Ion transport by primary cultures of canine tracheal epithelium: Methodology,morphology, and electrophysiology

Summary Canine tracheal epithelial cells were isolated by enzymatic and mechanical dispersion and cultured on permeable supports. The cells formed confluent monolayers and retained most of the morphologic characteristics of the intact epithelium, including apical microvilli, apical tight junctions, and a moderately interdigitated lateral intercellular space. The cells also retained the functional properties of the epithelium. The monolayer responded to addition of isoproterenol with the characteristic changes in cellular electrical properties expected for stimulation of Cl secretion: isoproterenol increased transepithelial voltage, depolarized apical membrane voltage, and decreased both transepithelial resistance and the ratio of apical-to-basolateral membrane resistance. Examination of the cellular response to ion substitutions and inhibitors of Cl secretion indicate that the cultured monolayers retain the same cellular mechanisms of ion transport as the intact epithelium. Thus, primary cultures of tracheal epithelium may provide a useful preparation for future studies of the mechanism and regulation of Cl secretion by airway epithelia.     

Adrenergic regulation of ion transport by primary cultures of canine tracheal epithelium: Cellular electrophysiology

Summary We examined the effect of adrenergic agents on the cellular electrical properties of primary cultures of canine tracheal epithelium. Both isoproterenol and epinephrine stimulated Cl secretion, as evidenced by an increase in transepithelial voltage and a fall in transepithelial resistance. Moreover, both agents appear to increase the conductance of apical and basolateral membranes. However, the pattern of response was different. Isoproterenol initially depolarized apical voltage _a and decreased the fractional resistance of the apical membranef _R. These changes are consistent with an initial increase in apical Cl conductance. In contrast, epinephrine acutely hyperpolarized _a and increasedf _R, changes consistent with an initial increase in basolateral K conductance. Following the acute effect of epinephrine, _a depolarized andf _R decreased to values not significantly different from those observed with isoproterenol. The acute increase in basolateral K conductance produced by epinephrine appeared to result from stimulation of adrenergic receptors because it was reproduced by addition of the agonist phenylephrine, and blocked by the antagonist phentolamine. The ability of prazosin but not yohimbine to block the acute epinephrine-induced increase in K permeability indicates the presence of ₁ adrenergic receptors. The acute adrenergic-induced increase in basolateral K conductance may be mediated by an increase in cell Ca because the response was mimicked by addition of the Ca ionophore A23187. In contrast, the response to isoproterenol was similar to that observed with addition of 8-bromo-cAMP and theophylline. These results indicate that both and adrenergic agents mediate the ion transport processes in canine tracheal epithelium. adrenergic agents have their primary effect on the apical Cl conductance, probably via an increase in cAMP. adrenergic agents exert their primary effect on the basolateral K conductance, possibly via an increase in cell Ca.     

Scanning and transmission electron microscopy of the squamose gill-filament epithelium from fresh- and seawater adaptedTilapia

Synopsis The architecture of the gill structure of variousTilapia species was studied in relation to their adaptability to hypersaline media. Using SEM and EM, it was shown that the squamose epithelial cells of the gills have species-typical patterns of ridges on their outer surfaces. These have previously been misinterpreted by other authors as microvilli or stereocillia. The ridges are more dense and better developed in euryhaline species, likeT. zillii, and less so in stenohaline species likeSarotherodon niloticus. Comparing freshwater and seawater-adapted individuals ofT. zillii, S. niloticus, S. galflaeus, andTristramella sacra, it was shown that in fresh water the surface cells are slightly swollen, extending over the openings of the chloride cells. During adaptation to sea water, these ridges become higher and denser and the cell surface shrinks, exposing the underlying orifices of the apical crypts of the chloride cells. The more euryhaline the species, the less change there is in the ridge pattern of the cells during passage from fresh to sea water. This evidence implicates the gill epithelium, together with the chloride cells, in the process of osmoregulation.     

