Signaling mechanisms and the activation of leukocyte integrins.

Myeloid cells and lymphocytes primarily circulate in the vascular system but move into the tissues in response to inflammatory signals. Such a mobile lifestyle necessitates the continual making and breaking of cell and cell matrix contacts; the receptors known as the integrins are well suited as mediators of this transient adhesiveness. In general, leukocyte integrins are not constitutively active but become adhesive in response to signaling through other membrane receptors. Candidate receptors for receiving the activating signals are the seven membrane-spanning receptors that are found on all leukocytes. The identity of the signals, however, that are responsible for triggering integrin adhesion in vivo and for promoting directed leukocyte movement into tissues remains incompletely resolved.

Peyronie's disease is associated with Paget's disease of bone.

Peyronie's disease is an idiopathic disorder in which an inflammatory fibrosis occurs in the tunica albuginea of the corpora cavernosa which causes the erect penis to become deformed. Peyronie's disease has a prevalence of 1% in men over age 50 years. Paget's disease of bone is a chronic skeletal disease with areas of increased bone turnover leading to pain, deformity, and in some cases arthritis. Because of a high rate of Peyronie's disease in subjects in a Paget's disease industry-sponsored drug trial, we asked whether there was an association between Peyronie's disease and Paget's disease of bone. We evaluated 61 men with Paget's disease attending our clinic for metabolic bone disease in a tertiary referral hospital, reviewed hospital records of all men discharged from our three hospitals with the diagnosis of Peyronie's disease, and mailed a validated questionnaire about shape of the erect penis to 1500 male members of the Paget Foundation. In the clinic population of men with Paget's disease of bone, 51 of 61 (83.6%) reported having normal erections; 10 patients (16.4%) were impotent. Sixteen of the 51 men (31.4%) had developed a bend or deformity in their erect penis which was confirmed by a urologist's examination to be Peyronie's disease. When the men with Paget's disease with and without Peyronie's disease were compared, there was no difference in their ages, years with Paget's disease, or serum alkaline phosphatase level. Upon medical record review, 1 patient of 262 (0.4%) with Peyronie's disease was found to have Paget's disease of bone. The men with Paget's disease returned their questionnaires for a response rate of 44.8% and reported Peyronie's disease with a prevalence of 14.5%. We suggest that Peyronie's disease is associated with Paget's disease of bone. Furthermore, we suggest that Peyronie's disease may be a previously unrecognized complication of Paget's disease of bone.

An assessment of pain responses to thermal stimuli during stages of pregnancy.

A study utilizing visual analogue scale (VAS) responses to 43-51 degrees C nociceptive thermal stimuli was carried out to determine whether women demonstrate a change in pain responsiveness across stages of pregnancy. Fifteen women tested in late pregnancy, labor, and post partum showed no significant differences in VAS responses to thermal stimuli across these stages. The results indicate that no central pain inhibitory system is active during late pregnancy or labor.

Nerve injury-associated kinase: a sterile 20-like protein kinase up-regulated in dorsal root ganglia in a rat model of neuropathic pain.

Partial injury of the rat sciatic nerve elicits a variety of characteristic chemical, electrophysical and anatomical changes in primary sensory neurons and constitutes a physiologically relevant model of neuropathic pain. To elucidate molecular mechanisms that underlie the physiology of neuropathic pain, we have used messenger RNA differential display to identify genes that exhibit increased ipsilateral expression in L4/5 dorsal root ganglia, following unilateral partial ligation of the rat sciatic nerve. One set of partial complementary DNA clones identified in this screen was found to encode a protein kinase, nerve injury-associated kinase. Cloning of the full-length human nerve injury-associated kinase complementary DNA, together with recombinant expression analysis, reveal nerve injury-associated kinase to be a functional member of a subgroup of sterile 20-like protein kinases characterised by the presence of a putative carboxy terminal autoregulatory domain. Induction of nerve injury-associated kinase expression in dorsal root ganglia in the rat neuropathic pain model was confirmed by quantitative reverse transcription-polymerase chain reaction, and RNA in situ hybridization analysis revealed enhanced levels of nerve injury-associated kinase within neurons.Together, our data implicate nerve injury-associated kinase as a novel upstream component of an intracellular signalling cascade that is up-regulated in dorsal root ganglia neurons in response to sciatic nerve injury.

