Biochemical and phenotypical correction of an envelope mutant of Salmonella typhimurium

Salmonella typhimurium DA 361 bears an env D1 mutation with the following abnormal phenotypical and biochemical characteristics: a) it autolyses at stationary phase in nutrient broth; b) it grows in chains of short rods; c) it is a poor maltose fermenter and d) it has a diphosphatidylglycerol (DPG) content twice as high than its isogenic non-lytic pair DA 362 (env D+) and LT2, of which both are derivatives. Growth of DA 361 in the presence of 400 mM ethanol leads on a 50% decrease of DPG level, thereby equalling its PG/DPG ratio with those of the control strain. Consequently, a correction on the other phenotypical and biochemical anomalies are induced since the DA 361 strain decreases its autolytic activity, ferments normally maltose and appear as rods undifferentiated from DA 362.     

Characteristics of the maintenance of the upright posture in subjects touching an external object while standing on a moving or immobile platform

Postural sway was compared for humans touching an external object while standing on an immobile or slowly moving posturographic platform. When the platform moves, the central nervous system may interpret the movement of the point of the contact with the external object as the movement of the body relative to the support or as the movement of the support itself. Thus, the information concerning the body position that is provided by the touch becomes ambiguous. It was demonstrated that contact with an external object during standing on an unstable support leads to a decrease in support sway. When a subject stands on a moving platform, this decrease is smaller than in the case of an immobile platform. Contact with an external object causes a decrease in postural responses to shank muscle vibrations on an immobile platform. On a moving platform, this decrease is nonsignificant. The change in postural sway depending on the unambiguity of afferent information is discussed in terms of the interaction between afferent signals of different modalities on the basis of the body scheme in subjects maintaining balance.Translated from Fiziologiya Cheloveka, Vol. 31, No. 1, 2005, pp. 59–65.Original Russian Text Copyright © 2005 by Kazennikov, Shlykov, Levik.     

Human skin chymotrypsin-like proteinase chymase. Subcellular localization to mast cell granules and interaction with heparin and other glycosaminoglycans

The subcellular localization of human skin chymase to mast cell granules was established by immunoelectron microscopy, and binding of chymase to the area of the dermo-epidermal junction, a basement membrane, was demonstrated immunocytochemically in cryosections incubated with purified proteinase prior to immunolabeling. Because heparin and heparan sulfate proteoglycans are major constituents of mast cell granules and basement membranes, respectively, the ability of chymase to bind to glycosaminoglycans (GAG) was investigated. Among a variety of GAGs, only binding of chymase to heparin and heparan sulfate appears physiologically significant. Binding was ionic strength-dependent, involved amino groups on the proteinase, and correlated with increasing GAG sulfate content, indicating a predominantly electrostatic association. Interaction with heparin was observed in solutions containing up to 0.5 M NaCl, and interaction with heparan sulfate was observed in solutions containing up to 0.3 M NaCl. Binding of heparin did not detectably affect catalysis of peptide substrates, but may reduce accessibility of proteinase to protein substrates. Measurements among a series of serine class proteinases indicated that heparin binding was a more common property of mast cell proteinases than proteinases stored in other secretory granules. Binding of chymase to heparin is likely to have a storage as well as a structural role within the mast cell granule, whereas binding of chymase to heparan sulfate may have physiological significance after degranulation.     

Biological and structural properties of MIP-1 alpha expressed in yeast.

The murine macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MIP-1 alpha have not been well studied, although it has been identified as being identical to an inhibitor of haemopoietic stem cell growth. We have expressed MIP-1 alpha in yeast cells and purified it to sequence homogeneity. Structural analysis of this biologically active material by circular dichroism and fluorescence spectroscopy confirms that MIP-1 alpha has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. The in-vitro stem cell inhibitory properties have been confirmed using a range of murine progenitor cells including purified bone marrow progenitor cells (FACS-1), the FDCP-mix A4 cell line, and spleen colony forming unit (CFU-S) populations. Plateau levels of inhibition of stem cell growth were achieved using concentrations of 0.15 micrograms/ml MIP-1 alpha. We have also demonstrated that MIP-1 alpha is active in vivo: 5 micrograms of MIP-1 alpha per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1 alpha was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage-colony forming cells (GM-CFC) in response to granulocyte macrophage-colony stimulating factor (GM-CSF).     

An apparatus to measure in vivo biomechanical behavior of dorsi- and plantarflexors of mouse ankle.

We developed an apparatus to quantify the biomechanical behavior of the dorsi- and plantarflexor muscles of the ankle of an anesthetized mouse. When the dorsi- or plantarflexor muscle group is activated by electrical stimulation of either the peroneal or tibial nerve, the apparatus measures the moment developed about the ankle during isometric, isovelocity shortening, or isovelocity lengthening contractions. Displacements may be performed over the full 105 degrees range of ankle motion with an angular resolution of 0.09 degrees. Bidirectional isovelocity ramps in ankle angle up to 1,100 degrees/s are possible and are equivalent to maximum velocities of 2.3 fiber lengths/s (Lf/s) for the fibers in tibialis anterior muscle and 11.9 Lf/s for those in gastrocnemius muscle. During single contractions of the dorsi- and plantarflexor muscle groups at 37 degrees C and with both knee and ankle joint at 90 degrees neutral position, the isometric tetanic force developed was 1.4 and 3.3 N, power output at 2.2 Lf/s was 3.1 and 5.9 mW, and power absorption at 0.5 Lf/s was 4.9 and 9.0 mW, respectively. These values are in reasonable agreement with data from the same muscle groups tested in situ. We conclude that the apparatus provides valid measurements of force and power of the dorsi- and plantarflexor muscle groups.     

Insulin release independent of a rise in cytosolic free Ca2+ by forskolin and phorbol ester

The role of cytosolic free Ca2+ in insulin release was evaluated using isolated rat pancreatic islets permeabilized with digitonin and incubated in Ca-EGTA buffers to fix free Ca2+ concentration at arbitrary levels. Ca2+ induced insulin release in a concentration-dependent manner with the threshold being between 0.1 and 1 microM. The hormone release was increased by forskolin and 12-O-tetradecanoyl phorbol-13-acetate (TPA), a potent activator of adenylate cyclase and that of protein kinase C, respectively. The findings suggest that activation of both protein kinase A and protein kinase C modulate insulin release without a concomitant increase in cytosolic free Ca2+.     

Scanning electron microscopic study of uropathogen adherence to a plastic surface

We used a polyethylene surface to study the adherence of various urinary pathogens to a representative inert surface. The bacteria were suspended in filter-sterilized urine during this adhesion study, and differential adhesion was clearly demonstrated. Pseudomonas aeruginosa adhered most avidly and formed large microcolonies that were surrounded by an extensive amorphous matrix. Staphylococcus saprophyticus also formed microcolonies on the surface of the plastic droppers. In general, piliated strains of Escherichia coli adhered less avidly than the other organisms, but more avidly than nonpiliated strains; however, one piliated strain of E. coli adhered very poorly and behaved like a nonpiliated strain.