Outbreak of Salmonella Braenderup infections associated with Roma tomatoes, northeastern United States, 2004: a useful method for subtyping exposures in field investigations.

Salmonella Braenderup is an uncommon serotype in the United States. In July 2004, a multistate outbreak of Salmonella Braenderup diarrhoeal infections occurred, with 125 clinical isolates identified. To investigate, we conducted a case-control study, enrolling 32 cases and 63 matched controls. Cheese, lettuce and tomato eaten at restaurants all appeared to be associated with illness. To further define specific exposures, we conducted a second study and asked managers of restaurants patronized by patients and controls about cheese, lettuce and tomato varieties used in dishes their patrons reported consuming. This information was obtained for 27 cases and 29 controls. Roma tomatoes were the only exposure significantly associated with illness (odds ratio 4.3, 95% confidence interval 1.2-15.9). Roma tomatoes from two restaurants were traced back to a single tomato packing house. The methods used in this field investigation to define specific exposures may be useful for other foodborne outbreaks.

[Urological complications after kidney transplantation]. / Urologische Komplikationen nach Nierentransplantation.

Between August 1981 and May 2005, 1065 consecutive kidney transplants were performed at our center; 393 patients (36.9%) developed urological complications in the first 60 postoperative days. Urinary tract infections occurred in 28.5% of all patients. The major urological problems seen were urinary leakage and ureteral obstruction in 6.2% and 1.4% of the patients. Two grafts were lost due to severe urinary leakage. No patient death occurred due to urological complications. The incidence of urological complications is mainly influenced by the surgical procedure of organ retrieval and ureteroneocystostomy. With double-J stenting of the extravesical ureteroneocystostomy, we observed a significantly lower rate of urinary leakage but a higher rate of urinary tract infections in our series. Early diagnosis and treatment of urological complications may prevent further morbidity of our transplant patients.

Apple ripening-related cDNA clone pAP4 confers ethylene-forming ability in transformed Saccharomyces cerevisiae.

The apple ripening-related cDNA insert of clone pAP4 (G.S. Ross, M.L. Knighton, M. Lay-Yee [1992] Plant Mol Biol 19: 231-238) has previously been shown to have considerable nucleic acid and predicted amino acid sequence similarity to the insert of a tomato ripening-related cDNA clone (pTOM13) that is known to encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase (A.J. Hamilton, G.W. Lycett, D. Grierson [1990] Nature 346: 284-287; A.J. Hamilton, M. Bouzayen, D. Grierson [1991] Proc Natl Acad Sci USA 88: 7434-7437). The cDNA insert from the clone pAP4 was fused between the galactose-inducible promoter and the terminator of the yeast expression vector pYES2. Transformation of Saccharomyces cerevisiae strain F808- with this DNA construct and incubation of the yeast in the presence of D[+]-galactose allowed these cells to convert ACC to ethylene. The transformed yeast converted 1-amino-2-ethylcyclopropane-1-carboxylate isomers to 1-butene with the same 1R,2S-stereoselectivity as achieved by the native ACC oxidase from applies. Both ascorbate and Fe2+ ions stimulated the rate of the production of ethylene from ACC by the transformed yeast, whereas Cu2+ and Co2+ were strongly inhibitory; these are features of ACC oxidase. Northern analysis of the total RNA from nontransformed and transformed yeast showed that the ability to convert the ACC to ethylene was correlated with the synthesis and accumulation of a novel 1.2-kb mRNA that hybridized to the cDNA clone pAP4. We conclude that the cDNA sequence of the clone pAP4 encodes ACC oxidase.

Microtubule-based filopodium-like protrusions form after axotomy.

The growth cone at the front of a growing neurite often has F-actin-rich structures--digitate filopodia and sheet-like veils and lamellipodia--whose protrusion advances the leading edge. Microtubules and other cytoplasmic constituents later fill the protruded area, transforming it into new neuritic length. Growth can be initiated from an axon by transecting it. We have used video-enhanced contrast-differential interference contrast microscopy to observe the early events following transection of Aplysia axons in culture. Many filopodium-like protrusions (FLPs) grew rapidly (average instantaneous velocity of 1.6 microns/sec) from the sides and end of the axon stump within minutes of transection. Some of these displayed bidirectional transport of swellings, at a rate similar to fast axonal transport. Dihydrocytochalasin B, which blocks actin polymerization, only halved the number of FLPs that formed within 10 min of transection, and actually increased the number of transporting FLPs. Nocodazole, a microtubule-specific drug, also halved the number of FLPs, but none of them displayed transport of swellings. No FLPs formed in the presence of both drugs. In transected axons that had not been exposed to either drug, removal of the plasma membrane revealed fibers in many of the FLPs; immunofluorescence showed these fibers to be microtubules. Thus, a substantial number of the FLPs that form soon after axotomy are microtubule based, rather than actin based, underscoring the potential of microtubules to drive the rapid extension of neuritic precursors.

