Major Motility Research Articles

Laminin-332 (Laminin-5) is the major motility ligand for B cell chronic lymphocytic leukemia

Cell adhesion and motility are central aspects in the pathophysiology of B cell chronic lymphocytic leukemia (B-CLL), but the role of specific extracellular matrix proteins is still to be completely unveiled. Purified peripheral blood neoplastic cells of B-CLL patients migrated poorly on laminins-111,-411,-511, but showed pronounced motility on laminin (LM)-332 in a high percentage of cases. B-CLL cell motility on LM-332 was mediated by the α3β1 integrin and was preferentially observed in cells carrying a mutated IgV H gene profile. Within normal lymph nodes, LM-332 was circumscribed around blood vessels and to areas corresponding to marginal zones, where it was deposited in a pattern reminiscent of reticular fibers. Conversely, in B-CLL involved lymph nodes, a positive LM-332 reticular mesh was diffusely evident, throughout the disrupted nodal architecture. In the present study we identified LM-332 as a crucial motility-promoting factor for B-CLL lymphocytes and as a potential constituent favoring the dissemination of B-CLL lymphocytes through vascular basement membranes and possibly lymph node compartments.

A Ser/Thr Kinase Required for Membrane-associated Assembly of the Major Sperm Protein Motility Apparatus in the Amoeboid Sperm of Ascaris

Leading edge protrusion in the amoeboid sperm of Ascaris suum is driven by the localized assembly of the major sperm protein (MSP) cytoskeleton in the same way that actin assembly powers protrusion in other types of crawling cell. Reconstitution of this process in vitro led to the identification of two accessory proteins required for MSP polymerization: an integral membrane phosphoprotein, MSP polymerization-organizing protein (MPOP), and a cytosolic component, MSP fiber protein 2 (MFP2). Here, we identify and characterize a 34-kDa cytosolic protein, MSP polymerization-activating kinase (MPAK) that links the activities of MPOP and MFP2. Depletion/add-back assays of sperm extracts showed that MPAK, which is a member of the casein kinase 1 family of Ser/Thr protein kinases, is required for motility. MPOP and MPAK comigrated by native gel electrophoresis, coimmunoprecipitated, and colocalized by immunofluorescence, indicating that MPOP binds to and recruits MPAK to the membrane surface. MPAK, in turn, phosphorylated MFP2 on threonine residues, resulting in incorporation of MFP2 into the cytoskeleton. Beads coated with MPAK assembled a surrounding cloud of MSP filaments when incubated in MPAK-depleted sperm extract, but only when supplemented with detergent-solubilized MPOP. Our results suggest that interactions involving MPOP, MPAK, and MFP2 focus MSP polymerization to the plasma membrane at the leading edge of the cell thereby generating protrusion and minimizing nonproductive filament formation elsewhere.

Identification and characterization of a sperm motility promoting glycoprotein from buffalo blood serum

Early investigators reported the occurrence of unidentified protein factors in biological fluids that may regulate sperm motility essential for fertility potential. This study reports for the first time purification of a forward motility stimulating protein (FMSF-I), to apparent homogeneity, from a biological fluid (buffalo blood serum) and its characterization. FMSF-I is the major motility protein of buffalo serum: a rich source of the factor. FMSF showed high protein specificity and affinity for activating forward motility of goat cauda epididymal spermatozoa. The motility promoter at 0.5 microM level showed maximal activity when nearly 60%-70% of spermatozoa expressed forward motility. It is a 66 kDa monomeric acidic protein rich in aspartate, glutamate, and leucine with isoelectric point of 3.7. FMSF: a Mg2+ -dependent protein binds to concanavalin A-agarose and the glycoprotein nature of FMSF has been confirmed by PAS staining. The factor lost activity completely when treated with alpha-mannosidase showing that the sugar part of the protein is essential for its biological activity. FMSF has no species specificity for its motility-activating potential. Sperm surface has specific receptors of FMSF, which is strongly immunogenic. The factor is present in testis and epididymis although liver is its richest source. Motility promoting efficacy of FMSF is markedly higher than the well-known non-protein motility activators: theophylline and bicarbonate or their combination. FMSF is a physiological activator of sperm motility and as a slaughterhouse byproduct it has potentiality for solving some of the problems of animal breeding, conservation of endangered species, and human infertility: a global social problem.

Characterization of Bacillus subtilis mutants resistant to cold shock-induced autolysis.

