Microtubule array formation during root hair infection thread initiation and elongation in the Mesorhizobium-Lotus symbiosis.

Nuclear migration during infection thread (IT) development in root hairs is essential for legume-Rhizobium symbiosis. However, little is known about the relationships between IT formation, nuclear migration, and microtubule dynamics. To this aim, we used transgenic Lotus japonicus expressing a fusion of the green fluorescent protein and tubulin-α6 from Arabidopsis thaliana to visualize in vivo dynamics of cortical microtubules (CMT) and endoplasmic microtubules (EMTs) in root hairs in the presence or absence of Mesorhizobium loti inoculation. We also examined the effect of microtubule-depolymerizing herbicide, cremart, on IT initiation and growth, since cremart is known to inhibit nuclear migration. In live imaging studies of M. loti-treated L. japonicus root hairs, EMTs were found in deformed, curled, and infected root hairs. The continuous reorganization of the EMT array linked to the nucleus appeared to be essential for the reorientation, curling, and IT initiation and the growth of zone II root hairs which are susceptible to rhizobial infection. During IT initiation, the EMTs appeared to be linked to the root hair surface surrounding the M. loti microcolonies. During IT growth, EMTs dissociated from the curled root hair tip, remained linked to the nucleus, and appeared to surround the IT tip. Lack or disorganized EMT arrays that were no longer linked to the nucleus were observed only in infection-aborted root hairs. Cremart affected IT formation and nodulation in a concentration-dependent manner, suggesting that the microtubule (MT) organization and successive nuclear migration are essential for successful nodulation in L. japonicus by M. loti.

Endoplasmic reticulum-targeted GFP reveals ER remodeling in Mesorhizobium-treated Lotus japonicus root hairs during root hair curling and infection thread formation.

The endoplasmic reticulum (ER) of the model legume Lotus japonicus was visualized using green fluorescent protein (GFP) fused with the KDEL sequence to investigate the changes in the root hair cortical ER in the presence or absence of Mesorhizobium loti using live fluorescence imaging. Uninoculated root hairs displayed dynamic forms of ER, ranging from a highly condensed form to an open reticulum. In the presence of M. loti, a highly dynamic condensed form of the ER linked with the nucleus was found in deformed, curled, and infected root hairs, similar to that in uninoculated and inoculated growing zone I and II root hairs. An open reticulum was primarily found in mature inoculated zone III root hairs, similar to that found in inactive deformed/curled root hairs and infected root hairs with aborted infection threads. Co-imaging of GFP-labeled ER with light transmission demonstrated a correlation between the mobility of the ER and other organelles and the directionality of the cytoplasmic streaming in root hairs in the early stages of infection thread formation and growth. ER remodeling in root hair cells is discussed in terms of possible biological significance during root hair growth, deformation/curling, and infection in the Mesorhizobium-L. japonicus symbiosis.

Health and safety management system audit reliability pilot project.

This pilot study assessed occupational health and safety (OHS) management system audit finding reliability using a modified test-retest method. Two industrial hygienists with similar training and education conducted four, 1-day management system audits in four dissimilar organizational environments. The researchers examined four auditable sections (employee participation, training, controls, and communications) contained in a publicly available OHS management system assessment instrument. At each site, 102 auditable clauses were evaluated using a progressive 6-point scale. The team examined both the consistency of and agreement between the scores of the two auditors. Consistency was evaluated by calculating the Pearson r correlations for the two auditors' scores at each site and for each section within each site. Pearson correlations comparing overall scores for each site were all very low, ranging from 0.206 to 0.543. Training and communication system assessments correlated the highest, whereas employee participation and control system scores correlated the least. To measure agreement, t-tests were first calculated to determine whether the differences were statistically significant. Aggregate mean scores for two of the four sites were significantly different. Of the 16 total sections evaluated (i.e., 4 sections per site), seven scores were significantly different. Finally, the agreement of the scores between the two auditors for the four sites was evaluated by calculating two types of intraclass correlation coefficients, all of which failed to meet the minimum requirement for agreement. These findings suggest that opportunities for improving the reliability of the instrument and the audit process exist. Future research should include governmental and commercial OHS program assessments and related environmental management systems and their attendant audit protocols.