Rapid effects of laminin on the growth cone.

To gain insight into how laminin promotes neurite growth, high resolution video microscopy was used to determine the rapid effects of laminin on growth cone structure. Sympathetic growth cones in serum-free medium on polylysine substrate displayed extensive motility and protrusive activity and often had large lamellipodia. However, their neurites grew slowly because membranous organelles from the central region advanced into the lamellipodium only slowly. Acute addition of laminin accelerated growth severalfold and had visible effects on the growth cone within minutes. Laminin dramatically accelerated the advance of membranous organelles, which, with microtubules, rapidly filled the lamellipodium. Retraction of individual protrusions (filopodia and veils) was rapidly reduced. These effects of laminin are important in accelerating growth and suggest a mechanism for pathway selection by growing neurites.

Substrate-bound factors stimulate engorgement of growth cone lamellipodia during neurite elongation.

The surfaces on which neurons grow greatly affect neurite elongation, but it is unclear how substrates influence the events within the growth cone that bring about elongation. Neurite elongation by Aplysia californica neurons in culture occurs through a series of transformations of the structures of the growth cone (Goldberg and Burmeister, J. Cell Biol., 103:1921-1931, 1986). The growth cone produces actin-rich protrusions, veils, and lamellipodia, which can then mature into the central body of the growth cone through the net advance of microtubules and membranous organelles from contiguous central regions, a process called "engorgement." Aplysia neurons form growth cones on poly-l-lysine-treated substrates, but their rate of neurite elongation is greatly enhanced on substrates additionally exposed to Aplysia hemolymph. The acute application of hemolymph to slowly growing neurites brings about a rapid acceleration of neurite elongation and engorgement. The enhancement of engorgement was effected with material eluted from hemolymph-treated substrates and was not seen when hemolymph was added to neurons cultured on hemolymph-treated substrates inactivated by exposure to UV radiation. Thus, we conclude that the rapid acceleration of engorgement caused by hemolymph is, in large part, a substrate-mediated effect. We propose that extracellular substrate molecules can modulate the rate of neurite growth through the regulation of the engorgement of lamellipodia. The microtubule disrupters colcemid and nocodazole inhibit the advance of vesicular elements into the lamellipodia following hemolymph treatment, but taxol, which promotes the polymerization and stabilization of microtubules, does not itself enhance engorgement. The microfilament disrupter cytochalasin B, however, stimulates engorgement. Our results suggest that regulating the resistance of the peripheral actin meshwork to penetration by microtubules and vesicles may be a mechanism by which substrate-attached molecules regulate neurite advance.

Early changes in nuclear proteins following axotomy.

Changes in protein synthesis are thought to be important in the response of the neuron to axotomy. Certain axonally transported proteins whose synthesis increases probably play important roles in regeneration of the axon. Although little is known about the regulation of these changes, the cell often controls its production of proteins at the nuclear level, where transactivating proteins modulate the transcription of specific genes. Thus, changes in nuclear proteins might be expected to be among the early events following axotomy, but such changes have not yet been described. We have addressed this issue by dissecting out single nuclei from [35S]methionine-labeled giant R2 neurons of Aplysia and analyzing the proteins by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This procedure was used to avoid contamination with nonneuronal and nonnuclear proteins. Our results demonstrate large increases in two nuclear proteins (56 kDa and 41 kDa) and decreases in two others (77 kDa and 46 kDa) 5 h after axotomy. These are the earliest postaxotomy changes in [35S]methionine-labeled proteins that have been reported.

Effect of target removal on goldfish optic nerve regeneration: analysis of fast axonally transported proteins.