Bacillus subtilis vegetative cells undergo autolysis when exposed to cold shock treatment. A mutant (CA1) resistant to cold shock was isolated, and its DNA was used for the transformation of B. subtilis 168AR. The transformant (TR1) and CA1 had almost completely lost major vegetative autolysins (Cw1B and Cw1G) and motility, and showed a filamentous cell morphology during the exponential phase. Expression of the sigD-lacZ fusion was reduced in TR1. But the introduction of a SigD overproducing plasmid, pHYSigD, into TR1 led to a considerable increase in the amount of autolysin, a normal cell morphology (short rod), and the cold shock-sensitive phenotype. However, motility was not restored in the transformant. The roles of pleiotropic genes in cold shock-induced autolysis are discussed.

Retraction in amoeboid cell motility powered by cytoskeletal dynamics.

Cells crawl by coupling protrusion of their leading edge with retraction of their cell body. Protrusion is generated by the polymerization and bundling of filaments, but the mechanism of retraction is less clear. We have reconstituted retraction in vitro by adding Yersinia tyrosine phosphatase to the major sperm protein-based motility apparatus assembled from Ascaris sperm extracts. Retraction in vitro parallels that observed in vivo and is generated primarily by disassembly and rearrangement of the cytoskeleton. Therefore, cytoskeletal dynamics alone, unassisted by conventional motors, are able to generate both of these central components of amoeboid locomotion.

Motility disorders in childhood: Working Group Report of the First World Congress of Pediatric Gastroenterology, Hepatology, and Nutrition.

Motility disorders in childhood: Working Group Report of the First World Congress of Pediatric Gastroenterology, Hepatology, and Nutrition.

Effect of Osmolality and Oxygen Tension on the Survival of Mouse Sperm Frozen to Various Temperatures in Various Concentrations of Glycerol and Raffinose

Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding numbers of lines of mutant mice. However, successful and reproducible cryopreservation has proven to be a difficult problem. Furthermore, the underlying factors responsible for success or failure are mostly obscure. Several contributors to these difficulties have been identified. Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses associated with pipetting, mixing, and centrifugation, and others have found that they are severely limited in their tolerance to osmotic volume changes. We have hypothesized two other contributors to the difficulties. One is that the concentrations of glycerol used in published protocols are substantially lower than those found to be optimal for most mammalian cells. The other hypothesis relates to the fact that mouse sperm membranes are especially susceptible to damage from oxygen-derived free radicals. That damage may reduce their ability to survive freezing. If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure. To achieve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media. A previous report showed a protective effect. That is confirmed here under a broader range of conditions. The conditions studied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility after freezing at 21°C/min to −70°C. Cryoprotection increased with increasing raffinose concentration, provided that the concentration of PBS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits. Surprisingly, the best results were achieved in the total absence of glycerol. The highest motilities to date (68 ± 8%) after freezing to −70°C have been achieved using media containing Oxyrase, 0 M glycerol, and 18% raffinose in 14× strength modified PBS. We also determined the motility loss after freezing to intermediate temperatures, i.e., −10 and −30°C. The major motility loss occurred by −10°C, especially in the absence of Oxyrase. These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation. Oxyrase appears to lessen that damage substantially.

Diurnal motor activities of the esophagus in conscious dogs.

Diurnal motor activities of the upper, middle, and lower thirds of the thoracic esophagus, the lower esophageal sphincter, and the gastric body were recorded in six conscious dogs, using extraluminal force transducers. The motor activities of the thoracic esophagus and the lower esophageal sphincter were divided into three major motility patterns: feeding, digestive, and interdigestive. Each motility pattern was basically composed of repetitive bursts of contractions that were clearly classified into type I and II according to their contractile properties. Type I bursts were peristaltic contractions initiated at the upper thoracic esophagus and sequentially propagated distally to include the sphincter. Propagation velocity and duration of type I contractions were similar in all three motility patterns, and these contractions never were propagated into the stomach. Type II bursts were nonperistaltic simultaneous contractions of the thoracic esophagus and lower sphincter appearing synchronously with phase III of gastric interdigestive migrating contractions. Amplitude and duration of type II contractions were maximal at the sphincter, suggesting initiation at that site. In nonfeeding patterns, type I contractions would clear the esophagus of refluxed gastric contents, while type II contractions would prevent reflux.

Esophageal pharmacology and treatment of primary motility disorders.