Reversible binding of heparin to the loop peptide of endotoxin neutralizing protein.

Endotoxin neutralizing protein (ENP) from Limulus polyphemus is an amphipathic, 11.8 kDa protein with an isoelectric point of 10.2. ENP neutralizes lipopolysaccharide (LPS) and possesses antibacterial activity against Gram-negative bacteria. Heparin binds to ENP and blocks its LPS-neutralizing activity. The relative blocking activity of heparin is equal to low molecular weight heparin and polyanetholsulfonic acid > heparan sulfate > chondroitin sulfate A > chondroitin sulfate C. Endoproteinase Glu-C hydrolysis of recombinant ENP results in four major peptides, three of which are seen following separation on reversed phase HPLC. Heparin binds to the loop peptide (31-72), which includes the heparin binding consensus sequence XBBXBX between the two cysteine residues of ENP. When heparin is added to the digest and then applied to a C18 column, the loop peptide is bound; however, it dissociates and elutes with either 5 M NaCl or 0.1 M sodium phosphate, demonstrating reversible binding to heparin. LPS and lipid A both bind to the loop peptide and remove it from digests of ENP; however, neither complex could be dissociated by salt or sodium phosphate. Heparin, LPS, and lipid A individually bind to the same site on ENP.

The relationship between multidisciplinary discharge outcomes and functional status after total hip replacement.

PURPOSE: To determine changes in functional status after primary total hip replacement and to explore the relationship between functional status and pain and mobility at time of hospital discharge. DESIGN: Descriptive correlational with a prospective cohort. SAMPLE: 21 elective primary total hip arthroplasty patients, ages 40 to 78, in an academic health center. METHOD: Subjects' functional status was measured using the Sickness Impact Profile and the hip outcome tool (Revised Hip Type Specification Tool 13.1), completed by face-to-face interview approximately 2 weeks prior to hospitalization. Pain and mobility were assessed at time of discharge, and the functional status measures were repeated at 3 months after hospital discharge by mail. FINDINGS: Statistically significant changes were found for physical and psychosocial dimensions of functional status, using the Sickness Impact Profile and the hip outcome tool, between the preoperative and postoperative measures. A significant relationship between mobility and pain at the time of discharge and functional status was not established. IMPLICATIONS: Hospital discharge criteria related to pain and mobility should be used with caution. In addition to pain and mobility information, patient education should reflect knowledge of what can be expected by 3 months postsurgery, including expected and potential improvements in mobility, pain, and ambulation and in nonphysical dimensions, such as sleep, home management, and social interaction. Case management and discharge planning should reflect the wide variance in pain and mobility across patients at time of discharge and the expected gains in specific aspects of functional status after discharge. Research attention should be increased on the identification and application of specific discharge criteria such as pain and mobility levels, and the further development and refinement of nursing-sensitive and practical functional status outcome measures.

Developmental transitions and dynamics of the cortical ER of Arabidopsis cells seen with green fluorescent protein.

Arabidopsis thaliana plants were stably transformed with DNA encoding green fluorescent protein and with sequences ensuring retention in the endoplasmic reticulum (ER). Confocal laser scanning microscopy shows fluorescent ER in many cells of seedlings so allowing developmental changes to be documented. The arrangement of the cortical ER changes as cells mature in the hypocotyl and root epidermis. In the root, cells that have completed expansion have reticulate cortical ER resembling the ER described in many previous studies. Expanding cells, however, show extensive perforated sheets of cortical ER which transform quite abruptly into a loose reticulum at the basipetal end of the elongation zone. The reticulum compacts in trichoblasts beginning at sites where root hairs are about to emerge. The compacted form is maintained throughout the hair until growth ceases and the open reticulate form returns. All forms of cortical ER are dynamic and we use a color overlay method to distinguish stable and moving structures in a single composite image. Reticulate ER continuously rearranges its polygonal layout and perforations move and change their shape in the ER sheets of younger cells. ER deeper in the cell (i.e. not close to the plasma membrane) moves more actively so that almost no tubules remain stable even over short periods of less than one minute. The function of the perforated sheets of cortical ER present in growing cells is unknown.

