Correlation between microtubular number and transport activity of hypothalamo-neurohypophyseal secretory neurons

Summary The lower pituitary stalk has been studied in rats given hypertonic saline for four days and in rats with congenital diabetes insipidus (GDI), that is, in two groups of animals in which there is evidence of an increase in the transport of polypeptides down the axons of the tractus hypophyseus.A quantitative ultrastructural analysis of this tract, the first yet made, has demonstrated that in normal animals it is composed of unusually small axons, over half of which are less than 300 nm in diameter.In this tract there is a significant increase in axonal diameter and microtubular content in animals given hypertonic saline for four days. In adult rats with CDI, these changes are even more marked, a feature which is correlated with the fact that in these animals the transport of polypeptides has probably been abnormal since birth.An alteration in the microtubular content of neurons has not previously been observed either in physiologically stressed animals or in diseased animals; such alterations suggest the participation of microtubules in polypeptide transport. The narrow diameter of axons in the tractus hypophyseus of the normal animal raises the possibility of the extravesicular transport of such polypeptides.Abbreviations used ADH antidiuretic hormone - AF aldehyde fuchsin - CDI congenital diabetes insipidus - HNS hypothalamo-neurohypophysiel system - NSM neurosecretory material - NSV neurosecretory vesicle - PVN paraventricular nucleus - SON supraoptic nucleus This paper is dedicated to Professor W. Bargmann.We are grateful to Miss Pauline Chapman for technical assistance.This project is supported by the Medical Research Council.     

The formation of free axonal sprouts from a dorsal root ganglion-nerve preparation maintained in organotypic culture,and the effects of demecolcine

Summary Nerve-ganglion preparations from rat dorsal spinal nerve roots were maintained in organotypic culture for 20 h. Free axonal sprouts formed at the cut tips. Clear and dense-core vesicles, mitochondria and smooth endoplasmic reticulum accumulated in the axons for a distance of 500 m behind the cut, as has previously been described in dorsal roots sectioned in vivo. Sprouting did not occur in dorsal roots maintained in culture without their ganglia attached. Sprouting was also prevented by demecolcine (3 × 10^-7 M) which reduced the number of microtubules in non-myelinated, small myelinated and large myelinated axons to respectively 45, 30 and 20% of control values. The sprouts contained several types of vesicle including small clear vesicles, large and small dense-core vesicles and flattened vesicles. The possible relevance of the vesicles to transmitter mechanisms in these neurones is discussed.I.R.D. is supported by the Medical Research Council; P.K. thanks the Mental Health Trust for a project grant     

Anion Transport in Red Blood Cells II. Kinetics of Reversible Inhibition by Nitroaromatic Sulfonic Acids

The anion exchange system of human red blood cells is highly inhibited and specifically labeled by isothiocyano derivatives of benzene sulfonate (BS) or stilbene disulfonate (DS). To learn about the site of action of these irreversibly binding probes we studied the mechanism of inhibition of anion exchange by the reversibly binding analogs p-nitrobenzene sulfonic acid (pNBS) and 4,4′-dinitrostilbene-disulfonic acid (DNDS). In the absence of inhibitor, the self-exchange flux of sulfate (pH 7.4, 25°C) at high substrate concentration displayed self-inhibitory properties, indicating the existence of two anion binding sites: one a high-affinity transport site and the other a low-affinity modifier site whose occupancy by anions results in a noncompetitive inhibition of transport. The maximal sulfate exchange flux per unit area was J_A = (0.69 ± 0.11) × 10^-10 moles · min^-1 · cm^-2 and the Michaelis-Menten constants were for the transport site K_S = 41 ± 14 mM and for the modifier site K_S' = 653 ± 242 mM. The addition to cells of either pNBS at millimolar concentrations or DNDS at micromolar concentrations led to reversible inhibition of sulfate exchange (pH 7.4, 25°C). The relationship between inhibitor concentration and fractional inhibition was linear over the full range of pNBS or DNDS concentrations (Hill coefficient n ? 1), indicating a single site of inhibition for the two probes. The kinetics of sul- fate exchange in the presence of either inhibitor was compatible with that of competitive inhibition. Using various analytical techniques it was possible to determine that the sulfate trans- port site was the target for the action of the inhibitors. The in- hibitory constants (Ki j for the transport sites were 0.45 ± 0.10 PM for DNDS and 0.21 ± 0.07 mM for pNBS. From the similarities between reversibly and irreversibly binding BS and DS inhibitors in structures, chemical properties, modus oper- andi, stoichiometry of interaction with inhibitory sites, and relative inhibitory potencies, we concluded that the anion trans- port sites are also the sites of inhibition and of labeling of co- valent binding analogs of BS and DS.     