Characterization of the 5-hydroxytryptamine2A receptor-activated cascade in rat C6 glioma cells.

We have investigated the identity and intracellular cascade of responses resulting from activation of the endogenous 5-hydroxytryptamine receptor in the C6 rat glioma cell line. Sequence analysis of reverse transcription-polymerase chain reaction products derived from C6 glioma cell messenger RNA revealed complete homology with a portion of the rat 5-hydroxytryptamine2A receptor. The binding of [3H]ketanserin to cell membranes demonstrated a significant correlation with the 5-hydroxytryptamine2A receptor in rat frontal cortex. On intact cells, 5-hydroxytryptamine stimulated a concentration-dependent increase in phosphatidyl inositide turnover and intracellular [Ca2+] mediated by 5-hydroxytryptamine2A receptors. In whole-cell patch-clamp recordings, 5-hydroxytryptamine induced an outward current mediated predominantly by K+ ions (reversal potential = -80 mV). Using caged molecules containing Ca2+ or inositol 1,4,5-trisphosphate in the patch electrode solution, we found that rapid photolytic release of Ca2+ and particularly inositol 1,4,5-trisphosphate within the cytosol induced an outward current with characteristics similar to those seen after application of 5-hydroxytryptamine. Comparison between differentiated and undifferentiated cells revealed significantly higher receptor density and maximal phosphoinositide response to 5-hydroxytryptamine in undifferentiated cells but the associated rise in [Ca2+]i and activation of an outward current was observed more frequently in differentiated cells. Prolonged exposure of the cells to 5-hydroxytryptamine led to a decrease in all responses and to the down-regulation of receptor number. We conclude that the rat C6 glioma cell expresses a 5-hydroxytryptamine2A receptor identical to that found in rat brain and that stimulation of the receptor in C6 cells leads to the activation of Ca2+ activated K+ channels via phosphoinositide hydrolysis and subsequent rise in cytosolic Ca2+ ion concentration. However, the contrasting effects of differentiation on receptor number and phosphoinositide response to 5-hydroxytryptamine compared to Ca2+ release and conductance change indicate that a complex relationship exists between the component parts of the receptor-activated cascade.

Identification of differentially expressed genes in dorsal root ganglia following partial sciatic nerve injury.

Partial sciatic nerve injury, a model of neuropathic pain, elicits a variety of neurochemical, electrophysiological and neuroanatomical changes in primary sensory neurons. We have used the technique of messenger RNA differential display to identify genes with altered expression in these neurons which may contribute to the development of aberrant sensation following such peripheral nerve damage. This approach identified 14 distinct complementary DNA clones, representing transcripts with increased ipsilateral expression in L4/5 dorsal root ganglia, two weeks after unilateral partial ligation of the rat sciatic nerve. Both Zucker diabetic fatty rats and their lean counterparts were used in this study but none of the transcripts identified showed an induction that was confined to one of the two groups. The majority of the clones did not show significant sequence similarity to previously reported genes and therefore may represent novel messenger RNA sequences or, alternatively, unknown regions of partially characterised messenger RNAs. Two of the clones represented transcripts for the known proteins muscle LIM protein and acidic epididymal glycoprotein, neither of which had previously been associated with expression in the nervous system. Reverse transcriptase-polymerase chain reaction analysis and in situ hybridization confirmed that the messenger RNA expression of both muscle LIM protein and acidic epididymal glycoprotein was induced in an ipsilateral-specific manner. Their localisations, examined with in situ hybridization in L5 dorsal root ganglia, were limited in each case to a sub-population of neuronal profiles. Those neuronal profiles that demonstrated muscle LIM protein hybridization were distributed across the profile size range, whereas the distribution of acidic epididymal glycoprotein-positive profiles appeared to be skewed towards smaller profiles. The induction of muscle LIM protein and acidic epididymal glycoprotein in dorsal root ganglia may play an important functional role in the adaptive response of primary sensory neurons following partial sciatic nerve injury.

Humoral opsonins of the tunicate, Pyura stolonifera.