Understanding and assessing the motivations of volunteers: a functional approach.

The authors applied functionalist theory to the question of the motivations underlying volunteerism, hypothesized 6 functions potentially served by volunteerism, and designed an instrument to assess these functions (Volunteer Functions Inventory; VFI). Exploratory and confirmatory factor analyses on diverse samples yielded factor solutions consistent with functionalist theorizing; each VFI motivation, loaded on a single factor, possessed substantial internal consistency and temporal stability and correlated only modestly with other VFI motivations (Studies 1, 2, and 3). Evidence for predictive validity is provided by a laboratory study in which VFI motivations predicted the persuasive appeal of messages better when message and motivation were matched than mismatched (Study 4), and by field studies in which the extent to which volunteers' experiences matched their motivations predicted satisfaction (Study 5) and future intentions (Study 6). Theoretical and practical implications are discussed.

Motor units of juvenile rat lumbrical muscles and fibre type compositions of the glycogen-depleted component.

1. Fourth deep lumbrical muscles were dissected out, with their nerve supply, from juvenile rats aged 8-15 days (a period corresponding to maximal rate of decline of polyneuronal innervation), and aged 28, 29 and 30 days (when developmental synapse elimination is complete). Preparations were superfused with rat Ringer solution at 25 degrees C. 2. Isometric twitches and tetani were recorded from the whole muscles and from a single motor unit in the muscle. Unit isolation was by partial section of the sural or lateral plantar nerves. The axon of a single unit occurred naturally in the sural nerve in some cases. 3. Fibres in single units were depleted of glycogen by repetitive stimulation, and studied histologically in frozen midbelly sections of the muscle, stained for glycogen with periodic acid-Schiff's reagent (PAS). Most fibre counts were based on transmittance measurements made with an image analysis system. Contralateral muscles were unstimulated and acted as controls. 4. Motor unit sizes were estimated from tetanic tensions and from muscle fibre cross-sectional area measurements. Comparison of the two methods indicated that in most units glycogen depletion was not complete. This effect was maximal at 8 and 10 days postnatally. It is suggested that this is due to weak neuromuscular transmission at synapses in the process of natural elimination during development. 5. Other sections (serial and semi-serial) were immunostained with a polyclonal antibody raised against slow myosin. Fibres staining for the antibody (slow; S-fibres) contribute about 12-9% of muscle fibres depending on age. In some muscles, fibre types were determined by myosin ATPase staining following alkali pre-incubation. Fast fibres (F-fibres) contained no slow myosin. 6. Some units had no S-fibres (i.e. they were homogeneous), and many units had a small proportion of S-fibres, though less than in the whole muscle (i.e. they were heterogeneous but composition was biased in favour of F-fibres). 7. One unit from a 10-day-old rat contained more S- than F-fibres. Many of the F-fibres were small. It is proposed that this was a developing IIC/IIA unit, a type known to occur in adults. 8. It is concluded that mismatched connections in developing motor units possibly become weak early (by 8 days postnatally) in the process of synapse elimination (which is complete by 20 days postnatally), but that the time course of actual withdrawal cannot be followed by the technique of glycogen depletion.

Synaptic vesicle movements monitored by fluorescence recovery after photobleaching in nerve terminals stained with FM1-43.

We used the fluorescence recovery after photobleaching technique to monitor movements of synaptic vesicles in top views of living frog motor nerve terminals that had been prestained with the fluorescent dye FM1-43. In each experiment, a small portion of a single stained vesicle cluster was bleached with a laser and monitored subsequently for signs of recovery as neighboring, unbleached vesicles moved into the bleached region. In resting terminals, little or no recovery from photobleaching occurred. Repetitive nerve stimulation, which caused all fluorescent spots to grow dim as dye was released from exocytosing vesicles, did not promote recovery from photobleaching. Pretreatment with botulinum toxin (type A, C, or D) blocked exocytosis and destaining, but intense nerve stimulation still did not cause significant recovery in bleached regions. These results suggest that lateral movements of synaptic vesicles are restricted severely in both resting and stimulated nerve terminals. We tested for laser-induced photodamage in several ways. Bleached regions could be restained fully with FM1-43, and these restained regions could be destained fully by nerve stimulation. Partially bleached regions could be destained, although the rate of destaining was lower than normal. Brisk recovery from photobleaching occurred after treatment with okadaic acid, which disrupts synaptic vesicle clusters and causes vesicles to spread throughout the nerve terminal. These results suggest that vesicle translocation and recycling machinery was intact in photobleached regions.

