Measurement of the positive muon lifetime and determination of the Fermi constant to part-per-million precision.

We report a measurement of the positive muon lifetime to a precision of 1.0 ppm; it is the most precise particle lifetime ever measured. The experiment used a time-structured, low-energy muon beam and a segmented plastic scintillator array to record more than 2×10(12) decays. Two different stopping target configurations were employed in independent data-taking periods. The combined results give τ(µ(+)) (MuLan)=2 196 980.3(2.2) ps, more than 15 times as precise as any previous experiment. The muon lifetime gives the most precise value for the Fermi constant: G(F) (MuLan)=1.166 378 8(7)×10(-5) GeV(-2) (0.6 ppm). It is also used to extract the µ(-)p singlet capture rate, which determines the proton's weak induced pseudoscalar coupling g(P).

Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.

An in vitro nuclear translocation system is described in which isolated rat liver nuclei were incubated in a defined buffered medium containing radiolabeled or fluorescently labeled exogenous proteins. The nuclei were rapidly recovered, extracted, and analyzed for the presence of associated radiolabeled or fluorescently labeled proteins. The isolated nuclei exhibited the same specificity for protein uptake as seen previously in vivo, accumulating simian virus 40 wild-type large-T antigen and p53 while excluding a cytoplasmic variant of large-T antigen (d10) and bovine serum albumin. The rapid nuclear accumulation of wild-type large-T antigen was shown to be selective and dependent upon the recognition of a wild-type nuclear location signal, ATP and temperature dependent, and unidirectional. Taken together, the data suggest that in our in vitro system the nuclear translocation of wild-type large-T antigen exhibits some of the characteristics of an active transport process.

Exercise enhances axonal growth and functional recovery in the regenerating spinal cord.

We investigated whether enhancing locomotory activity could accelerate the axonal growth underlying the significant recovery of function after a complete spinal transection in the eel, Anguilla. Eels with low spinal transections (at about 60% body length) were kept in holding tanks, where they were inactive, or made to swim continually against a water current at about one body length/s. Their locomotion was periodically assessed by measuring tail beat frequencies at different swimming speeds. Axonal growth was determined from anterograde labeling with 1,1'-diotadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, inserted postmortem into the spinal cord, just rostral to the transection. Twenty days after surgery, there were significantly more labeled growth cones more than 2 mm caudal from the transection in the exercised fish (74.6+/-2.3%; cf. 34.5+/-1.1%). This difference was still observed at 40 days (57.9+/-1.6% cf. 42.1+/-2% >2 mm), but the regenerated axons were of similar maximum lengths by 120 days (9.8+/-0.3 cf. 7.7+/-2.8 mm). After surgery, each eel undulated its whole body faster at any given swimming speed, thus changing the linear relationship between tail beat frequency and forward speed established before transection. The slope increased by up to 112.5+/-27.4% over the first 8 days post-surgery in inactive animals, while a smaller rise (45.6+/-10.5%) was observed in exercised fish during this period. Thereafter, the slope progressively declined to pre-surgery levels in both groups of animals, but the recovery occurred within 20+/-4 days in exercised eels, as opposed to 40+/-5 days in inactive fish. The locomotory performance of sham-operated fish was unaffected by 10 days of continual locomotion and remained similar to that of naïve eels, pre-transection. These data show that elevated locomotory activity enhances axonal growth and accelerates recovery of locomotory function.

A cancer gene therapy approach utilizing an anti-erbB-2 single-chain antibody-encoding adenovirus (AD21): a phase I trial.

The purpose of this Phase I study was to determine the feasibility of using an anti-erbB-2-encoding adenovirus (Ad21) to treat erbB-2-overexpressing ovarian cancer. Recurrent ovarian cancer patients were treated i.p. with Ad21 in dosages ranging from 1 x 10(9) to 1 x 10(11) pfu. Patients were monitored after treatment for evidence of clinical toxicity and efficacy. Peritoneal aspirates and serum samples were obtained to assess for evidence of gene transfer/expression, for generation of wild-type vector, and antiadenoviral humoral response. Fifteen patients were treated per study specifications. Treatment-specific grade 1/2 fever was experienced by 9 of 15 (60%) patients. Other transient grade 1/2 constitutional, pain, and gastrointestinal symptoms were also experienced. No dose-limiting vector-related toxicity was experienced. Of 13 patients evaluable for response, 5 (38%) had stable disease and 8 (61%) had evidence of progressive disease. One patient with nonmeasurable disease normalized her CA125 at the 8-week evaluation, and one patient with nonmeasurable disease remained without clinical evidence of disease for 6 months after treatment. PCR analysis of peritoneal aspirates demonstrated the presence of Ad21 in 84.6%, 84.6%, and 61.6% of evaluable specimens at days 2, 14, and 56 after treatment, respectively. No wild-type virus was detected. Reverse transcription-PCR analysis demonstrated expression of the anti-erbB-2 sFv-encoding gene in 10 of 14 evaluable patients at day 2. Five of six evaluable patients had an increase in antiadenovirus antibody titer. This study suggests that adenoviral-mediated gene therapy using an anti-erbB-2-directed intrabody is feasible in the context of human ovarian cancer.

Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13.

Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage.

Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors.

We report display of the complete protease inhibitor (Kunitz) domain, BPTI, on the surface of bacteriophage M13 as a fusion to the gene III product. Phage that display BPTI bind specifically to anti-BPTI antibodies, trypsin and anhydrotrypsin. A point mutation of BPTI [Lys15-->Leu(K15L)] alters the binding specificity of fusion phage such that a human neutrophil elastase-binding phenotype is conferred while a trypsin-binding phenotype is eliminated. Phage were eluted from an immobilized protease with step gradients of decreasing pH. Phage that display Kunitz domains having higher affinity for the immobilized protease exhibit characteristic pH elution phenotypes, indicating that bound display phage can be selectively recovered from an affinity matrix. Utilization of this technology should enable the selection of remodeled protease inhibitors exhibiting novel binding specificities.

Distribution and morphological characteristics of efferent neurons innervating end organs in the ear and lateral line of the European eel.

Neurons that provide the efferent innervation to the labyrinthine and lateral line sense organs of the eel were located by applying horseradish peroxidase to branches of the appropriate cranial nerves. Retrogradely labeled neurons were found in a single median column, the octavolateralis efferent nucleus (OEN), located immediately rostral to and overlapping with the facial nucleus of the branchiomotor column. We estimate that on each side of the brain the efferent nucleus contains about 60-70 neurons, most of which supply the ear and the lateral line system of the head. Most neurons (approximately 90%) are ipsilateral to the targets they innervate. There is no crisp topographical order within the nucleus because neurons supplying different end organs intermingle. However, the head lateral line is supplied by rostrally located neurons, and the body system by more caudal neurons. There are no marked differences in cell form between neurons supplying different targets. Most are multipolar, relatively uniform in size, and have extensive dendrites. The dendrites of some cells extend to the contralateral side of the brain. Efferent axons are of small diameter (approximately 3 micron). Two neurons are sufficiently constant in size and location that they can be consistently recognized from fish to fish. Their axons branch to supply more than one target. Nearly all efferent neurons stain for acetylcholinesterase and some, bordering the midline, consistently stain weakly.

Medullary and cerebellar projections of the statoacoustic nerve of the dogfish, Scyliorhinus canicula.

The statoacoustic nerve of the dogfish, Scyliorhinus canicula, was transected medial to the ganglion for the purpose of elucidating its central pathways and terminal fields. Following two to six weeks postoperative survival times, transverse, horizontal, and sagittal sections of the brain stem were stained by the Fink-Heimer silver-impregnation method to reveal degenerating axons and terminals. Fragmented axons enter the medulla and give rise to medial, descending, and ascending pathway. Fibers of the medial pathway terminate about the soma and lateral dendrites of the large cells that comprise nucleus magnocellularis; descending and ascending fibers terminate on the dendrites of the cells of ventral and superior nuclei respectively. In addition, fibers emanate from fascicles of the descending pathway to form a large field of degenerating axons and terminals within the ventromedial part of the medulla, and a substantial proportion of the fibers of the ascending pathway continues beyond the superior nucleus to terminate among the granule cells of the medial part of the vestibulolateral lobe of the cerebellum. No fragmented axons are traceable to the lateral part (auricles) of the vestibulolateral lobe, cerebellar nucleus or corpus, or those nuclei associated with the lateral-line lobes. It appears therefore that octavus terminal fields are separate from those of the lateral line at both cerebellar and medullary levels, at least at the level of the first-order neuron.

Choline acetyltransferase immunoreactive neurons innervating labyrinthine and lateral line sense organs in amphibians.