This study characterizes humoral opsonins from the tunicate, Pyura stolonifera. The predominant opsonic components in P. stolonifera hemolymph were found to be calcium-dependent lectins with broad carbohydrate specificities. The opsonic lectins were purified by carbohydrate affinity chromatography which eluted a complex pattern of proteins ranging in molecular mass from 80 to >200kDa. Reducing and two dimensional SDS-PAGE indicated that the diversity of mature lectins evident under non-reducing conditions resulted from the differential oligomerization of two polypeptide sub-units (35 and 22kDa). In addition to lectin-mediated opsonic activity, hemolymph was also found to contain proteolytically activated opsonins. These data suggest that multiple, possibly interactive opsonic systems co-exist in P. stolonifera.

A complement component C3-like protein from the tunicate, Styela plicata.

This study identifies a complement component C3-like protein in the solitary tunicate, Styela plicata. Three different polyclonal antibodies raised against C3 molecules from two species (humans and the tunicate, Halocynthia roretzi) were used to identify the C3-like protein in S. plicata hemolymph. The C3 cross-reactive protein is a 170kDa heterodimer composed of polypeptides (116 and 80kDa) that have molecular weights comparable to those of C3alpha and C3beta from other species. Amino acid sequencing and amino acid composition analysis confirmed that the C3-like protein from S. plicata is closely related to C3 from H. roretzi.

Chemotactic responses of hagfish (Vertebrata, Agnatha) leucocytes.

The chemotactic responses of hagfish leucocytes were tested using a variety of chemoattractants. Leucocyte migration was significantly enhanced by purified mammalian complement anaphylotoxin (C5a) and LPS-activated hagfish plasma. Checkerboard analyses confirmed that the responses of leucocytes to both of these chemoattractants were directed along concentration gradients (chemotaxis) and did not result from accelerated random movement (chemokinesis). Chemotaxis was undertaken by leucocyte fractions that were enriched in granulocytes, the predominant phagocytic cells of hagfish. The data suggest that chemotactic mechanisms may have been conserved during evolution to such a degree that mammalian chemoattractants can bind and activate chemotactic receptors on hagfish leucocytes. Moreover, hagfish appear to express plasma proteins that are structurally and functionally homologous to mammalian complement anaphylotoxins.

Acute administration of cocaine, but not amphetamine, increases the level of synaptotagmin IV mRNA in the dorsal striatum of rat.

Synaptotagmin IV (Syt IV) is an inducible member of a multi-gene family of synaptic vesicle proteins that participate in Ca2+-dependent and Ca2+-independent interactions during membrane trafficking. We have examined the pattern of expression of Syt IV mRNA following the administration of cocaine and amphetamine. A single acute dose of cocaine, but not amphetamine, resulted in a transient increase, as determined by in situ hybridization, in the steady-state level of Syt IV mRNA in the dorsal striatum of rats 1 h after the administration of the drug. No change in the hybridization pattern of the Syt IV-specific probe to other regions of the rat brain were observed following cocaine or amphetamine administration at the time points examined (1, 3, 6, 12 and 24 h). The pattern of synaptotagmin I-(Syt I) specific hybridization remained constant, relative to controls, for both the cocaine- and amphetamine-treated animals. Northern hybridization analysis of mRNA isolated from striatal tissue using oligonucleotide probes specific to Syt I and Syt IV demonstrated that the probes hybridized exclusively to transcripts of the sizes previously reported for these two synaptotagmins and confirmed that the relative level of Syt IV to Syt I mRNA increased following the administration of cocaine but not amphetamine. These results indicate that these drugs have different effects on altering the levels of Syt IV mRNA. This work, in conjunction with earlier work that demonstrated that cocaine and amphetamine have different effects on the expression of immediate early genes such as c-Fos, supports the hypothesis that these psychotropic agents evoke different patterns of gene expression which may lead to alteration in synaptic efficacy.

Dorsal root ganglion neurons show increased expression of the calcium channel alpha2delta-1 subunit following partial sciatic nerve injury.