The smooth endoplasmic reticulum in neurohypophysial axons of the rat: Possible involvement in transport,storage and release of neurosecretory material

Summary The intra-axonal organization of the smooth endoplasmic reticulum was studied in the neurohypophysis of rats during and after water deprivation. Parallel to conventional electron microscopy, the material was treated with a double impregnation staining technique specifically designed to contrast the intracellular membranous system. In conventionally stained ultrathin sections from severely dehydrated rats most axons appeared to be free of membranous organelles, whereas corresponding axons treated with the double-impregnation technique generally exhibited a highly developed system of smooth endoplasmic reticulum. In axonal endings, both techniques revealed a profusion of microvesicles in intimate relationship with tubular elements of the smooth endoplasmic reticulum. In short-term (12 h) rehydrated rats, a similarly developed system of smooth endoplasmic reticulum was still observed at all axonal levels with both procedures. After 24 to 48 h of rehydration the tubules of the smooth endoplasmic reticulum exhibited, in double impregnated material, numerous dilatations which resembled the adjacent neurosecretory granules. In conventionally stained ultrathin sections, an accumulation of electron dense material occurred within tubules of the smooth endoplasmic reticulum in the more proximal axonal segments, while in the more terminal segments, which contained numerous elongated granules, membrane continuity was frequently observed between newly formed granules and the smooth endoplasmic reticulum. After 7 days of rehydration the general pattern of the axonal smooth endoplasmic reticulum was comparable to that in untreated rats. These results are discussed in the light of a suggested involvement of the axonal smooth endoplasmic reticulum in the non-granular transport of neurosecretory material in connection with (1) storage in distally formed granules, and (2) release via microvesicles. Acknowledgements: The authors wish to express their gratitude to Mrs. M. Balmefrézol for her skillful technical assistance     

Enhanced detection and retrograde axonal transport of PrPc in peripheral nerve

Neuroinvasion of the CNS during orally acquired transmissible spongiform encephalopathies (TSEs) may involve the transport of the infectious agent from the periphery to the CNS via the peripheral nerves. If this occurs within axons, the mechanism of axonal transport may be fundamental to the process. In studies of peripheral nerve we observed that the cellular prion protein (PrPc) is highly resistant to detergent extraction. The implication of this is an underestimation of the abundance of PrPc in peripheral nerve. We have developed nerve extraction conditions that enhance the quantification of the protein in nerve 16-fold. Application of these conditions to evaluate the accumulation of PrPc distal to a cut nerve now reveals that PrPc is retrogradely transported from the axon ending. These results provide a potential cellular mechanism for TSE infectivity to gain entry to the CNS from the periphery.     

Mechanisms of axon guidance: membrane dynamics and axonal transport in semaphorin signalling

The intricate geometry of neuronal networks poses many unique cell-biological problems regarding the way a growing axon responds to its environment. Several groups of ligand-receptor pairs have been identified to regulate such processes. In this study, we take class 3 semaphorins as an example and review what is known about the intracellular movements of semaphorins throughout neuronal cells, transport support structures and location of release sites. We discuss how their receptor trafficking may contribute to regulate membrane dynamics underlying growth cone motility and the physiological contribution made by class 3 semaphorins-induced acceleration of axoplasmic transport on neurite development.     

Effect of weightlessness on colloidal particle transport and segregation in self-organising microtubule preparations

Weightlessness is known to effect cellular functions by as yet undetermined processes. Many experiments indicate a role of the cytoskeleton and microtubules. Under appropriate conditions in vitro microtubule preparations behave as a complex system that self-organises by a combination of reaction and diffusion. This process also results in the collective transport and organisation of any colloidal particles present. In large centimetre-sized samples, self-organisation does not occur when samples are exposed to a brief early period of weightlessness. Here, we report both space-flight and ground-based (clinorotation) experiments on the effect of weightlessness on the transport and segregation of colloidal particles and chromosomes. In centimetre-sized containers, both methods show that a brief initial period of weightlessness strongly inhibits particle transport. In miniature cell-sized containers under normal gravity conditions, the particle transport that self-organisation causes results in their accumulation into segregated regions of high and low particle density. The gravity dependence of this behaviour is strongly shape dependent. In square wells, neither self-organisation nor particle transport and segregation occur under conditions of weightlessness. On the contrary, in rectangular canals, both phenomena are largely unaffected by weightlessness. These observations suggest, depending on factors such as cell and embryo shape, that major biological functions associated with microtubule driven particle transport and organisation might be strongly perturbed by weightlessness.     