Luminal responses to bradykinin on the isolated canine tracheal epithelium: effects of bradykinin antagonists

We have tested the ability of several B₂ antagonists on the responses of the open-circuited isolated canine tracheal epithelium to the luminal addition of Bradykinin (BK), Lys-BK, and substance P (SP). All three peptides produced biphasic changes in transmural potential difference (PD), an initial decrease (dip) followed by an increase (rise). The B₂ antagonists -Arg^o [Hyp³,Thi^5,8, -Phe⁷]BK (B5630) reversibly inhibited both the dips and the rise with IC₅₀ values of 2.01 · 10⁻⁸ and 1.54 · 10⁻⁷ M, respectively. The responses to SP were unaffected even with high concentrations of the antagonist. Other antagonists tested [ -Phe^1,7,Thi^5,8]BK (B4158), [ -Phe^2,7]BK (B4404), and [ -Phe⁷,Hyp⁸]BK (B5092) were ineffective.     

Organ culture of adult Rat and mouse tracheal epithelium: I. Ultrastructure following various culture periods

Summary Electron microscopic studies of adult rat and mouse tracheal epithelium maintained in organ culture for a period of up to 6 days were performed. In specimens cultured for 60 minutes no conspicuous micromorphological alterations could be observed. Following culture periods from 1–6 days the number of cilia in some of the ciliated cells was reduced while their structure and the other ultrastructural details of the epithelial cells were preserved. In specimens cultured for 5–6 days some additional alterations could be noticed: polymorphism of mitochondria, increased number of lysosomes, appearance of intracellular vacuoles, exhaustion of goblet cells and disappearance of granulated mast-cell like cells in the rat tracheal epithelium.I want to thank Miss J. Selbmann and Mrs. S. Kolassa for technical help and Mr. H. Wagner for preparing the micrographs; I am indebted to Dr. D. Kerjaschki and to Mr. H. Hörandner for performing preparations for scanning electron microscopy and to Mr. P. Scholze (Österreichische Studiengesellschaft für Atomenergie, Institut für Metallurgie, Abteilung Fremdkörperphysik) for preparing the scanning electron micrograph.This work was supported by the Fond zur Förderung der wissenschaftlichen Forschung: Project 2099.This paper is dedicated to Prof. Bargmann on the occasion of his 70th birthday.The author wishes to express her appreciation to Prof. Stockinger for suggestion and advice.     

Fine structure and function of the gut epithelium of pike eel larvae

The development of the alimentary canal of pike eel, Muraenesox cinereus , that were hatched and reared in the laboratory, was examined. The larvae did not feed on zooplankton or phytoplankton and survived for 10 days. Histological and histochemical investigations showed that the alimentary canal was morphologically and functionally differentiated in 3 days after hatching and before exhaustion of the yolk. The foregut was not functional in absorption. The midgut absorptive cell was characterized by a well-developed lamellar membranous structure in the basal portion of the cytoplasm. Na¹-K¹ ATPase activity was demonstrated on the lamellar membrane of the structure. It suggests that water and solute transport is quite active in the midgut epithelium. Orally administered HRP (horseradish peroxidase) was ingested and intracellularly digested in the hindgut absorptive cells. Some of the HRP was transported, without complete digestion, into tissue spaces under the epithelium. Evidence suggests that pike eel larvae possibly utilize dissolved organic matter, including soluble macromolecules, as their nutrition sources.     

Secretion of Na+, K+ and fluid by the Malpighian (renal) tubule of the larval cabbage looper Trichoplusia ni (Lepidoptera: Noctuidae)

The Malpighian (renal) tubules play important roles in ionic and osmotic homeostasis in insects. In Lepidoptera, the Malpighian tubules are structurally regionalized and the concentration of Na⁺ and K⁺ in the secreted fluid varies depending on the segment of tubule analyzed. In this work, we have characterized fluid and ion (Na⁺, K⁺, H⁺) transport by tubules of the larval stage of the cabbage looper Trichoplusia ni; we have also evaluated the effects of fluid secretion inhibitors and stimulants on fluid and ion transport. Ramsay assays showed that fluid was secreted by the iliac plexus but not by the yellow and white regions of the tubule. K⁺ and Na⁺ were secreted by the distal iliac plexus (DIP) and K⁺ was reabsorbed in downstream regions. The fluid secretion rate decreased > 50% after 25 μM bafilomycin A1, 500 μM amiloride or 50 μM bumetanide was added to the bath. The concentration of K⁺ in the secreted fluid did not change, whereas the concentration of Na⁺ in the secreted fluid decreased significantly when tubules were exposed to bafilomycin A1 or amiloride. Addition of 500 μM cAMP or 1 μM 5-HT to the bath stimulated fluid secretion and resulted in a decrease in K⁺ concentration in the secreted fluid. An increase in Na⁺ concentration in the secreted fluid was observed only in cAMP-stimulated tubules. Secreted fluid pH and the transepithelial electrical potential (TEP) did not change when tubules were stimulated. Taken together, our results show that the secretion of fluid is carried out by the upper regions (DIP) in T. ni Malpighian tubules. Upper regions of the tubules secrete K⁺, whereas lower regions reabsorb it. Stimulation of fluid secretion is correlated with a decrease in the K⁺/Na⁺ ratio.     