Swallowing is a complex mechanism based on the coordinated collaboration of tongue, pharynx and esophagus. Disturbances of this interplay or disorders of one or several of these components lead to dysphagia, non-cardiac chest pain or regurgitation. The major primary esophageal motility disorders--achalasia, diffuse esophageal spasm, hypercontractile esophagus ('nutcracker esophagus') and non-specific motility disorder--are of unknown etiology. Other esophageal diseases, such as cervical diverticula or gastroesophageal reflux disease, might also be caused by a primary esophageal motility disorder. Medical treatment of esophageal disorders with esophageal hyper- or dysmotility requires agents that reduce esophageal contractile force (anticholinergic agents, nitrates, calcium antagonists). Despite the beneficial effect of the various drugs on esophageal motility parameters, the clinical benefit of medical treatment of esophageal motility disorders is rather disappointing. Calcium channel antagonist, alone or in combination with anticholinergics or nitrates, can be used as a medical trial, especially in mild achalasia. However, medical therapy is clearly inferior to pneumatic balloon dilation therapy. Recently, botulinum toxin injection was suggested as a therapeutic option in achalasia patients with good results on lower esophageal sphincter pressure (LESP) and symptom scores that were similar to the results achieved by pneumatic balloon dilation. Hypercontractile esophagus shows a good manometric response to calcium channel antagonists, but only little clinical effect in terms of improvement of symptoms. Diffuse esophageal spasm is a relatively rare disease and few clinical studies are available. The use of calcium channel antagonists can be beneficial, at least in some patients with diffuse esophageal spasm. From clinical and epidemiological studies, there is some evidence of a 'psychological' component in the pathogenesis or perception of esophageal symptoms. There is some clinical benefit from centrally acting drugs such as benzodiazepines or antidepressants. With the exception of botulinum toxin for achalasia, medical therapy of primary esophageal motility disorders is rather limited and the clinical results are poor. Further understanding of esophageal pathophysiology as well as development of new receptor-selective drugs might increase our chances of a successful treatment of primary esophageal motility disorders.

Characterization of Bacillus subtilis mutants resistant to cold shock-induced autolysis

Bacillus subtilis vegetative cells undergo autolysis when exposed to cold shock treatment. A mutant (CA1) resistant to cold shock was isolated, and its DNA was used for the transformation of B. subtilis 168AR. The transformant (TR1) and CA1 had almost completely lost major vegetative autolysins (CwlB and CwlG) and motility, and showed a filamentous cell morphology during the exponential phase. Expression of the sigD-lacZ fusion was reduced in TR1. But the introduction of a SigD overproducing plasmid, pHYSigD, into TR1 led to a considerable increase in the amount of autolysin, a normal cell morphology (short rod), and the cold shock-sensitive phenotype. However, motility was not restored in the transformant. The roles of pleiotropic genes in cold shock-induced autolysis are discussed.

Characterization of monoclonal antibodies raised to C-terminal peptides of pS2: A major trefoil peptide and motility factor expressed in adenocarcinomas and regions of mucosal injury

Two novel monoclonal antibodies, GE1 and GE2 raised against the C-terminal 31 and 28 amino acids of the estrogen-inducible trefoil peptide pS2, are described. Both antibodies are able to detect pS2 in formalin-fixed, paraffin-embedded tissues. Conditions are presented under which pS2 can be shown in cell lines by immunohistochemistry that has previously been problematic. The antibodies can specifically show the presence of pS2 in cell lysates by Western blotting and immunoprecipitation. In the form of an affinity column, the GEl monoclonal antibody can be used to purify pS2 from MCF-7 supernatants. The eluted peptide from the GEl affinity column shows a single band at 6,600 Da (predicted size for pS2) on Western blotting. These antibodies are valuable reagents in the analysis of the role of trefoil peptides in the maintenance of mucosal integrity, and may have applications in the assessment of pS2 expression in chronic gastrointestinal ulceration and adenocarcinomas that secrete pS2, where it may serve as a prognostic marker.

Immunocytes and abnormal gastrointestinal motor activity during ileitis in dogs.

Infiltration of specific immunocytes and stimulation of abnormal gastrointestinal motor activity during ileal inflammation induced by mucosal exposure to ethanol and acetic acid were investigated in 17 dogs. Ileal inflammation significantly increased the frequency of giant migrating contractions (GMCs) and decreased the frequency of migrating motor complexes (MMCs). The frequency of retrograde giant contractions (RGCs) increased only on the day of ethanol and acetic acid treatment. Diarrhea, urgency of defecation, and apparent abdominal discomfort were related to the increased frequency of GMCs. Ileal inflammation also prolonged the duration of postprandial MMC disruption. Histological and immunohistochemical findings indicated transmural inflammation with marked increase in polymorphonuclear cells in the lamina propria and muscularis externa layers. Myeloperoxidase activity increased severalfold in both layers. Cells containing interleukin-2 receptor (IL-2R) increased in the lamina propria. Other immunocytes, such as B and T lymphocytes, dendritic cells, and human leukocyte antigen DR-1 (HLADR)-positive cells, did not exhibit a significant increase in the inflamed ileum compared with the normal proximal jejunum. We conclude that stimulation of GMCs may be the major motility marker of intestinal inflammation.

Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells.