How is axonal transport in regenerating neurons affected by contact with their synaptic target? We investigated whether removing the target (homotopic) lobe of the goldfish optic tectum altered the incorporation of 3H-proline into fast axonally transported proteins in the regenerating optic nerve. Regeneration was induced either by an optic tract lesion (to reveal the changes in the original axon segment that remained connected to the cell body) or by an optic nerve lesion (to reveal the changes in the newly formed axon segment). Of 26 proteins analyzed by 2-dimensional gel electrophoresis and fluorography, all but one showed increased labeling as a result of tectal lobe ablation. By 2 d after the lesion, significantly increased labeling of some proteins was seen with a 6-hr labeling interval, but not with a 24-hr labeling interval. This is probably indicative of an increased velocity of transport, which may have been a nonspecific consequence of the surgery. Otherwise, tectal lobe removal had relatively little effect until 3 weeks, when there was a transitory increase in labeling of transported proteins in the new axon segments of the tectum-ablated animals. Beginning at 5 weeks, tectal lobe ablation caused considerably higher labeling of many of the proteins in the original axon segments. Because this was seen with both 6-hr and 24-hr labeling intervals, it is probably indicative of increased protein synthesis. The increased synthesis lasted until at least 12 weeks, though some proteins were beginning to show a diminished effect at this time. In the late stages of regeneration (8-12 weeks), there was also increased labeling of proteins in the new axon segments as a result of the absence of the target tectal lobe. This included a disproportionately large increase in the relative contribution of cytoskeletal proteins and of protein 4, which is the goldfish equivalent of the growth-associated protein GAP-43 (neuromodulin). We conclude that, after the regenerating axons begin to innervate the tectum, the expression of most of the proteins in fast axonal transport is down-regulated by interaction between the axons and their target. However, the changes in expression may be preceded by a modulation of the turnover and/or deposition of proteins in the newly formed axon segment.

Effect of conditioning lesions on regeneration of goldfish optic axons: time course of the cell body reaction to axotomy.

The time course of the cell body reaction to axotomy was determined in goldfish retinal ganglion cells by measuring cell body size and the amount of labelled protein conveyed by fast axonal transport to the optic tectum, both of which increase during regeneration of the optic axons. Following a single testing lesion of the optic nerve, the regenerating axons began to innervate the tectum at about 14 days after the lesion and the cell body reaction began to decline 2-3 weeks thereafter. If the testing lesion had been preceded by a conditioning lesion 2 weeks earlier, the time for the regenerating axons to arrive in the tectum was reduced by a week, because of the faster rate of axonal outgrowth, but the interval between their arrival and the beginning of the decline of the cell body reaction was unchanged. Electrophysiological measurements showed that synaptic transmission was initiated earlier when the axons reached the tectum faster. These results indicate that the mechanisms initiating the recovery of cell body metabolism are independent of those governing the rate of axonal outgrowth. The recovery of the cell body may begin shortly after synapses are established, regardless of whether they are correctly or incorrectly targetted. The correctness of the target may be a separate factor in determining how rapidly and completely the cell body recovers.

Looking into growth cones.

The growth cone is a crucial structure in effecting neurite elongation and guiding the neurite onto correct pathways by responding to environmental cues. Recently developed techniques in light and electron microscopy have greatly improved our understanding of the dynamic organization of membrane and cytoskeleton within the growth cone. The growth cone can now be directly observed to undergo a sequence of developmental changes to produce the neurite. The importance, in elongation and steering, of pulling of growth cone protrusions against adhesive contacts on the substrate is re-evaluated in the light of these findings.

The distribution and movement of organelles in maturing growth cones: correlated video-enhanced and electron microscopic studies.

The morphology of growth cones from identified neurons of Aplysia californica was analysed both with video-enhanced contrast differential-interference contrast (VEC-DIC) microscopy, and through serial electron microscopic reconstructions of the same growth cones. The largest structures seen in the living growth cones, the large irregular refractile bodies (LIRBs), were shown in electron micrographs to be unique structures, composed predominantly of dense-core vesicles but including mitochondria and smooth membrane profiles. The LIRBs were stratified in the growth cones, occurring predominantly in sections distant from the substrate and relatively devoid of microtubules. VEC-DIC observations showed that LIRBs formed in the peripheral regions of the organelle-rich central growth cone, and grew in size through fusion with other LIRBs, accumulating into a large central mass in more proximal regions. The distribution of microtubules and LIRBs and the movements of LIRB suggest that there is an overall circulatory pattern in the growth cones, with the delivery of new vesicles occurring at distal areas close to the substrate, and the accumulation and retrograde processing of organelles occurring in more proximal areas away from adhesive contacts.

