Stop-flow: a new technique for measuring axonal transport, and its application to the transport of dopamine-beta-hydroxylase.

An apparatus was devised which utilizes local cooling to reversibly interrupt the axonal transport of dopamine-beta-hydroxylase (DBH) in rabbit sciatic nerves in vitro. Lowering the temperature of a short region of nerve to between 1 and 3 degrees C, while keeping the remainder at 37 degrees C, caused DBH activity to accumulate in and proximal to the cooled region. This accumulation was evident after 0.5 hr of cooling and increased in a nearly linear fashion with time for about 3 hr. The cooling-induced interruption in transport was rapidly reversed when nerves were rewarmed to 37 degrees C. Upon rewarming after local cooling for 1.5 hr, a peak of accumulated DBH activity migrated toward the distal end of the nerve at a velocity of 300 +/- 17 mm/day. This velocity was maintained for as long as the peak could be followed and was four times greater than the average velocity estimated from the rate of accumulation of DBH activity above a ligature at the distal end of these same nerves. It is concluded that ligation experiments grossly underestimate the true velocity of axonal transport of DBH and that the present technique offers great advantages in permitting direct study of the migration of separate axonal compartments of transported materials.     

Direct comparison of the rapid axonal transport of norepinephrine and dopamine-β-hydroxylase activity

Stop-flow techniques were used to examine the rapid axonal transport of norepinephrine in rabbit sciatic nerves. When the midpoint of a nerve incubated in vitro was cooled to 2°C while the remainder was kept at 37°C, norepinephrine accumulated proximal to the cooled region at a rate corresponding to an average transport velocity between 5 and 6 mm/hr in a distal direction. Since only about half of the norepinephrine appeared to be free to move, the mean velocity of the moving fraction was probably twice as great. No norepinephrine accumulated distal to a broad cooled region under conditions in which there would have been a significant accumulation of dopamine-β-hydroxylase activity. Therefore, unlike dopamine-β-hydroxylase, norepinephrine may not be subject to rapid retrograde transport. When nerves that had been locally cooled for 1.5 hr were rewarmed uniformly to 37°C, a wave of norepinephrine moved exclusively in a distal direction. The peak of this wave moved at a velocity of 12.2 ± 0.5 mm/hr or 293 ± 12 mm/day; the front of the wave moved at about 18 mm/hr. or 430 mm/day; and the tail probably moved faster than 6 mm/hr. This spectrum of velocities was virtually identical to the one displayed by the wave of dopamine-β-hydroxylase activity that was generated under the same conditions. Our results are consistent with the conclusion that all axonal structures containing norepinephrine also contain dopamine-β-hydroxylase, but they are not consistent with the converse.     

New technique for measuring dynamic axonal transport and its application to temperature effects

A new technique was devised for the dynamic detection of the axoplasmic transport of β-radioactively labeled materials in which a semiconductor radiation detector was used as the β-ray counter. The detector element is a silicon p-n junction diode and has a diameter of 2.0 mm. With this detector, the β-radioactive distribution of axoplasmic transport could be measured in an axon maintained physiologically without cutting nerves. This method makes possible determination of the transport rate using one bundle of peripheral nerves. The rate in the bullfrog was 6.4 mm per hour at 24.0 °C. Temperature effects on the bullfrog axoplasmic transport were also observed at different temperatures, ranging from 5.0 to 24.0 °C. At these temperatures the rate increased as an exponential function of temperature from 1.1 to 6.4 mm per hour. Within this temperature range, the Q₁₀ is 2.5 and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 Kcal. This technique offers great advantages in permitting direct study of the axoplasmic flow of the axon in a physiological condition.     

New technique for measuring dynamic axonal transport and its application to temperature effects.

A new technique was devised for the dynamic detection of the axoplasmic transport of beta-radioactively labeled materials in which a semiconductor radiation detector was used as the beta-ray counter. The detector element is a silicon p-n junction diode and has a diameter of 2.0 mm. With this detector, the beta-radioactive distribution of axoplasmic transport could be measured in a axon maintained physiologically without cutting nerves. This method makes possible determination of the transport rate using one bundle of peripheral nerves. The rate in the bullfrog was 6.4 mm per hour at 24.0 degrees D. Temperature effects on the bullfrog axoplasmic transport were also observed at different temperatures, ranging from 5.0 to 24.0 degrees C. At these temperatures the rate increased as an exponential function of temperature from 1.1 to 6.4 mm per hour. Within this temperature range, the Q10 is 2.5 and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 Kcal. this technique offers great advantages in permitting direct study of the axoplasmic flow of the axon in a physiological condition.     