Neuropathic pain is associated with changes in the electrophysiological and neurochemical properties of injured primary afferent neurons. A mRNA differential display study in rat L(4/5) dorsal root ganglia (DRGs) revealed upregulation of the calcium channel alpha2delta-1 subunit 2 weeks after partial sciatic nerve ligation (Seltzer model of neuropathic pain). The upregulated transcript appeared to represent previously unidentified sequence from the 3'-untranslated region of rat alpha2delta-1 mRNA. In situ hybridization using L(5) DRGs from sham operated rats showed that 73, 40 and 19% of small (<700 microm(2)), medium (700-1100 microm(2)) and large (>1100 microm(2)) neuronal profiles, respectively, expressed alpha2delta-1 mRNA. Two weeks following nerve injury there was a significant ipsilateral increase, both in the percentage of DRG neurons expressing alpha2delta-1 mRNA and in the intensity of the hybridization signal. Comparison of this ipsilateral expression with that in sham animals, revealed that for small, medium and large neurons, respectively, the proportion of neurons labelled increased by 1.2-, 1.8- and 2.7-fold, while the hybridization signal in alpha2delta-1-labelled neurons increased by 2.8-, 2.5- and 3.7-fold. The most intensely labelled neuronal profiles in ipsilateral, sham and contralateral DRGs, were generally those with small cross-sectional areas. The alpha2delta-1 auxiliary subunit is known to modulate calcium channel function in heterologous expression systems via its association with the pore-forming alpha1 calcium channel subunit. Therefore the increased levels of this subunit in the populations of primary afferents described may, via modulation of calcium-dependent processes such as neurotransmitter release and neuronal excitability, influence the processing of sensory information.

Genetic association and cellular function of MC1R variant alleles in human pigmentation.

We have examined MC1R variant allele frequencies in the general population of South East Queensland and in a collection of adolescent dizygotic and monozygotic twins and family members to define statistical associations with hair and skin color, freckling, and mole count. Results of these studies are consistent with a linear recessive allelic model with multiplicative penetrance in the inheritance of red hair. Four alleles, D84E, R151C, R160W, and D294H, are strongly associated with red hair and fair skin with multinomial regression analysis showing odds ratios of 63, 118, 50, and 94, respectively. An additional three low-penetrance alleles V60L, V92M, and R163Q have odds ratios 6, 5, and 2 relative to the wild-type allele. To address the cellular effects of MC1R variant alleles in signal transduction, we expressed these receptors in permanently transfected HEK293 cells. Measurement of receptor activity via induction of a cAMP-responsive luciferase reporter gene found that the R151C and R160W receptors were active in the presence of NDP-MSH ligand, but at much reduced levels compared with that seen with the wild-type receptor. The ability to stimulate phosphorylation of the cAMP response element binding protein (CREB) transcription factor was also apparent in all stimulated MC1R variant allele-expressing HEK293 cell extracts as assessed by immunoblotting. In contrast, human melanoma cell lines showed wide variation in the their ability to undergo cAMP-mediated CREB phosphorylation. Culture of human melanocytes of known MC1R genotype may provide the best experimental approach to examine the functional consequences for each MC1R variant allele. With this objective, we have established more than 300 melanocyte cell strains of defined MC1R genotype.

Validity of the multi-directional reach test: a practical measure for limits of stability in older adults.

BACKGROUND: Falls occur not only in the forward direction, but also to the side and backward. The purpose of this study was to develop a portable and valid tool to measure limits of stability in the anterior-posterior and medial-lateral directions. METHODS: Two hundred fifty-four community-dwelling older persons were administered the Berg Balance Test (BBT), the Timed Up & Go Test (TUG), and the Multi-Directional Reach Test (MDRT). For the MDRT, subjects performed maximal reaches with the outstretched arm forward (FR), to the right (RR), to the left (LR), and leaning backward (BR), with feet flat on the floor. Reach was measured by the subject's total hand excursion along a yardstick affixed to a telescoping tripod. RESULTS: Mean scores on the MDRT were FR = 8.89 +/- 3.4 in., BR = 4.64 +/- 3.07 in., RR = 6.15 +/- 2.99 in., and LR = 6.61 +/- 2.88 in. Interclass Correlation (ICC2,1) for the reaches were greater than.92. Reliability analysis (Cronbach's Alpha,.842) demonstrated that directional reaches measure similar but unique aspects of the MDRT. The MDRT demonstrated significant correlation with the BBT sum and significant inverse relationship with the scores on the TUG. Regression analysis revealed that activity level contributed to scores in the forward, right, and left direction and that fear of falling contributed to scores in the backward direction. CONCLUSION: The Multi-Directional Reach Test is an inexpensive, reliable, and valid tool for measuring the limits of stability as derived by reach in four directions. Values obtained on relatively healthy community-dwelling older adults serve as norms for screening patient populations.