The goal of the present study was to investigate aspects of the central organization of the neurons belonging to the octavolateralis efferent system of amphibians. The perikarya of three genera, Pleurodeles, Xenopus, and Discoglossus, were located in the brainstem by applying retrograde tracers to the appropriate cranial nerves and choline acetyltransferase immunohistochemistry was used to identify cholinergic neurons. The efferent neurons supplying lateral line (Pleurodeles, Xenopus) and labyrinthine (Pleurodeles, Xenopus, and Discoglossus) end organs were found to intermingle in a single octavolateralis efferent nucleus. The neurons lie bilateral to the labelled nerves in Pleurodeles and ipsilateral in Xenopus and Discoglossus. Separate labelling of the anterior and posterior octavus rami provided no evidence for distinct groupings of efferent neurons that could be associated with auditory and vestibular end organs. In all three species many if not all octavolateral efferent neurons displayed immunoreactivity for choline acetyltransferase. They could be distinguished from the cholinergic facial motoneurons, with which they sometimes intermingle, on the basis of either their distinctive size and shape (Pleurodeles, Xenopus) or their location (Discoglossus). Double labelling in Xenopus confirmed the cholinergic nature of the efferent neurons.

Distribution of afferent fibers in the brainstem from end organs in the ear and lateral line in the European eel.

Sensory nerve fibers from the lateral line system and labyrinth of Anguilla anguilla were labeled with horseradish peroxidase and traced to various targets in the ipsilateral brainstem. The three rami of the anterior lateral line nerve and the supratemporal ramus of the posterior lateral line nerve form overlapping terminal zones in the ventral portion of nucleus medialis. The posterior lateral line nerve on the body is represented exclusively in the dorsal half of the nucleus medialis. Eighth nerve fibers from the otolithic end organs in the inner ear send fibers into dorsal portions of three octavus nuclei: anterior, magnocellular, and descending, and saccular fibers lie most medial and utricular fibers most lateral. Fibers from vestibular organs, especially the semicircular canals and utricle, end densely in ventral portions of these nuclei and in the tangential nucleus. All labyrinthine sense organs send fibers into the region of a Mauthner-like neuron, and all except the saccule terminate in the reticular formation, tangential nucleus, and eminentia granularis of the cerebellum. Primary sensory input to the octavolateralis efferent nucleus comes only from the labyrinth, and fibers from the saccule alone penetrate the region of efferent neuronal somata. Fibers from labyrinthine end organs except the saccule project to the reticular formation where they may contact the dendrites of efferent somata. Fibers from the lateral line and the eighth nerve overlap most extensively at the rostral pole of the nucleus medialis and in the eminentia granularis of the cerebellum.

Fos-like immunohistochemical identification of neurons active during the startle response of the rainbow trout.

Activity-dependent Fos-like expression was investigated immunohistochemically in rainbow trout (Oncorhynchus mykiss) that had performed vibratory-evoked startle responses. We found significantly higher numbers of Fos-like-immunoreactive neurons in the reticular formation, in the octavolateral area, and in several cranial nerve motor nuclei in the brain and in the motor column of the spinal cord of startled fish than in control fish. In one fish, in which stimulation did not evoke startle responses, substantial numbers of positive cells occurred in the brain, primarily in the magnocellular octavolateral nucleus. We observed Fos-like-immunoreactive neurons in cell groups that are known to participate in the startle response (e.g., the Mauthner cell) as well as in cell groups that have been proposed but until now not shown to be involved.

The topographical organization of the vagal motor column in the elasmobranch fish, Scyliorhinus canicula L.

The location within the brainstem of vagal preganglionic motoneurons has been determined in the dogfish Scyliorhinus canicula L. by means of the retrograde transport of horseradish peroxidase and cobalt applied to the vagus nerve and its component branches. Labelled vagal motoneurones were located in the ipsilateral caudal rhombencephalon from 2.1 mm caudal to 2.73 mm rostral to obex. The motoneurons of the vagal motor column are arranged as four distinct groups. Caudal to obex the column contains dorsomedial and ventromedial divisions, whilst rostrally it consists of a single rostromedial division and a short lateral division. The cells in the ventromedial division are approximately twice the size (mean area 1,094 microns 2) of the other vagal neurons. The dorsomedial division contains neurons that supply the heart and viscera; the ventromedial division supplies the viscera. The heart is also innervated by the neurons of the lateral division and the visceral nerve also receives axons from the rostromedial division. All neurons supplying axons to the gill arches are located in the rostromedial division. There is a sequential topographical representation of the vagus nerve in the vagal motor column. Neurons supplying the gastrointestinal tract are located caudally; those supplying the cardiac nerves lie in the midportion of the column, and the proximal supply to the gills is given by the most rostral neurons. There is some overlap between the pools of neurons supplying adjacent branches of the vagus.