Catecholamines and dopamine-β-hydroxylase activity during therapeutic abortion induced by sulprostone

To evaluate the changes in circulating norepinephrine (NE), epinephrine (E) and dopamine-β-hydroxylase (DBH) caused by an intravenous infusion of a derivate of PGE₂, sulprostone, in connection with legal termination of pregnancy, serial plasma samples were analyzed for six gravidae. Plasma catecholamines were measured by a sensitive radioenzymatic method (9,10) and DBH activities by a photometric assay (11). Intravenous infusion of sulprostone, in abortifacient doses as an intravenous infusion of 3–4 μg per minute for six to eight hours produced a decrease in circulating norepinephrine. No significant alteration was found in plasma epinephrine or dopamine-β-hydroxylase activity. The finding suggests an inhibitory effect of sulprostone on the release of norepinephrine from the adrenergic terminals without inhibition of the adrenal medulla.     

A model for slow axonal transport and its application to neurofilamentous neuropathies

A model for slow axonal transport is developed in which the essential features are reversible binding of cytoskeletal elements and of soluble cytosolic proteins to each other and to motile elements such as actin microfilaments. Computer simulation of the equations of the model demonstrate that the model can account for many of the features of the SCa and SCb waves observed in pulse experiments. The model also provides a unified explanation for the increase and decrease of neurofilament transport rates observed in various toxicant-induced neuropathies.     

Biochemical evidence for the presence of small and large noradrenergic storage vesicles isolated from cat ovary in isoosmotic conditions. Distribution of dopamine-β-hydroxylase activity

The cat ovary presents unusually high levels of noradrenaline that change according to the endocrine status of the animal. Their functional meaning remains unknown. The cat ovary innervation, unlike that of other organs receiving noradrenergic innervation, has been poorly characterized on biochemical grounds. We present here a biochemical characterization of the neurotransmitter storage. By using hyperosmotic and isoosmotic gradients evidence is presented that noradrenaline is associated to two different populations of vesicles. In hyperosmomolarity conditions (sucrose gradients) “light” vesicles (density 1.12 g/ml) and “heavy” vesicles (density 1.17 g/ml) appeared. In both vesicles, noradrenaline and dopamine-β-hydroxylase were found. In isoosmotic Percoll gradients distribution of the markers also suggested the presence of two vesicle populations. Light vesicles (density 1.033 g/ml) with high dopamine-β-hydroxylase activity but very low levels of noradrenaline and adenosine triphosphate; [³H]noradrenaline, used as a specific exogenous vesicle marker, was feebly incorporated in this fraction. Heavy vesicles (density 1.041 g/ml) containing high levels of noradrenaline, adenosine triphosphate, low levels of dopamine-β-hydroxylase activity are able to incorporate high amounts of [³H]noradrenaline. In these gradients, Mg²⁺ activated ATPase activity was present in both vesicle fractions.

Sedimentation analysis by analytical differential centrifugation also disclosed two types of vesicles: large vesicles with a sedimentation coefficient between 348 and 308 and small vesicles with a sedimentation coefficient of 96 . Large vesicles were associated with noradrenaline-β-hydroxylase activity, while small vesicles were associated only with noradrenaline.

In isoosmotic conditions the use of other microsomal markers allowed us to define the degree of contamination of the vesicle fractions. It was found that the noradrenergic heavy vesicles fraction presented under 11% of 5′-nucleotidase activity of the total activity present in the gradient and less than 5% of acid phosphatase, NADH-cytochrome c reductase and monoaminooxidase of the total activities in the gradients.