Modulation of cortical and pyramidal tract induced motor responses by electrical stimulation of the basal ganglia.

Two general mechanisms based on anatomical studies are possible for modulation of motor activity by the caudate nucleus and globus pallidus. These mechanisms are: (1) modulation of the output of cortical neurons that exert motor influences; and (2) modulation of subcortical neurons that exert motor influences. Differentiation between these two mechanisms was accomplished in the present study by two experimental approaches, both of which employed the conditioning-test paradigm. The first approach was an investigation of caudate nucleus or globus pallidus modulation (conditioning stimulus) of flexor responses of the anterior tibialis muscle elicited by electrical stimulation of the sensorimotor cortex (test stimulus) or pyramidal tract (test stimulus). These investigations were carried out in the intact and in decorticate cats. The second approach was an analysis of modulation or cortically induced pyramidal tract responses (direct and indirect, D-I potentials) by conditioning shock trains delivered to various loci within the caudate nucleus or globus pallidus. Both approaches were designed to determine whixh inhibitory and facilitatory motor influences of the basal ganglia occurred at a cortical or subcortical level. Simultaneous stimulation of a locus within the caudate nucleus and the sensorimotor cortex evoked either an enhancement, reduction or no alteration of the cortically induced increase in flexor responses (measured by Ia afferent activity, EMG, myogram). In contrast, no inhibitory influences occurred from caudate nucleus stimulation upon pyramidal tract induced flexor responses in either the intact or decorticate preparation. Inhibitory loci were distributed toward the rostral portion of the caudate nucleus, whereas facilitatory loci were distributed throughout; this distribution was statistically significant (chi2; P less than 0.01). Only enhancement or no influence upon cortical induced or pyramidal tract induced responses were obtained by conditioning stimuli to the globus pallidus. In the unanesthetized but immobilized cat, trains of shocks delivered to the caudate nucleus enhanced, reduced or had no influence upon the cortically evoked direct (D) and indirect (I) potentials recorded in the bulbar pyramidal tract. The distribution of facilitatory and inhibitory loci was organized in a similar fashion as in theanesthetized preparation. From these observations, a model was proposed in which the output of the caudate nucleus exerts both facilitatory or inhibitory modulation of the tonically active globus pallidus cells. The latter in turn predominantly or exclusively facilitate output of pyramidal tract neurons as well as the output of subcortical structures; both effects facilitate motor responses at the spinal level.

Efficacy and safety of sildenafil citrate for treatment of erectile dysfunction in a population with associated organic risk factors.

The objective of this study was to determine the efficacy and safety of sildenafil in patients with erectile dysfunction (ED) and associated organic risk factors in a multispecialty clinic. Patients (n = 521) were diagnosed with ED based on self-assessment. Associated risk factors were managed by medication or life-style modifications, or both, before treatment with sildenafil for ED. Patients received a 50-mg dose of sildenafil that could be adjusted to 100 mg or 25 mg based on tolerability and efficacy. Patients recorded the number of successful intercourse encounters for 6 to 8 weeks, and the number of adverse events. Overall, there was an 82% successful intercourse rate with sildenafil treatment. The predominant associated risk factors for ED were hypertension (39%), hypogonadism (37%), and multiple medications (34%). Common adverse events due to sildenafil treatment were mild to moderate in nature and resulted in <2% patient discontinuation. Clinicians should be particularly careful to evaluate patients presenting with ED because the condition can be accompanied by a wide spectrum of risk factors requiring monitoring and treatment. However, with adequate treatment and control of these risk factors, the use of sildenafil in a representative population of men with ED in a multispecialty clinic can achieve a higher efficacy rate than previous studies have indicated.

A collectin-like protein from tunicates.

