ileum or jejunum with intact blood supply in rats prepared with a biliary fistula. [14C]Taurocholic acid was perfused simultaneously with each glucuronide to compare glucuronide absorption with that of an actively transported bile acid. Intestinal absorption was determined by measuring the rate of secretion of labeled bile acid in bile. The absorption of [3H]cholic acid glucuronide by the ileum and jejunum was one fortieth and one eighth, respectively, that of [14C]taurocholic acid. Comparison of the two glucuronides show that [3H]lithocholic acid glucuronide absorption was 18 and 10 times greater than [3H]cholic acid glucuronide absorption from the jejunum and ileum, respectively. Collectively, the above observations suggest that glucuronidation of bile acids markedly reduces absorption from the small intestine.