Dopaminergic and GABAergic cerebrospinal fluid-contacting neurons along the central canal of the spinal cord of the eel and trout.

In anamniote vertebrates the central region of the spinal cord has been implicated in its regeneration. This is a complex region and so as a first step in understanding its possible regenerative role we have examined the organization of the cells that contact the lumen of the spinal cord in two teleost fishes, eel and trout, using immunohistochemical procedures and light and electron microscopy. Cell bodies immunoreacting positively with antibodies for tyrosine hydroxylase and for dopamine were located at the ventral rim of the central canal, whereas cell bodies reacting for an antibody for gamma-aminobutyric acid were more laterally located. None of the canal-contacting cells were positively immunoreactive for choline acetyltransferase. All immunopositive cells have a similar morphology: the amphora-shaped perikaryon is bipolar and has a single process that extends to the lumen of the canal, and another that branches and forms extensive lateral and ventral plexuses. Electron microscopic investigations of the ventral dopaminergic cells showed that the apical processes bear one or more cilia, which protrude into the canal lumen and which originate from within a superficial rosette of nonciliated processes. The ventral process was occasionally seen to form synapses; the cell body was also the target of synapses.

Affinity maturation of proteins displayed on surface of M13 bacteriophage as major coat protein fusions.

This chapter described the preparation and fractionation of libraries of M13 phage displaying proteins as fusions to the major coat protein. High titer (10(13) pfu/ml) phage libraries can readily be generated using a single vector and the level of display surpasses that of gene III fusion phage. Since the synthetic VIII fusion gene can be customized, this system should provide the flexibility required to construct phage libraries displaying a variety of different peptides and proteins and to select variants possessing the highest affinity for target molecules of a diverse chemical nature.

Central organization of the efferent supply to the labyrinthine and lateral line receptors of the dogfish.

Neurons that provide the efferent innervation of the inner ear and lateral line were located in the brain by applying horseradish peroxidase to appropriate cranial nerves. The efferent neurons are found in a rhombencephalic nucleus, called here the octavolateralis efferent nucleus, which lies at the rostral pole of the visceromotor column. Up to 40% of these neurons are contralateral. The location of efferent cells is not topographically related to the sense organs they innervate. Their axons leave the brain together with other motor axons in the facial and glossopharyngeal nerves and there is evidence that some neurons innervate both the ear and the lateral line. The efferent nucleus receives direct sensory input from the labyrinth, but not from the lateral line. The organization of the efferent neurons and the distribution of their axons indicates that their effect on the sense organs is probably widespread and nonspecific.

Recovery of locomotion correlated with axonal regeneration after a complete spinal transection in the eel.

This research has examined the relationship between axonal regeneration and the return of normal movement following complete transection of the spinal cord. We made measurements of tail beat frequency and amplitude of the caudal body wave from video recordings of eels (Anguilla anguilla) swimming in a water tunnel at several speeds. Each eel was then anaesthetised and the spinal cord cut caudal to the anus; in some animals the resulting gap was filled with a rubber block. All animals were kept at 25 degrees C for recovery periods ranging from 7 to 128 days, during which their swimming performance was monitored regularly. Each fish was then re-anaesthetised and perfused with fixative and the regrowing descending axons labelled with 1,1'-diotadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. For all animals and at all speeds after surgery, tail beat frequency increased, while amplitude decreased. In non-blocked animals, an improvement in performance was first seen from 8 days following transection and thereafter tail beat frequency decreased progressively until it had returned to normal after 35 to 45 days, while amplitude remained below baseline until at least 45 days. In these animals, few axonal growth cones had penetrated the caudal stump by 7 days, but some had extended as much as 3 mm by 15 days. Many had reached as far as 6 mm between 25 and 36 days, while by 128 days they had progressed up to 10.5 mm. Contralateral crossing was never observed. Functional recovery was never witnessed in animals in which the cord had been blocked and these eels swam at all times with elevated tail beat frequency and reduced caudal amplitude. No labelled axons could be traced into the caudal spinal cord at any recovery stage in such animals. We conclude that re-innervation of only 1-2 segments caudal to the injury is necessary for functional recovery, although continued axonal growth may be important for the refinement of some aspects of movement.