Partial N-terminal amino acid sequence of the anti-neoplastic urinary protein (ANUP) and the anti-tumour effect of the N-terminal nonapeptide of the unique cytokine present in human granulocytes.

The N-terminal amino acid sequence of the anti-neoplastic urinary protein (ANUP), a unique cytokine present in human granulocytes, was determined to be: Pyroglu-Leu-Lys-X-Tyr-Thr-X-Lys-Glu-Pro-Met-Thr-Ser(Thr)-Ala-Ala... This sequence showed no significant homology with any other protein when used in database searches. Furthermore, a synthetic nonapeptide corresponding to the first nine residues, with Cys in positions 4 and 7, was found to be a biologically active in vitro anti-tumour agent. An alternate method for the purification of ANUP to that previously reported is also presented. This method involves differential Amicon Diaflo membrane filtration.

Infrared laser Doppler flowmeter in the determination of small bowel perfusion after ischemic injury: comparison with the clearance of locally generated hydrogen and fluorescein angiography.

The determination of small bowel perfusion after an ischemic insult is difficult. Regional perfusion was determined in an animal model of neonatal intestinal ischemia using the techniques of laser Doppler flowmetry and the clearance of locally generated hydrogen. Both methods reliably measured tissue perfusion in the areas of maximal ischemic injury. However, considerable variability, perhaps owing to motion artifact, was seen in areas of patchy necrosis. The results suggest that the laser Doppler flowmeter is a suitable technique to measure tissue perfusion in areas of maximal ischemia. However, efforts to reduce motion artifact will be necessary if the laser Doppler is to be used in tissue sites where blood flow is critical for safe anastomosis. This will be a subject of future study.

Comparison of FM1-43 staining patterns and electrophysiological measures of transmitter release at the frog neuromuscular junction.

Frog motor nerve terminals were stained with the activity-dependent dye FM1-43, which appears to stain recycled synaptic vesicles. Superficial end plates which could be visualized in their entirety were imaged and end plate potentials (EPPs) evoked by low frequency nerve stimulation were recorded from the muscle fibers which were innervated by the imaged terminals. Curare was present to block muscle contractions. The amplitude of the EPPs correlated reasonably well with the number of fluorescent spots counted in the terminals (r = 0.68). Each fluorescent spot probably represents a cluster of synaptic vesicles localized at an active zone. Several other morphological and electrophysiological values were measured and calculated. The results are consistent with the ideas that FM1-43 stains recycled synaptic vesicles, and that the number of vesicle clusters in a terminal is a good predictor of synaptic efficacy.

Intracellular movements of fluorescently labeled synaptic vesicles in frog motor nerve terminals during nerve stimulation.

We stained synaptic vesicles in frog motor nerve terminals with FM1-43 and studied changes in the shape and position of vesicle clusters during nerve stimulation. Each stained vesicle cluster appeared as a fluorescent spot. During repetitive nerve stimulation the spots gradually dimmed, most without changing shape or position. Occasionally, however, a spot moved, appearing in some cases to stream toward and coalesce with a neighboring spot. This suggests the existence of translocation mechanisms that can actively move vesicles in a coordinated fashion between vesicle clusters. Within single clusters, we saw no signs of such directed vesicle movements. Fluorescent spots in terminals viewed from the side with a confocal microscope did not shrink toward the presynaptic membrane during nerve stimulation, but dimmed uniformly. This suggests that vesicles continuously mix within a cluster during destaining and provides no evidence of active vesicle translocators within single vesicle clusters for moving vesicles to the presynaptic membrane.

Cortical and cap sedimentation in gravitropic Equisetum roots.