Collectins are a sub-family of C-type lectins from mammals and birds that are characterized by their collagen-like domains. The mammalian collectin, mannose binding lectin, has attracted considerable interest because it can activate complement components via a lectin-mediated complement pathway that is independent of immunoglobulins. In this study, we have identified a calcium-dependent lectin from the invertebrate (tunicate), Styela plicata, that bears substantial similarities to mammalian collectins. The tunicate lectin, which was isolated by carbohydrate affinity chromatography, has a reduced apparent molecular mass of 43 kDa. The 43 kDa reduced polypeptide appeared as dimers, trimers and hexamers when analyzed by non-reducing and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while gel filtration suggested that the native form of the protein was a nonamer. Amino acid sequence and amino acid composition analysis revealed obvious similarities between the tunicate lectin and mammalian collectins, notably the inclusion of a collagenous domain and a short, cysteine bearing N-terminal domain. The identification of a collectin-like protein in an invertebrate such as S. plicata, which does not express immunoglobulin, indicates that lectin-mediated complement pathways may predate the origin of antibodies.

Effects of vapocoolants on passive hip flexion in healthy subjects.

Vapocoolants have been documented clinically to increase range of motion limited by pain, but only one research study has been undertaken to investigate these observations. The purposes of this study were twofold: to replicate one existing quantitative study on Fluori-Methane Spray and to examine the effects of other brief, cold stimuli (ethyl chloride and isopropyl alcohol) to increase passive hip flexion in healthy adults. Three experimental groups and a control group were used; each subject served as his own control. Pretest and posttest measurements of passive hip flexion were measured in a gravity-minimized position. A specially designed table to ensure trunk stabilization was used. Brief, cold stimuli applied to the posterior region of the thigh were found ineffective in increasing passive hip flexion in healthy adults. The rationale for the findings is described in terms of the effects of brief, cold stimuli on a quiescent CNS as opposed to a CNS demonstrating heightened excitability in the pain-spasm-pain cycle.

Joint receptor contributions to reflexive and kinesthetic responses.

This article presents a review of the neuroanatomy and neurophysiology of joint receptors. The role of joint receptors in signaling position and movement has been studied since the early part of this century. Morphological descriptions and reflexive and kinesthetic contributions of articular receptors in the regulation of motor behavior have been identified in studies on anesthetized animals and to a lesser extent in studies on human subjects. Areas for future laboratory and clinical research include articular receptor input in motor learning and relearning tasks as well as standardization of methodology and responses for assessing joint position.

Kinesthetic awareness in subjects with multiple ankle sprains.

The purposes of this study were 1) to determine whether decreased kinesthetic awareness occurs in individuals with recurrent ankle sprains and 2) to determine whether the one-legged standing balance test can be used to differentiate between ankle instability in injured and uninjured ankles. Thirty athletes between 18 and 24 years of age with multiple sprains of one ankle and no reported sprains of the other ankle were tested to compare their ability to detect passive plantar flexion and standing balance in each ankle. Luce's choice theory was used to analyze subjects' responses. Subjects had significantly greater difficulty detecting passive motion in the ankle with sprains as compared with the uninjured ankle. Subjects also performed a one-legged standing balance test on both the injured and uninjured legs. In 20 subjects, either the subject or observer reported balance deficits on the injured side as compared with the uninjured side. The results of this study demonstrate the need for clinicians to evaluate kinesthetic deficits and to design exercise programs to improve kinesthetic awareness and decrease ankle instability in individuals with multiple ankle sprains.

Hand function in patients on maintenance hemodialysis.

The purpose of this study was to assess characteristics of hand function in 30 subjects on maintenance hemodialysis (MHD) with a forearm vascular access. Hand function was evaluated by measuring subjects' grip and pinch strength, range of motion, edema, and sensation and with the Grip Function Test and self-assessment scale. A difference in all hand function test results was found in the extremity with the vascular access compared with the contralateral extremity. Comparison of short-term subjects (on MHD less than two years) with long-term subjects (on MHD greater than two years) revealed significantly lower handgrip strength (p less than .05) and pinch strength (p less than .05) and significantly higher hand volume (edema) (p less than .05) in the long-term group than in the short-term group. We suggest that both the chronicity of renal failure and the presence of a vascular access may contribute to deterioration of hand function. The presence of hand deterioration in patients on MHD identifies a new patient population and a need for early baseline measurement and periodic assessment by physical therapists or hand therapists as renal rehabilitation team members.