Fine structure of the gastric epithelium of the ascidian botryllus schlosseri vacuolated and zymogenic cells

Summary Vacuolated and zymogenic cells, which are two of five cell types identified by electron microscopy in gastric epithelium of B. schlosseri, are described in detail. The vacuolated cells are characterized by one, or a few, supranuclear vacuoles containing myelin figures. A peculiar Golgi apparatus is consistently found at the base of the vacuoles; it consists of cisternae frequently containing small vesicles and tubules of constant diameter and/or a strong electron-opaque material. A variety of vesicles and multivesicular bodies are visible in the apical cytoplasm below long ribbon-like microvilli. The se findings suggest that the vacuolated cells are involved in absorptive and perhaps secretory activity. The zymogenic cells are characterized by a highly developed RER, numerous apical secretory granules and a well developed supranuclear Golgi apparatus. At the apical end, autophagosomes are frequently encountered, some of which contain also zymogen granules. Both cell types contain numerous lipid droplets, which are interpreted as an energy reserve available for the cells and for the entire colony during the change of generation. Correlation between structure and function in both cell types is discussed by taking into account the peculiar life cycle of B. schlosseri, as well as previously reported data on similar cells in other ascidians.We would like to dedicate this work to Prof. Giuseppe Reverberi on the occasion of his 70th birthday.The authors are indebted to Profs. A. Sabbadin and G. Mazzocchi for their most helpful suggestions and advice. We would also like to thank Mr. G. Tognon for technical assistance and the staff of the Stazione Idrobiologica di Chioggia for their assistance in collecting material. — This research was supported by a grant of C.N.R., contract from the Istituto di Biologia del Mare, Venezia, No. 7100396/04115542 and with the E.M. facilities of C. N. R. contract No. 70.01798.04.115.876.     

Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri. Mucous,endocrine and plicated cells

Summary The following five cell types have been recognized and defined on the basis of their fine structure in the gastric epithelium of B. schlosseri: vacuolated and zymogenic cells (described in a previous paper); ciliated mucous, endocrine and plicated cells. The ciliated mucous cells are distributed at the apex and the bottom of the gastric folds and along the dorsal groove. The mucus droplets appear to form from the Golgi complex as secretory granules of variable density and texture, which are released from the cell after fusion of their membranes with the apical plasma membrane. Holocrine or apocrine secretion has not been observed. The endocrine cells are scattered and are characterized by electron dense granules, especially numerous in the basal region of the cell. Finally, the plicated cells, present in the pyloric caecum, show rod-like microvilli, a well developed Golgi complex and abundant, deep infoldings of the basal plasma membrane, which are associated with numerous mitochondria. The possible role of the gastric cell types is discussed taking into account information concerning morphologically similar cells in other animals, as well as previously reported data on the biochemistry and physiology of digestion and excretion in ascidians.The authors are grateful to Mr. G. Tognon for technical help and to the Staff of the Stazione Idrobiologica di Chioggia for their assistance in collecting material. Work supported by a C.N.R. Grant from the Istituto di Biologia del Mare, Venezia, Contract n. 71.00396/04.115.542.     

Fine structure of the ependyma and intercellular junctions in the area postrema of the rat

Summary Ependymal cells and their junctional complexes in the area postrema of the rat were studied in detail by tracer experiments using horseradish peroxidase (HRP) and colloidal lanthanum and by freeze-etch techniques, in addition to routine electron microscopy. The ependyma of the area postrema is characterized as flattened cells possessing very few cilia, a moderate amount of microvilli, a well-developed Golgi apparatus and rough endoplasmic reticulum. Numerous vesicles or tubular formations with internal dense content were found to accumulate in the basal processes of ependymal cells; the basal process makes contact with the perivascular basal lamina. It is suggested that the dense material in the tubulovesicular formations is synthesized within the ependymal cell and discharged into the perivascular space. The apical junctions between adjacent ependymal cells display very close apposition, with a gap of 2–3 nm, but no fusion of adjacent plasma membranes; they thus represent a transitional form between the zonulae adhaerentes present in the ordinary mural ependyma and the zonulae occludentes in the choroidal epithelium. A direct intercommunication between the ventricular cerebrospinal fluid (CSF) and the blood vascular system indicates that a region exists lacking a blood-ventricular CSF barrier.     