Micropruning: the mechanism of turning of Aplysia growth cones at substrate borders in vitro.

Growth cones of Aplysia californica neurons were observed with video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy as they turned at a border between poly-L-lysine-treated and untreated glass. Growth cones that turned generally developed 2 distinct active areas of filopodial and veil formation, much in the way of growth cones undergoing branching. Both active areas advanced, but turning of the neurite occurred through the selective resorption of the incipient branches developing on the untreated substrate. Thus, micropruning of developing regions of the growth cone, rather than the asymmetric extension of filopodia or veils, was primarily responsible for directing neurite growth. We present the hypothesis that abrupt turns by growing neurites are mediated by 2 sets of signals, one causing growth cone splitting, and a second set regulating the survival of the separate branches.

Recovery of regenerating goldfish retinal ganglion cells is slowed in the absence of the topographically correct synaptic target.

When the axons of goldfish retinal ganglion cells are severed the cell bodies undergo a series of changes as the axons regenerate. These changes begin to reverse when the axons start to innervate the tectum and by 3 months after the lesion the cell bodies have nearly returned to normal. When the axons projecting to the caudal tectum were severed by a mediolateral transection of the tectum, only retinal ganglion cells in the nasal portion of the contralateral retina underwent the changes normally associated with regeneration, followed by a speedy return to normal. Because the injured fibers probably did not fully retract from the tectum, these results indicated that: (1) the complete removal of the axons from the tectal milieu was not essential for initiating the cell body changes, and (2) close proximity to the target sites would speed the recovery of the cells. When the caudal portion of the tectum was ablated the retinal ganglion cells of the nasal retina remained enlarged significantly longer than after tectal transection. During the time the cells remained enlarged the electrophysiological projection onto the remaining rostral part of the tectum revealed no significant 'compression' of the visual field. Compression of the visual field onto the rostral portion of the tectum can be accelerated if the caudal tectal ablation is accompanied by an optic nerve crush. However, under this condition the recovery of ganglion cells in the nasal retina was significantly slower than the recovery of cells in the temporal retina. This may reflect an element of topographical specificity in the regulation of the recovery of the cell body from axonal injury.

Fast axonally transported proteins in regenerating goldfish optic axons.

Fast axonal transport of protein was examined in regenerating goldfish optic axons after a lesion of either the optic tract or optic nerve, which revealed changes in the original intact optic axon segments or in the newly regenerated axon segments, respectively. In animals killed either 6 or 24 hr after injection of 3H-proline into the eye, labeling of total fast-transported protein in the original axon segments was increased by 2 d after the lesion, reached a peak of nearly 20 X normal at 2 weeks, and then declined to a level somewhat above normal at 12 weeks. When the labeling of individual transported proteins was examined by 2-dimensional gel electrophoresis, it was found that no new labeled proteins appeared during regeneration, but all proteins examined showed an increase in labeling. Among the various proteins, there was great variation in the magnitude and time course of the labeling increase. The largest increase, to nearly 200 X normal with 6 hr labeling, was seen in a protein with a molecular weight of 45 kDa and a pl of about 4.5, resembling a protein that has previously been designated a "growth-associated protein" (GAP-43; Skene and Willard, 1981a). The proteins showing increased labeling included a small fraction of cytoskeletal proteins (alpha-tubulin, beta-tubulin, and actin) that was apparently transported at a much faster rate than is usually expected of these constituents. In the new axon segments, the total protein labeling was increased by 1 week after the lesion, remained elevated at a nearly constant level of about 7 X normal from about 2 to 5 weeks, and then declined to levels somewhat above normal by 12 weeks. The 45 kDa protein again showed the largest increase, and became the single most prominently labeled constituent in the new axons. On the basis of the time course of labeling in both original and new axon segments during regeneration, the fast-transported proteins were tentatively separated into 5 classes that may represent groups of proteins that are coregulated during regeneration. They may conceivably correspond to different functional or structural entities within the neuron.

Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy.