Characterization of tryptophan high affinity transport system in pinealocytes of the rat. Day-night modulation

Summary.  Tryptophan is required in the pineal gland for the formation of serotonin, precursor of melatonin biosynthesis. The level of this amino acid in the serum and in the pineal gland of the rat undergoes a circadian rhythm, and reduced plasma tryptophan concentration decreases secretion of melatonin in humans. Tryptophan is transported into the cells by the long chain neutral amine acid system T and by the aromatic amino acid system T. The high affinity component of [³H]tryptophan uptake was studied in pinealocytes of the rat. Inhibition was observed in the presence of phenylalanine or tyrosine, but not in the presence of neutral amino acids, alanine, glycine, serine, lysine or by 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid, a substrate specific for system L. The transport of tryptophan was temperature-dependent and trans-stimulated by phenylalanine and tyrosine, but was energy-, sodium-, chloride-, and pH-independent. In addition, the sulphydryl agent N-ethylmaleimide did not modify the high affinity transport of tryptophan in pinealocytes. The kinetic parameters were not significantly different at 12:00 as compared to 24:00 h. The treatment with the inhibitor of tryptophan hydroxylase, p-chlorophenylalanine, produced an increase in the maximal velocity of the uptake and a reduction in the affinity at 12:00, but not at 24:00 h, probably indicating that during the day, the formation of serotonin in the pineal gland is favoured by elevating the uptake of tryptophan, whereas at 24:00 h other mechanisms, such as induction of enzymes are taking place. High affinity tryptophan uptake in the rat pineal gland occurs through system T and is upregulated during the day when the availability of serotonin is reduced. Received March 15, 2001 Accepted July 8, 2002 Published online January 20, 2003 Acknowledgements This work was supported by the Grant S1-3490 from Consejo Nacional de Investigaciones Cientificas y Tecnológicas (CONICIT), Venezuela. We appreciate the secretarial assistance of Mrs. Isabel Otaegui. Carmen I. Gutiérrez is a PhD Student from Ciencias Fisiológicas, Facultad de Medicina, Universidad Central de Venezueta (UCV), Caracas, and supported by Universidad Francisco de Miranda, Coro, Falcón, Venezuela. Joseph Glykys is a Medical Student from Universidad de Carabobo, Valencia, Venezuela, and an Assistant Student of Centro de Estudios Avanzados, IVIC. Authors' address: Dr. Lucimey Lima, Laboratorio de Neuroquímica, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Apdo. 21827, Caracas 1020-A, Venezuela, Fax: 58-212-504-1295, E-mail: llima@cbb.ivic.ve     

Changes in axonal transport in neurones of Asterias vulgaris and Asterias forbesei produced by colchicine and dimethyl sulfoxide

Summary Gas flow analysis, autoradiography, and electron microscopy were performed on radial nerve cords of sea stars injected intragastrically with [¹⁴C] leucine tracer, a precursor of axonal proteins. Neurotubules, neurofilaments, and axonal transport were demonstrated for the first time in sea stars. This transport occurred at two rates (fast phase: 240–480 mm/da, slow phase: 20–40 mm/da) moving in both the cellulifugal and centripetal directions. The rate in the centripetal direction was slightly slower. Displacement of peaks of radioactivity appeared cyclic.Transneuronal transport apparently occurred between several groups of neurones suggesting the possibility of metabolic communication, perhaps across the synapse, between groups of neurones or even areas of the nervous system. Regenerating nerve cords demonstrated an increase in the rate (400–600 mm/da) and quantity (6–11 times as much) of label transported to cord termini.The relationship between colchicine and the disruption of neurotubular integrity, resulting in disturbances in fast phase transport, was confirmed. More significantly, the association between nerve injury and site or concentration of treatment is emphasized. Treatment of nerve cords with dimethyl sulfoxide (DMSO) produced more subtle effects than did colchicine, reversible 4–6 hours following the local treatment of the involved nerves. Axon bundles swelled and the organization of axons within the bundles was distorted. Stereochemical structure was suggested from this work as playing a key role in effecting fast phase transport.The authors are indebted to Dr. B. Kiefer, Department of Biology, Wesleyan University, Dr. L. Ross, Department of Anatomy, Medical College of Pennsylvania, and Dr. M. Shelanski, Department of Molecular Biology, Harvard University for their help and constructive criticism.     