Effects of bicarbonate on fluid and electrolyte transport by guinea pig and rabbit gallbladder: Stimulation of absorption

Summary The effect of bicarbonate (HCO₃) on fluid absorption by guinea pig gallbladder was investigatedin vitro. Stimulation of fluid absorption was concentration dependent resulting in a fourfold increase in transport over the range 1 to 50mm. Phosphate, Tris, glycodiazine and glutamine buffers failed to substitutte for HCO₃ in stimulating absorption. Unidirectional²²Na fluxes were measured across short-circuited sheets of guinea pig and rabbit gallbladders mounted in Ussing-type chambers. In both species the net Na flux was unaffected by serosal HCO₃ alone but was stimulated by addition of HCO₃ to the mucosal bathing solution. Transepithelial electrical potential difference in rabbit gallbladder was about 1.4 mV (lumen positive) when HCO₃ was present in the mucosal or in both compartments. This fell to 0.2 mV under HCO₃-free conditions or when HCO₃ was present only in the serosal solution. The respective values for guinea pig gallbladder were –1.6 and –0.6 mV (lumen negative). HCO₃ stimulation of Na absorption by guinea pig gallbladder was abolished by increasing the bathing pH from 7.4 to 7.8, an effect resulting mainly from a reduction inJ _mis ^Na . Tris buffer (25mm) inhibited HCO₃-dependent fluid absorption in this species completely at pH 8.5 and partially at 7.5. These results indicate that HCO₃ stimulates gallbladder transport in both species by an action from the mucosal side. This effect cannot be attributed to simple buffering of H⁺ but may be explained by the participation of HCO₃ in the maintenance of intracellular H⁺ for a Na/H-exchange.     

KCl transport across and insect epithelium: I. Tracer fluxes and the effects of ion substitutions

Summary Models for active Cl transport across epithelia are often assumed to be universal although they are based on detailed studies of a relatively small number of epithelia from vertebrate animals. Epithelial Cl transport is also important in many invertebrates, but little is known regarding its cellular mechanisms. We used short-circuit current, tracer fluxes and ion substitutions to investigate the basic properties of Cl absorption by locust hindgut, an epithelium which is ideally suited for transport studies. Serosal addition of 1mm adenosine 35-cyclic monophosphate (cAMP), a known stimulant of Cl transport in this tissue, increased short-circuit current (I _sc) and net reabsorptive³⁶Cl flux (J _net ^Cl ) by 1000%. Cl absorption did not exhibit an exchange diffusion component and was highly selective over all anions tested except Br. Several predictions of Na- and HCO₃-coupled models for Cl transport were tested: Cl-dependentI _sc was not affected by sodium removal (<0.05mm) during the first 75 min. Also, a large stimulation ofJ _net ^Cl was elicited by cAMP when recta were bathed for 6 hr in nominally Na-free saline (<0.001 to 0.2mm) and there was no correlation between Cl transport rate and the presence of micromolar quantities of Na contamination. Increased unidirectional influx of³⁶Cl into rectal tissue during cAMP-stimulation was not accompanied by a comparable uptake of²²Na.J _net ^Cl was independent of exogenous CO₂ and HCO₃, but was strongly dependent on the presence of K. These results suggest that the major fraction of Cl transport across this insect epithelium occurs by an unusual K-dependent mechanism that does not directly require Na or HCO₃.     