Dose-response to inhaled aerosolized prostacyclin for hypoxemia due to ARDS.

STUDY OBJECTIVES: This study was carried out to determine the efficacy of and dose-response relationships to inhaled aerosolized prostacyclin (IAP), when used as a selective pulmonary vasodilator (SPV) in patients with severe hypoxemia due to ARDS. DESIGN: Unblinded, interventional, prospective clinical study. SETTING: A general ICU in a university-affiliated, tertiary referral center. PATIENTS: Nine adult patients with severe ARDS (lung injury score, > or = 2.5). INTERVENTIONS: All patients received IAP over the dose range 0 to 50 ng/kg/min. The IAP was delivered via a jet nebulizer placed in the ventilator circuit. Dose increments were 10 ng/kg/min every 30 min. MEASUREMENTS AND RESULTS: Cardiovascular parameters (cardiac index and mean pulmonary and systemic pressures), indexes of oxygenation (PaO(2)/fraction of inspired oxygen [FIO(2)] ratio and alveolar-arterial oxygen partial pressure difference [P(A-a)O(2)]) and shunt fraction were measured or calculated at each dose interval, as were platelet aggregation and systemic levels of prostacyclin metabolite (6-keto prostaglandin F1(alpha)). A generalized linear regression model was used to determine a dose effect of IAP on these parameters. The Wilcoxon rank sum test for related measures was used to compare the effects of various doses of IAP. IAP acted as an SPV, with a statistically significant dose-related improvement in PaO(2)/FIO(2) ratio (p = 0.003) and P(A-a)O(2) (p = 0.01). Systemic prostacyclin metabolite levels increased significantly in response to delivered IAP (p = 0.001). There was no significant dose effect on systemic or pulmonary arterial pressures, or on platelet function, as determined by platelet aggregation in response to challenge with adenosine diphosphate. CONCLUSIONS: IAP is an efficacious SPV, with marked dose-related improvement in oxygenation and with no demonstrable effect on systemic arterial pressures over the dose range 0 to 50 ng/kg/min. Despite significant systemic levels of prostacyclin metabolite, there was no demonstrable platelet function defect.

A polyclonal antibody against mammalian FOS can be used as a cytoplasmic neuronal activity marker in a teleost fish.

A polyclonal antibody raised against a conserved region of a mammalian FOS sequence was tested for its use as an activity marker in the rainbow trout. The FOS-like expression in the trout is entirely cytoplasmic and appears in a Nissl-like pattern. The reaction is specifically induced by both orthodromic and antidromic electrical stimuli and during motor responses evoked by natural stimulation, although some positive neurons are found at locations that are not obviously related to the presented stimuli. Following spinal nerve stimulation, antidromically activated motoneurons were found to be positive in the ipsilateral spinal cord. Orthodromic driving of spinal moto- and interneurons by stimulation of the medial longitudinal fasciculus (MLF) in the hindbrain evoked FOS-like immunoreactivity throughout the motor column in the spinal cord, but not in regions lying caudal to a lesion of the MLF-axons. Evoking about 25 startle responses by natural auditory stimulation gives FOS-like immunoreactivity in the Mauthner cell, which initiates the response, whereas positive Mauthner cells were never observed in control fish. The stimulation protocols that were used strongly activated the stimulated cells and so the observed FOS-like immunoreactivity might be related to an increase protein synthesis needed to restore their depleted transmitter levels.

Differences in the dopaminergic innervation of the electroreceptive and mechanoreceptive medullary lateral line nuclei of the ray, Raja radiata.

An immunohistochemical study was made of the octavolateral centres in the medulla of a cartilaginous fish, using an antibody specific for dopamine. No immunopositive cell bodies were observed but dopaminergic fibres were present in parts of the region. The overlying cerebellar crest was devoid of dopamine; the dorsal (electroreceptive) nucleus was poorly innervated, but the medial (mechanoreceptive) nucleus received a rich dopaminergic innervation comprising very fine, varicose fibres.

Immunohistochemical study of a dopaminergic system in the spinal cord of the ray, Raja radiata.

Using a specific antibody we have demonstrated the presence of an intrinsic dopamine (DA)-containing system in the spinal cord of a cartilaginous fish. The DA neurons are distributed throughout the spinal cord in the subependymal layer that surrounds the central canal. These cells extend a thick process into the canal and thinner neurites throughout the gray matter; they resemble the liquor-contacting neurons reported for a range of vertebrates.