The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, is markedly modulated in cells during growth activation, differentiation, motility and cell transformation. The stimulation of quiescent cells by serum factors and the culturing of cells on highly adhesive matrices induce vinculin gene expression, whereas the transformation of fibroblast and epithelial cells often results in decreased vinculin expression (reviewed in Rodríguez Fernández, J. L., B. Geiger, D. Salomon, I. Sabanay, M. Zöller, and A. Ben-Ze'ev. 1992. J. Cell Biol. 119:427). To study the effect of reduced vinculin expression on cell behavior, 3T3 cells were transfected with an antisense vinculin cDNA construct, and clones displaying decreased vinculin levels down to 10-30% of control levels were isolated. These cells showed a round phenotype with smaller and fewer vinculin-positive plaques localized mostly at the cell periphery. In addition, they displayed an increased motility compared to controls, manifested by a faster closure of "wounds" introduced into the monolayer, and by the formation of longer phagokinetic tracks. Moreover, the antisense transfectants acquired a higher cloning efficiency and produced larger colonies in soft agar than the parental counterparts. The results demonstrate that the regulation of vinculin expression in cells can affect, in a major way, cell shape and motility, and that decreased vinculin expression can induce cellular changes reminiscent of those found in transformed cells.

Naegleria: A Research Partner For Cell and Developmental Biology1

ABSTRACT. Organisms in the genus Naegleria offer special opportunities for research in contemporary biology. the dramatic cell differentiation from amebae to flagellates is unique among eukaryotes in the rapidity, synchrony, reproducibility, homogeneity, and accessibility of a major phenotypic change. Environmental signals initiate a progressive signal transduction pathway in which genes are turned on, including those for several calcium‐binding proteins, and newly synthesized proteins become localized in newly assembled organelles, including the centriole‐like basal bodies, with the overall consequence that the cell changes its shape, motility, and behavior. This essay reviews research opportunities for which Naegleria excels, as well as interesting aspects of its biology that provide challenges for future investigations. Because these organisms alternate between two major eukaryotic motility forms, their phylogenetic position is also provocative. Although there are hints that Naegleria is capable of sexual reproduction in nature, mating has not yet been observed in the laboratory. In order to fully exploit the opportunities offered by this wonderful experimental system we are working to develop means to do genetic manipulation, in particular via DNA‐mediated transformation.

Medical treatment of esophageal motility disorders.

Swallowing is a complex mechanism that is based on the coordinated interplay of tongue, pharynx, and esophagus. Disturbances of this interplay or disorders of one or several of these components lead to dysphagia, non-cardiac chest pain, or regurgitation. The major esophageal motility disorders include achalasia, diffuse esophageal spasm, hypercontractile esophagus ("nutcracker esophagus"), and hypocontractile esophagus ("scleroderma esophagus"). Other esophageal diseases such as hypopharyngeal (Zenker's) diverticula or gastroesophageal reflux disease also may be sequelae of primary esophageal motility disorder. Finally, a substantial group of patients referred for evaluation of possible esophageal motor disorders have milder degrees of dysmotility--referred to as nonspecific esophageal motor disorder--that are of unclear clinical significance. Medical treatment of esophageal motility disorders involves the uses of agents that either reduce (anti-cholinergic agents, nitrates, calcium antagonists) or enhance (prokinetic agents) esophageal contractility. Despite the beneficial effect of the various drugs on esophageal motility parameters, the clinical benefit of medical treatment is often disappointing. From clinical and epidemiological studies there is some evidence for a "psychological" component in the pathogenesis or perception of esophageal symptoms. Further understanding of esophageal pathophysiology, as well as development of new receptor selective drugs, might increase our chances of successful treatment of esophageal motility disorders.

Gastrointestinal myoelectric activity in an infant with congenital idiopathic motility disorder

We investigated myoelectric activity in an 8-month-old male who presented with a perinatal bowel obstruction, duodenal band, congenital short small intestine, and persistent feeding intolerance. Serosal electrodes were surgically implanted on stomach, duodenum, and jejunum during Nissen fundoplication and ileostomy. A 5-cm ileal specimen was taken for in vitro studies. Spontaneous migrating myoelectric complexes (MMC) were present in stomach and small intestine. Bethanechol increased electrical response activity (ERA) in stomach and duodenum. Morphine induced intense ERA and distinct phase III activity. Pentagastrin infusion did not disrupt MMC cycling. Feeding disrupted MMC complex cycling 30-40 min after the meal. Metoclopramide before feeding delayed disruption of the MMC cycling after the feeding. Intermittent gastric arrhythmias were present after the fifth postoperative day. In vitro muscle strips showed spontaneous contractions and electrical control activity (ECA). Bethanechol, McNeil A-343, motilin, and cholecystokinin induced contractions, but pentagastrin had no effect. We conclude that in spite of a major clinical motility dysfunction, several of our findings were normal. The abnormalities include short MMC period, absence of disruption of MMC by pentagastrin, and gastric arrhythmias.