Although the rootcap is required for gravitropic sensing, various classical and contemporary data raise the question of whether additional sensing occurs away from the cap in roots. Roots of Equisetum hyemale L. (horsetail) were examined by light and electron microscopy to determine which cell components were distributed with respect to gravity both in and away from the rootcap. Adventitious roots from stem cuttings were gravitropic in a vertical orientation or if reoriented to the horizontal. Obvious amyloplast sedimentation was found in vertical and in reoriented roots 1) in cells in the center of the rootcap and 2) in young, elongating cortical cells located in two to three layers outside the endodermis. These cortical amyloplasts were smaller than cap amyloplasts and, unlike central cap amyloplasts, were occasionally found in the top of the cell. The nucleus was also sedimented on top of the amyloplasts in both cell types, both in vertical and in reoriented roots. Sedimentation of both organelles ceased as cortical cells elongated further or as cap cells became peripheral in location. In both cell types with sedimentation, endoplasmic reticulum was located in the cell periphery, but showed no obvious enrichment near the lower part of the cell in vertical roots. This is the first modern report of sedimentation away from the cap in roots, and it provides structural evidence that gravitropic sensing may not be confined to the cap in all roots.

The importance of competition between motoneurones in developing rat muscle; effects of partial denervation at birth.

1. The number of motor units in developing fourth deep lumbrical muscles was reduced by unilateral partial denervation of the muscle at birth, by cutting the lateral plantar nerve. A minority of the motor axons arrive via the sural nerve, and were thus not cut. Those muscles that contained one motor unit (one-unit muscles) after partial denervation developed in the absence of competition between motoneurones. Muscles with two motor units had little competition. A few four-unit muscles were studied for comparison. 2. Isometric twitch and tetanic tensions of single motor units were recorded in vitro at 60 days of age in response to stimulation of the sural nerve. On average, units in partially denervated muscles generated more tension than normal units. The isometric tension characteristics of the units in the one-unit and two-unit muscles were different from the normal units (e.g. slower contracting and more fatiguable). The units of four-unit muscles had properties similar to those of normal muscles. 3. Fibres of an individual unit were identified by glycogen depletion and S (slow) fibres were identified in cross-section that bound a polyclonal antibody to slow type I myosin. Those fibres that did not bind the antibody were designated F fibres. The units of one-unit muscles had the same total number of fibres and fibre type composition (both S and F fibres in the same unit) as estimated from previous work to exist at birth. The units of two-unit muscles contained the same total number of fibres, but apparently fewer S fibres, though this may have been as a result of incomplete glycogen depletion of some fibres. 4. It is concluded that competition between motoneurone terminals is necessary for the withdrawal of mismatched connections on muscle fibres present at birth; or, alternatively, that such withdrawal cannot take place if it would result in denervation of the muscle fibre.

Volunteers' motivations: a functional strategy for the recruitment, placement, and retention of volunteers.

A psychological strategy for understanding the motivational underpinnings of volunteerism is described. In a presentation that merges the theoretical interests of researchers with the practical interests of volunteer administrators, six different motivational functions served by volunteerism are identified, and an inventory designed to measure these motivations is presented. The implications of this functional approach for the recruitment, placement, and retention of volunteers are then elaborated. Finally, recommendations are provided for volunteer administrators who seek to increase the number of people who volunteer and to improve their human resource management.

Motor units of the fourth deep lumbrical muscle of the adult rat: isometric contractions and fibre type compositions.