Hydrodynamic properties of mucins secreted by primary cultures of Guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the physical properties of the purified mucins. The weight average molecular mass (M _w ) for three different preparations ranged from 3.3×10⁶ to 4.7×10⁶ g/mol (corresponding to an average structure of 1 – 2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging from 4.5×10^–8 to 6.4×10^–8 cm²/s were calculated using the Svedberg equation. A polydispersity index (M _z /M _w ) of ∼1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution. A median diffusivity value of 8.05×10^–8 cm²/s (inter-quartile range = 1.11×10^–7 to 6.08×10^–8 cm²/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98×10^–9 cm²/s (inter-quartile range = 1.47×10^–8 to 3.25×10^–9 cm²/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D _20,w ) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting D _37,g was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory cell. Received: 10 March 1998 / Accepted: 27 March 1998     

Orthogonal arrays of particles in non-pigmented cells of rat ciliary epithelium: Relation to distribution of filipin- and digitonin-induced alterations of the basolateral membrane

Summary It has been suggested that orthogonal arrays of particles may increase the rigidity of plasma membrane, as does cholesterol. Therefore, using freeze-fractured non- pigmented ciliary epithelium, the distribution of such arrays was compared to the distribution of membrane deformations induced by the sterol-probes filipin and digitonin in different domains of the basolateral plasma membrane. The distribution of orthogonal arrays of particles was homogeneous between different regions of the basolateral membrane of the non-pigmented ciliary epithelium, while the number of filipin-induced alterations was nearly 4 times higher in the membrane domains not in contact with the basal lamina than in domains in contact with it. Contrary to the homogeneous distribution of arrays, digitonin-induced deformations also differed markedly in these two basolateral membrane domains. Considering that a marked positive response to sterol probes implies a high sterol content, we conclude that orthogonal arrays of particles can occur in plasma membrane regions well-provided with cholesterol and not in direct contact with the basal lamina. Other possible roles of these arrays are discussed.This paper was presented in part at the ARVO meeting, April– May 1986, Sarasota, USA     

Impact of fine or large needle aspiration on the dog's testis: in vitro ultrasonographic, bacteriological, gross anatomy and histological assessment

Despite its extensive use for evaluation of spermatogenesis and assisted reproduction, the safety and consequences of fine (FNA) and large needle aspiration (LNA) to the testicular parenchyma and its normal function have not been established. This study was performed in order to accurately assess, by serial in vitro ultrasonographic, bacteriologic, gross anatomic and histological examinations, the type and extent of the effect of FNA or LNA on the dog's testis. Twenty three sexually mature, 1 to 2 years old, healthy laboratory Beagles were randomly assigned to 2 groups: (1) 5 dogs without testicular aspiration (control group) and (2) 18 dogs in which one of their testes was aspirated using a 23 G butterfly needle and the other using a 19 G butterfly needle (experimental group). Two dogs at a time were castrated 10 minutes, 60 minutes, 2, 14, 29, 63, 76, 90 or 180 days post-aspiration. The control group was also castrated 2, 29, 63, 90 or 180 days after the beginning of the experiment. Following castration, in vitro ultrasonographic, gross anatomic, cytological examinations of epididymal sperm, bacteriologic and histological examinations of the testes were performed. Following testicular FNA and LNA bacteriologic, gross anatomic, histologic, epididymal sperm findings and the in vitro ultrasonographic appearance of the testis were normal, except of intratesticular haemorrhage, detected the first days post-aspiration, and degeneration of less than 1.5% of the seminiferous tubules. Within the parameters of this experiment, testicular FNA and LNA have no ill effect on the canine testis and therefore, both FNA and LNA should be considered safe.     

Directional protein secretion by the retinal pigment epithelium: roles in retinal health and the development of age‐related macular degeneration

The structural and functional integrity of the retinal pigment epithelium (RPE) is fundamental for maintaining the function of the neuroretina. These specialized cells form a polarized monolayer that acts as the retinal–blood barrier, separating two distinct environments with highly specialized functions: photoreceptors of the neuroretina at the apical side and Bruch's membrane/highly vascularized choriocapillaris at the basal side. The polarized nature of the RPE is essential for the health of these two regions, not only in nutrient and waste transport but also in the synthesis and directional secretion of proteins required in maintaining retinal homoeostasis and function. Although multiple malfunctions within the RPE cells have been associated with development of age‐related macular degeneration (AMD), the leading cause of legal blindness, clear causative processes have not yet been conclusively characterized at the molecular and cellular level. This article focuses on the involvement of directionally secreted RPE proteins in normal functioning of the retina and on the potential association of incorrect RPE protein secretion with development of AMD. Understanding the importance of RPE polarity and the correct secretion of essential structural and regulatory components emerge as critical factors for the development of novel therapeutic strategies targeting AMD.     