In isoosmotic conditions the physical properties of presumed vesicles were apparently undisturbed supporting the current morphometric observations. Our results then suggest prevailing roles for each type of vesicle: synthesis for light vesicles, and storage and/or release for heavy ones.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

β‐Tubulin mutations that cause severe neuropathies disrupt axonal transport

Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed β‐tubulin mutations that cause neuronal diseases and we have identified mutations that strongly inhibit axonal transport of vesicles and mitochondria. These mutations are in the H12 helix of β‐tubulin and change the negative charge on the surface of the microtubule. This surface is the interface between microtubules and kinesin superfamily motor proteins (KIF). The binding of axonal transport KIFs to microtubules is dominant negatively disrupted by these mutations, which alters the localization of KIFs in neurons and inhibits axon elongation in vivo. In humans, these mutations induce broad neurological symptoms, such as loss of axons in the central nervous system and peripheral neuropathy. Thus, our data identified the critical region of β‐tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations.     

Study of the enhancement of a new chemiluminescence reaction and its application to determination of β‐lactam antibiotics

An enhanced thiosemicarbazide(TSC)–H₂O₂ chemiluminescence (CL) system was established and proposed as a new analytical method for determination of β‐lactam antibiotics, ampicillin sodium and amoxicillin at microgram levels. The method is based on the inhibition of CL emission accompanying oxidation of TSC by H₂O₂ in alkaline medium. The effect of anionic, cationic, and non‐ionic surfactants on the CL emission of the system was studied. Both N‐cetyl‐N,N,N‐trimethylammonium bromide (CTMAB) and Triton X‐100, unlike sodium dodecyl sulfate (SDS), reinforced the CL intensity and were efficient to approximately the same level. The effect of the presence of eight non‐aqueous solvents on the CL system was also investigated. Upon addition of both of the non‐ionic surfactant, Triton X‐100, and the non‐aqueous solvent, N,N‐dimethyl formamide (DMF), the intensity of the CL reaction was increased 100‐fold. This method allows the measurement of 25–545 µg amoxicillin, and 35–350 µg ampicillin sodium. The detection limits are 8 µg for amoxicillin and 9 µg for ampicillin sodium. The relative standard deviations of six replicate measurements of 200 µg amoxicillin and 200 µg ampicillin sodium were 1.9 and 2.1%, respectively. The effect of foreign species on the determination of amoxicillin and ampicillin sodium was also examined. The proposed method was successfully applied to the determination of ampicillin sodium and amoxicillin in some pharmaceutical dosage forms. Copyright © 2008 John Wiley & Sons, Ltd.     

Linkers, packages and pathways: new concepts in axonal transport

The molecular mechanisms that generate efficient and directed transport of proteins and organelles in axons remain poorly understood. In the past year, many studies have identified specific transmembrane or scaffold proteins that might link motor proteins to their cargoes. These studies have also identified previously unsuspected pathways and raised the intriguing possibility that pre-packaged groups of functionally related proteins are transported together in the axon. Evidence suggests that fast molecular motor proteins have a role in slow axonal transport, and the axonal transport machinery has been implicated in the genesis of neurodegenerative diseases.     

Kinesin‐1/Hsc70‐dependent mechanism of slow axonal transport and its relation to fast axonal transport

Cytoplasmic protein transport in axons (‘slow axonal transport’) is essential for neuronal homeostasis, and involves Kinesin‐1, the same motor for membranous organelle transport (‘fast axonal transport’). However, both molecular mechanisms of slow axonal transport and difference in usage of Kinesin‐1 between slow and fast axonal transport have been elusive. Here, we show that slow axonal transport depends on the interaction between the DnaJ‐like domain of the kinesin light chain in the Kinesin‐1 motor complex and Hsc70, scaffolding between cytoplasmic proteins and Kinesin‐1. The domain is within the tetratricopeptide repeat, which can bind to membranous organelles, and competitive perturbation of the domain in squid giant axons disrupted cytoplasmic protein transport and reinforced membranous organelle transport, indicating that this domain might have a function as a switchover system between slow and fast transport by Hsc70. Transgenic mice overexpressing a dominant‐negative form of the domain showed delayed slow transport, accelerated fast transport and optic axonopathy. These findings provide a basis for the regulatory mechanism of intracellular transport and its intriguing implication in neuronal dysfunction.     