1. Isometric twitch and tetanic tensions were recorded from whole muscles and single motor units in fourth deep lumbrical muscles isolated from young adult (60 days) rats. Muscles were superfused with oxygenated Ringer solution at 25 degrees C except where stated otherwise. 2. It was confirmed that the muscle is supplied most commonly by eleven motor axons, nine via the lateral plantar nerve (LPN), and two via the sural nerve (SN). Motor units whose axons were isolated from either LPN or SN were studied. There was no difference in mean motor unit size. 3. In their unfused tetani most units showed 'sag' and some 'no sag', with no segregation between LPN and SN. 'No sag' units were always small (unit tetanic tension less than 8% whole-muscle tetanic tension), tended to be relatively slowly contracting and relaxing during an isometric twitch, and tended to have relatively low twitch:tetanus ratios. Units showing sag ranged from large to small. 4. In some motor units muscle fibres were depleted of their glycogen by repetitive stimulation at 30 degrees C in glucose-free Ringer solution, and the muscle and its unstimulated control frozen and sectioned. Neighbouring sections were stained for glycogen and for binding of two myosin-specific antibodies, one specific for slow myosin and the other for type IIA myosin. Myosin ATPase and succinic dehydrogenase histochemistry were also carried out in some muscles. 5. Serial reconstructions showed that all or virtually all extrafusal fibres in the muscle were present in a midbelly section, and that the myosin type of individual fibres did not change significantly along their length. Spindle profiles were seen frequently and in two muscles eight and twelve spindles were identified. 6. Of twenty-six motor units examined twenty contained almost exclusively muscle fibres of the recently described type IIX. All these units showed sag in their isometric tetani. 7. Six units each contained 50% or more of slow myosin-containing fibres (IIC and a few type I). The remaining fibres in these units were IIA. All these units were therefore of mixed fibre composition, and are discussed as IIC/IIA units. In whole muscles slow-myosin-containing fibres were generally distributed evenly (non-randomly) throughout the muscle cross-section. 8. Whole muscles contained on average 970 fibres (S.D. +/- 70) of which 82 (+/- 9) were slow-myosin-containing. A few muscles from older rats (3-24 months) contained very few such fibres.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonia-regulated expression of a soybean gene encoding cytosolic glutamine synthetase in transgenic Lotus corniculatus.

A full-length cDNA clone encoding cytosolic glutamine synthetase (GS), expressed in roots and root nodules of soybean, was isolated by direct complementation of an Escherichia coli gln A- mutant. This sequence is induced in roots by the availability of ammonia. A 3.5-kilobase promoter fragment of a genomic clone (lambda GS15) corresponding to this cDNA was isolated and fused with a reporter [beta-glucuronidase (GUS)] gene. The GS-GUS fusion was introduced into a legume (Lotus corniculatus) and a nonlegume (tobacco) plant by way of Agrobacterium-mediated transformations. This chimeric gene was found to be expressed in a root-specific manner in both tobacco and L. corniculatus, the expression being restricted to the growing root apices and the vascular bundles of the mature root. Treatment with ammonia increased the expression of this chimeric gene in the legume background (i.e., L. corniculatus); however, no induction was observed in tobacco roots. Histochemical localization of GUS activity in ammonia-treated transgenic L. corniculatus roots showed a uniform distribution across all cell types. These data suggest that the tissue specificity of the soybean cytosolic GS gene is conserved in both tobacco and L. corniculatus; however, in the latter case, this gene is ammonia inducible. Furthermore, the ammonia-enhanced GS gene expression in L. corniculatus is due to an increase in transcription. That this gene is directly regulated by externally supplied or symbiotically fixed nitrogen is also evident from the expression of GS-GUS in the infection zone, including the uninfected cells, and the inner cortex of transgenic L. corniculatus nodules, where a flux of ammonia is encountered by this tissue. The lack of expression of GS-GUS in the outer cortex of the nodules suggests that ammonia may not be able to diffuse outside the endodermis.

Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily.

A bone-inductive protein has been purified from bovine bone and designated as osteogenic protein (OP). The purified OP induces new bone at less than 5 ng with half-maximal bone differentiation activity at about 20 ng/25 mg of matrix implant in a subcutaneous bone induction assay. The purified osteogenic protein is composed of disulfide-linked dimers that migrate on sodium dodecyl sulfate gels as a diffuse band with an apparent molecular weight of 30,000. Upon reduction, the dimers yield two subunits that migrate with molecular weights of 18,000 and 16,000. Both subunits are glycosylated. After chemical or enzymatic deglycosylation, the dimers migrate as a diffuse 27-kDa band that upon reduction yields two polypeptides that migrate at 16 kDa and 14 kDa, respectively. The carbohydrate moiety does not appear to be essential for biological activity since the deglycosylated proteins are capable of inducing bone formation in vivo. Amino acid sequences of peptides generated by proteolytic digestion show that the subunits are distinct but related members of the transforming growth factor-beta super-family. The 18-kDa subunit is the protein product of the bovine equivalent of the human OP-1 gene and the 16-kDa subunit is the protein product of the bovine equivalent of the human BMP-2A gene.