Effect of 2,5-hexanedione and 3,4-dimethyl-2,5-hexanedione on retrograde axonal transport in sciatic nerve

The effects of systemically introduced neurotoxic solvents 2,5-hexanedione (2,5-HD) and 3,4-dimethyl-2,5-hexanedione (DMHD) on retrograde axonal transport (RT) of¹²⁵I-labeled tetanus toxin (TT) was studied in rat and mouse sciatic nerves. The rate of retrograde transport of TT in control rat sciatic nerves was slightly higher (6.8±0.4 mm/h) than in mouse sciatic nerves (5.4±0.5 mm/h). A single high dose of 2,5-HD (1,000 mg/kg, i.p.) produced a time-dependent effect on RT in mouse sciatic nerves. 2,5-HD caused a gradual decrease in the velocity of RT (approximately 65% inhibition between 2.0–2.5 h) with a reversal to normal rate 3–5 h after the toxin administration. The effect of DMHD on RT was examined following semi-chronic treatment in rats. DMHD caused a significant decrease (approximately 50%) in the rate of TT transport, in addition, it produced weight loss and hind-limb paralysis.I had the good opportunity of being a member of Professor Alan N. Davison' research team during 1971–1977. This research paper is dedicated to his retirement.     

Metabolism and axonal transport of polyamines in a single identified neuron of Aplysia californica

Abstract: The metabolism of polyamines was investigated by injecting purified [³H]putrescine directly into the soma of the giant neuron R2 of Aplysia . Injected putrescine was rapidly metabolized to spermidine, spermine, and several catabolites, including GABA and monoacetylputrescine. Identification of these products was by comparison with the authentic compound using ion exchange chromatography. When R2 was injected with amounts of [³H]putrescine determined so that the intracellular content of labeled precursor was less than 6 × 10-⁶ M , metabolism was rapid and occurred via pathways similar to those in mammalian tissues. At concentrations of labeled precursor greater than 2 × 10⁻⁴ M , relatively little putrescine was converted to product. By 4 h after injection, putrescine and its labeled products appeared in R2's axon, where additional metabolism occurred. These results indicated that the enzymes involved in polyamine interconversion are not restricted to R2's cell body, and this suggestion was corroborated by finding ornithine decarboxylase and S -adenosylmethionine decarboxylase activities in Aplysia nerves. The distribution of the polyamines along R2's axon was compared with that of ³H-glycoproteins, with the finding that while the acid-soluble polyamines move by diffusion, labeled polyamines associated with protein are rapidly transported.     

An inducible,specific and derepressible transport of l-serine in Saccharomyces cerevisiae

Both rho⁺ and rho^? cells were capable of accumulating l-serine against a concentration gradient; however, the extent of serine accumulation differed between these two strains. About 60% of the total accumulation of serine was reduced in rho^? cells which were shown to lack functional mitochondria. The transport of serine was mediated via a specific and an inducible system. It was also derepressible under nitrogen-starved conditions. The derepression of l-serine uptake was also evident under conditions where general amino-acid permease is not expressed.     

Effect of dibutyryl cyclic adenosine monophosphate on active amino acid transport in Streptomyces hydrogenans

The active uptake of 2-aminoisobutyric acid (AIB) and several other amino acids in resting cells of Streptomyces hydrogenans was found to be stimulated by exogenously added adenosine cyclic monophosphate (cAMP). The uptake of glycerol, sorbose, and pyrimidine nucleosides remained unaffected. Among the various cAMP derivatives tested, the dibutyryl derivative was found to be most effective, followed by monobutyryl cAMP, and cAMP. Dibutyryl cGMP was also found to stimulate AIB transport, and its effectivity was as good as that of dibutyryl cAMP. The effect of dibutyryl cAMP is time dependent and attains its maximum after 40–60 min of incubation at 30°C in K-Na-phosphate buffer. Dibutyryl cAMP-dependent transport stimulation has a high temperature coefficient and is prevented by rifamycin SV or chloramphenicol. The rate of leucine incorporation into protein was rapidly increased upon addition of dibutyryl cAMP. Kinetic studies reveal that the stimulation of AIB transport is characterized by an increase in maximum uptake rate and an unaltered apparent Michaelis constant. Analysis of the unidirectional fluxes show that both influx and efflux are enhanced by dibutyryl cAMP. It is concluded that exogenous dibutyryl cAMP stimulates de novo synthesis of certain protein including the transport catalysts for various amino acids.     

Protein domains involved in nuclear transport of Fos.