The regenerative growth in culture of the axons of two giant identified neurons from the central nervous system of Aplysia californica was observed using video-enhanced contrast-differential interference contrast microscopy. This technique allowed the visualization in living cells of the membranous organelles of the growth cone. Elongation of axonal branches always occurred through the same sequence of events: A flat organelle-free veil protruded from the front of the growth cone, gradually filled with vesicles that entered by fast axonal transport and Brownian motion from the main body of the growth cone, became more voluminous and engorged with organelles (vesicles, mitochondria, and one or two large, irregular, refractile bodies), and, finally, assumed the cylindrical shape of the axon branch with the organelles predominantly moving by bidirectional fast axonal transport. The veil is thus the nascent axon. Because veils appear to be initially free of membranous organelles, addition of membrane to the plasmalemma by exocytosis is likely to occur in the main body of the growth cone rather than at the leading edge. Veils almost always formed with filopodial borders, protruding between either fully extended or growing filopodia. Therefore, one function of the filopodia is to direct elongation by demarcating the pathway along which axolemma flows. Models of axon growth in which the body of the growth cone is pulled forward, or in which advance of the leading edge is achieved by filopodial shortening or contraction against an adhesion to the substrate, are inconsistent with our observations. We suggest that, during the elongation phase of growth, filopodia may act as structural supports.

Changes in protein content of goldfish optic nerve during degeneration and regeneration following nerve crush.

After the goldfish optic nerve was crushed, the total amount of protein in the nerve decreased by about 45% within 1 week as the axons degenerated, began to recover between 2 and 5 weeks as axonal regeneration occurred, and had returned to nearly normal by 12 weeks. Corresponding changes in the relative amounts of some individual proteins were investigated by separating the proteins by two-dimensional gel electrophoresis and performing a quantitative analysis of the Coomassie Brilliant Blue staining patterns of the gels. In addition, labelling patterns showing incorporation of [3H]proline into individual proteins were examined to differentiate between locally synthesized proteins (presumably produced mainly by the glial cells) and axonal proteins carried by fast or slow axonal transport. Some prominent nerve proteins, ON1 and ON2 (50-55 kD, pI approximately 6), decreased to almost undetectable levels and then reappeared with a time course corresponding to the changes in total protein content of the nerve. Similar changes were seen in a protein we have designated NF (approximately 130 kD, pI approximately 5.2). These three proteins, which were labelled in association with slow axonal transport, may be neurofilament constituents. Large decreases following optic nerve crush were also seen in the relative amounts of alpha- and beta-tubulin, which suggests that they are localized mainly in the optic axons rather than the glial cells. Another group of proteins, W2, W3, and W4 (35-45 kD, pI 6.5-7.0), which showed a somewhat slower time course of disappearance and were intensely labelled in the local synthesis pattern, may be associated with myelin. A small number of proteins increased in relative amount following nerve crush. These included some, P1 and P2 (35-40 kD, pIs 6.1-6.2) and NT (approximately 50 kD, pI approximately 5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (approximately 45 kD, pI approximately 4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (approximately 60 and 65 kD respectively, pIs 4.5-5.0), that are carried mainly by slow axonal transport. Other proteins, including actin, that showed no net changes in relative amount (but presumably changed in absolute amount in direct proportion to the changes in total protein content of the nerve), are apparently distributed in both the neuronal and nonneuronal compartments of the nerve.

Removal of optic tectum prolongs the cell body reaction to axotomy in goldfish retinal ganglion cells.

Injury to the optic axons of goldfish elicits dramatic changes in the cell bodies of the neurons from which these axons arise, the retinal ganglion cells. The changes include a large increase in cell size and in synthesis and axonal transport of protein. The cells begin to return to normal about 3 weeks after the injury, when the axons invade the contralateral (homotopic) lobe of the optic tectum, and recovery is essentially complete by 8-10 weeks after the lesion. However, if the homotopic lobe of the tectum was removed at the time of nerve crush, we found that the cell body reaction was greatly prolonged. The cells remained enlarged, and [3H]proline incorporation and fast axonal transport of protein remained elevated, until at least 10-12 weeks after nerve crush, although by this time most of the regenerating axons had probably regained their normal length and many had entered the remaining ipsilateral (heterotopic) lobe of the tectum. The cells showed partial recovery by the latest time tested, 26 weeks after nerve crush, when the projections from the two eyes had segregated into separate bands in the heterotopic tectal lobe.