A new methodological approach for studying axonal transport: cytofluorometric scanning of nerves

A new technique for studying axonal transport has been developed. The technique, which is based on histofluorescence techniques, enables the measurement of several different accumulated substances and parameters within a single nerve in relation to a nerve crush or local cooling. Any substance that can be made to fluoresce can be measured. The tissue is treated according to the formaldehyde-induced fluorescence method of Hillarp and Falck for visualization of monoamines, or according to the indirect immunofluorescence method. For immunofluorescence the nerve is cryostat-sectioned and various sections can be incubated with primary antisera against different antigens. After incubation and mounting the sections are placed in a cytofluorimeter (Leitz MPV II). They are passed under a measuring slit at a steady speed by a motor driven cross-table. The fluorescence intensity passing through the measuring slit is continuously registered by a recording unit with an integrator. This recorder produces a graphical nerve accumulation profile, and the area under the profile, relating to the fluorescence, is expressed in arbitrary units. This article presents data on the accumulation of noradrenaline, dopamine beta-hydroxylase, and tyrosine hydroxylase in crush-operated rat sciatic nerve. The time-course accumulations for noradrenaline (visualized by the Falck and Hillarp method) and dopamine beta-hydroxylase (visualized by immunofluorescence) demonstrated a striking similarity, which is to be expected since the two substances are stored in the same organelle. Tyrosine hydroxylase (visualized by immunofluorescence) showed a slower accumulation with time, but faster than would be expected had the enzyme been 100% soluble. Colchicine but not lumi-colchicine blocked the transport of noradrenaline organelles. With the new scanning technique we have the potential to study accumulation profiles of several different substances within a single nerve. Morphometric data, morphological observations, and photograph documentation of the same nerve section are also available.     

Dopamine β-hydroxylase distribution in density gradients: Physiological and artefactual implications

Knowledge of the vesicular origin of circulating dopamine β-hydroxylase (DβH) is indispensable for any attempts to explain the parallelism or lack of it between circulating enzyme and catecholamines as they may relate to physiological stress, forms of hypertension, neurological disorders, and the response to pharmacological agents. The present study represents an effort to evaluate and to place in proper perspective data based on the DβH activity found in the region of the light vesicle peak of noradrenaline (NA), which is used as a quantitative measure of a population of small terminal vesicles. Distributions of vesicles and subvesicular components are compared with DβH and NA in sucrose-D₂O density gradients used to prepare relatively pure fractions of large dense cored vesicles (LDV) from bovine splenic nerve. Although NA in sedimentable particles of the light vesicle peak is likely to be a valid measure of a small vesicle population, the following is demonstrated: (1) A substantial fraction (25%–37%) of the total sedimentable DβH acitivity can be proven to distribute in the region of the light vesicle peak from a tissue with an insignificant small vesicle population. Based on studies of vesicles from sequential nerve segments, this enzyme activity probably corresponds to a population of “immature” LDV which are undergoing axoplasmic transport and have not synthesized their full complement of transmitter. (2) Physical lysis which depletes the matrix of LDV causes redistribution of DβH activity from the heavy vesicle peak into the region of the light vesicle peak. Analogously, DβH associated with exocytosed LDV and retrograde transport particles is also likely to contaminate the region of the light vesicle peak. (3) Based on available data, it can be calculated that each small dense cored vesicle could contain only 0.1–0.5 molecules of DβH and that a contamination of only 0.016% LDV can account for all of the DβH reported to occur in the light vesicle peak of normal rat vas deferens preparations.     

Extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by a benign testicular leydig cell tumor

We present an unusual case with bilateral testicular Leydig cell tumors displaying extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase. Histological examination of a 38-yr-old man infertile due to azoospermia showed him to have bilateral testicular Leydig cell tumors. The in vitro steroidogenic potential of the tumors and their adjacent testicular tissue was evaluated using organ culture. Tumor tissue was found to secrete deoxycorticosterone (DOC), corticosterone (B) and cortisol, which are not produced in normal adult testis, into the medium, while testicular tissue adjacent to the tumors secreted a small amount of DOC and B. Northern blot analysis with cytochrome P-450_C21 complementary DNA (cDNA) and P-450_11β cDNA as probes revealed that the tumor contained a considerable amount of mRNA for P-450_C21 and P-450_11β, while the mRNAs were not detected in the testicular tissues adjacent to the tumors. It is suggested that the high local levels of estrogen and/or progesterone within the Leydig cell tumors and their adjacent testicular tissues induced extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by the tumors and their adjacent testicular tissues.