The protein encoded by the proto-oncogene c-fos is constitutively nuclear in most cell types analyzed. It has a predicted molecular weight of about 55 kDa. Proteins with a molecular weight above 40 kDa cannot enter the nucleus passively. Our interest was to study which regions in the protein are involved in the nuclear transport. We prepared a series of deletions and point mutations of the protein and cloned the mutated genes into a eukaryotic expression vector. Cos-1 cells were used to express the mutants transiently. Using indirect immunofluorescence we studied the subcellular localization, analyzing the percentage of cells containing the protein in the nucleus, the cytoplasm, or both locations. Our studies showed that the Fos protein contains several regions which can act independently to translocate the protein into the nucleus.     

Kinetin-promoted transport of assimilates in stems of Phaseolus vulgaris L.

Kinetin, applied as a dispersion in aqueous lanolin to the stumps of decapitated stems of P. vulgaris plants with their roots removed, was found to promote the transport of ¹⁴C- and ³²P-labelled assimilates to the site of hormone application. Measurement of photosynthetic rate of, and assimilate export rate from the source leaves, indicated that kinetin was not acting to promote assimilate transport by stimulating these processes. Moreover, it was found that the time between kinetin application and detection of an enhanced transport flux was independent of the distance over which kinetin would need to move to be present throughout the length of the transport pathway. These observations, together with the finding that lateral applications of kinetin to the stems resulted in an enhanced localized accumulation of assimilates, provided evidence that kinetin acted locally at its point of application to stimulate assimilate transfer.Abbreviations GA₃ gibberellic acid - IAA indol-3yl-acetic acid     

Threshold distances and depths of nucleopolyhedrovirus in soil for transport to cotton plants by wind and rain

Two aspects of abiotic transport of nucleopolyhedrovirus from soil to cotton plants were examined in greenhouse experiments: the distance from the plants and depth in soil from which the virus could be transported under controlled conditions of soil type and moisture, wind, and precipitation. Transport distance and depth were tested separately under relatively conducive (precipitation/sandy soil and wind/clay soil) and non-conducive (precipitation/clay soil and wind/sandy soil) conditions, as determined in previous research. The amount of virus transported by precipitation generally decreased as distance from the plant increased, but in wind the amounts of virus transported were best described by polynomial models, with transport efficiency usually peaking at a distance of 60 cm. Depending on plant height and tissue, the farthest distances that virus was transported ranged from 30 to 60 cm by precipitation from clay soil, 60-75 cm in precipitation/sand, 60-80 cm in wind/clay, and 60-80 cm in wind/sand. In the depth experiments, transport by precipitation and wind generally decreased as the depth of virus in soil increased. The greatest depth from which NPV was transported ranged from 0 to 0.5 cm by precipitation from clay soil, 0.5-1.0 cm in precipitation/sand, 1.0-2.0 cm in wind/clay, and 0.5-1.0 cm in wind/sand. All of the experimental parameters (distance or depth, soil type, plant height, plant tissue) and all two-way interactions significantly (P<0.05) affected transport in all four experiments, except for the "soilxplant tissue" interaction in the depth/wind experiment. In all of the experiments, transport was significantly greater (P<0.05) to lower than to upper portions of plants and to leaves than to buds and squares. Transport was significantly greater from sandy soil than from clay in precipitation, and it was greater from clay than from sandy soil in wind. The results will contribute to NPV epizootiology, microbial control, and risk assessment.     

Structure and function of the microtubular cytoskeleton during megasporogenesis and embryo sac development in Gasteria verrucosa (Mill.) H. Duval

Summary Using immunocytochemical techniques, tubulin distribution in various stages of meiosis and embryo sac development was studied. In the archespore cell some microtubules appeared to be randomly oriented. During zygotene and pachytene, when the cell volume increases, a large number of microtubules in dispersed configurations and bundles were observed. During this stage the nucellar cells divide, and their parallel cortical microtubules play an important role in preparing the direction of cell enlargement. The protoderm cells show anticlinal-directed cortical microtubules. It can be concluded that the enlargement of the meiocyte during these early meiotic stages is influenced both by its own cytoskeleton and by growth of the nucellus. Thereafter, the microtubules function directly in meiosis and disappear for the greater part until the two-nucleate coenocyte is formed. In a four-nucleate coenocyte microtubules reappear around the nucleus; in a young synergid, randomly oriented microtubules are involved in cell shaping during the formation of the filiform apparatus; in the synergids of the mature embryo sac, many parallel arrays of microtubules are present. Microtubules are less abundant in other cells. It is concluded that the cytomorphogenesis of the developing coenocyte and embryo sac are due to cell growth of the nucellar cells together with vacuolation of the coenocyte.