Mechanisms of fluid and ion secretion by the parotid gland of the kangaroo,Macropus rufus,assessed by administration of transport-inhibiting drugs

Possible mechanisms of primary fluid formation by macropodine parotid glands were investigated in anaesthetized red kangaroos using ion transport inhibitors. Carotid plasma amiloride concentrations of 0.05–0.5 mmol·l^-1 progressively reduced a stable acetylcholine-evoked half-maximal flow rate of 2.0±0.04 to 0.22±0.024 ml·min^-1 (mean±SEM). Concurrently, saliva bicarbonate concentration and secretion fell (135±1.6 to 67±1.7 mmol·l^-1 and 272±7.6 to 15±2.6 mol·min^-1, respectively); [phosphate], [chloride] and [sodium] rose and [potassium] and osmolality were unaltered. High-rate cholinergic stimulation did not increase saliva flow beyond 11±1.0% of that for equivalent pre-amiloride stimulation. Equipotent levels of amiloride and methazolamide given concurrently were no more effective at blocking flow and bicarbonate secretion than when given separately. Furosemide (up to 2 mmol·l^-1), bumetanide (up to 0.2 mmol·l^-1) and ethacrynate (1 mmol·l^-1) in carotid plasma had no effect on salivary flow or ion concentrations. During methazolamide blockade, furosemide did not curtail the concurrent increase in salivary [chloride]. Chlorothiazide at 0.25–1.0 mmol·l^-1 caused progressive depression of saliva flow and [bicarbonate], and elevation of [chloride]. 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid at 0.1 mmol·l^-1 was without effect, whereas at 0.5 mmol·l^-1 it stimulated fluid secretion and increased saliva [protein], [sodium], [potassium], [bicarbonate] and osmolality. Concurrently, mean arterial blood pressure and pulse pressure fell and heart rate, haematocrit and carotid artery plasma flow rose. These responses were absent if saliva flow was kept constant by reduction in cholinergic stimulation during 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid administration. It is concluded that secretion of primary fluid by the kangaroo parotid is initiated mainly (>90%) by secretion of bicarbonate which is formed in the endpiece cells from CO₂ delivered by the circulation. No evidence was found for initiation of fluid secretion by chloride transport involving basolateral Na⁺-K⁺-2Cl^- symports, Na⁺-Cl^- symports or Cl^-/HCO ₃ ^- antiports.Abbreviations CA carbonic anhydrase - CAI carbonic anhydrase inhibitors - MAP mean arterial blood pressure - PAH p-aminohippurate - SITS 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid     

Leishmania in the chick embryo: IV. Effects of embryo age and hatching,and behavior of L. donovani in cultures of chick fibroblasts

On incubation Days 9, 11, 12, 14, or 15, chick embryos were injected intravenously with 4.0 × 10⁶L. donovani amastigotes. Embryos were incubated at 33 C immediately after infection. Numbers of amastigotes found in the liver 1 hr after injection increased as the age of embryo recipients increased. Most 14- or 15-day infected embryos hatched when allowed to do so, but many younger embryos were unable to survive at 33 C. Numbers of amastigotes in the liver of chicks, hatched after infection as embryos, decreased as the cloacal temperature of the chicks increased. Despite a 31 C incubation temperature, chicks exhibited a mean 38.3 C cloacal temperature 1 day after hatching.Chick fibroblast cultures were initiated as explants of embryo brain and infected with amastigotes from hamster spleen. Only amastigotes were seen in cultures kept at 37 C, but extracellular promastigotes and intracellular amastigotes were present in cultures at 33 C. Although promastigotes increased in number in the medium overlay at 33 C, amastigotes decreased in number at 33 C and 37 C. One intracellular amastigote was seen in a culture which had been incubated at 25 C after inoculation with promastigotes.     

When nutrient limitation places bacteria in the domains of slow growth: metabolics, morphologic and cell cycle behavior

Abstract Growth systems appropriate for studying mass transfer in different bacterial environments are reviewed. Fed batch and recycling fermentors are suited to modelling nutrient limitation and slow growth. Use of these two growth systems reveals the existence of three growth rate regions, or domains, defined by maintenance energy demands, nucleotide regulation, metabolism, and physiological behavior. They are exemplified in Escherichia coli by domain-dependent synthesis of attachment antigens, heat-labile toxin, and inducible enzymes. Distribution of the bacterial population among cell cycle stages changes with growth rate domain because lengths of the stages differ in their dependence on growth rate. This produces subpopulations whose ratios vary with growth rate and that are likely to differ in both molecular composition and stress resistance.