Retinoylation of proteins in cell-free fractions of rat tissues in vitro.
(33/631)
all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins both in vivo and in vitro. The relative rates of incorporation of all-trans-11,12-(3)H-retinoic acid into rat tissue homogenates in the presence of ATP and coenzyme A were testes>>lung> or =brain> or =kidney>liver. Although all studied cellular organelles of the testes incorporated (3)H-retinoic acid into protein, mitochondria were by far the most active; indeed, up to 25% of the added tritiated retinoic acid (RA) became covalently bound to protein in a 90 min incubation period. In the absence of ATP, coenzyme A, or both cofactors, the amount of RA incorporated into the proteins of testes mitochondria fell to 37%, 16%, and 11%, respectively, of that incorporated in their presence. N-Ethylmaleimide (5 mM) strongly inhibited the reaction. Boiled mitochondria were inactive. After extensive extraction with CHCl(3)-CH(3)OH, the protein-bound radioactivity, which proved largely to be retinoic acid, was released by treatment with proteinase K, hydroxylamine, and dilute base. Thus, retinoic acid is most probably linked to protein as a thiol ester. By SDS-polyacrylamide gel electrophoresis, four protein fractions with molecular masses of approx. 20, 24, 29, and 45 kDa, as well as smaller amounts of larger entities, were labeled in testes mitochondria. The possible identities and roles of these retinoylated proteins are currently being explored. (+info)
Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue.
(34/631)
A critical step in the development of Agrobacterium tumifaciens-mediated transformation is the establishment of optimal conditions for T-DNA delivery into tissue from which whole plants can be regenerated. The efficient transformation of inflorescence tissue from 'Baldus', a commercial wheat variety, using the Agrobacterium strain AGLI harbouring the binary vector pAL156 is reported here. The effects of various factors on delivery and the transient expression of the uidA gene were studied including the duration of preculture, vacuum infiltration, the effect of sonication treatments, and Agrobacterium cell density. Optimal T-DNA delivery (as measured by uidA activity) was obtained from inflorescence tissues precultured for 21 d and sonicated. Increasing Agrobacterium cell density, the duration of inoculation/co-cultivation, and vacuum pressure, up to a threshold, increased uidA expression. The investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of immature wheat inflorescence tissue. (+info)
A chemotactic role for prostaglandins released from polymorphonuclear leucocytes during phagocytosis.
(35/631)
1. Prostaglandin E1 is chemotactic at concentrations down to 10 ng/ml for rabbit polymorphonuclear (PMN) leucocytes. Prostaglandins E2 and F2alpha have little or no chemotactic effect at concentrations up to 10 mug/ml. 2. Washed PMN leucocytes produced a chemotactic agent during phagocytosis, but not in the presence of indomethacin (28 muM). 3. Phagocytosing PMN leucocytes produce up to ten times as much prostaglandin as do resting cells. Some of this is prostaglandin E1 as judged by thin layer chromatography and differential bioassay. This prostaglandin production by PMN leucocytes is abolished by indomethacin (28 muM). 4. Ultrasonicated suspensions of PMN leucocytes produced prostaglandin from arachidonic aicd. This synthesis is inhibited by indomethacin. 5. Homogenates of PMN leucocytes which have been pre-incubated withe bacteria for 30 min show more prostaglandin synthetase activity than homogenates from PMN leucocytes which have not been exposed to bacteria. 6. It is concluded that in some forms of inflammation, prostaglandin E1 may play a controlling role in cellular migration. 7. PMN leucocytes may contribute to the generation of prostaglandins found in some inflammatory lesions. (+info)
Granulocytes as effectors in cell-mediated cytotoxicity of adherent target cells.
(36/631)
In the microassay for cell-mediated immunity, detachment of adherent target cells from the wells occurs to an even greater extent when tested with granulocytes than when tested with lymphocytes. Intact cells are not necessary since the sonic extract from granulocytes causes the same effect. The reaction by granulocytes appears to be mediated by enzymes, and is inhibited by the presence in the wells of hyaluronic acid. Moreover, it is limited to the detachment of adherent target cells, since neither intact nor sonically disrupted granulocytes exhibit cytotoxic activity in the chromium release assay. The detachment of target cells is also inhibited by heparin which may be used to specifically mullify the effect of granulocytes contaminating lymphocyte suspensions. (+info)
Thermodynamic effects of the hydrophobic surfactant proteins on the early adsorption of pulmonary surfactant.
(37/631)
We determined the influence of the two hydrophobic proteins, SP-B and SP-C, on the thermodynamic barriers that limit adsorption of pulmonary surfactant to the air-water interface. We compared the temperature and concentration dependence of adsorption, measured by monitoring surface tension, between calf lung surfactant extract (CLSE) and the complete set of neutral and phospholipids (N&PL) without the proteins. Three stages generally characterized the various adsorption isotherms: an initial delay during which surface tension remained constant, a fall in surface tension at decreasing rates, and, for experiments that reached approximately 40 mN/m, a late acceleration of the fall in surface tension to approximately 25 mN/m. For the initial change in surface tension, the surfactant proteins accelerated adsorption for CLSE relative to N&PL by more than ten-fold, reducing the Gibbs free energy of transition (DeltaG(O)) from 119 to 112 kJ/mole. For the lipids alone in N&PL, the enthalpy of transition (DeltaH(O), 54 kJ/mole) and entropy (-T. DeltaS, 65 kJ/mole at 37 degrees C) made roughly equal contributions to DeltaG(O). The proteins in CLSE had little effect on -T. DeltaS(O) (68 kJ/mole), but lowered DeltaG(O) for CLSE by reducing DeltaH(O) (44 kJ/mole). Models of the detailed mechanisms by which the proteins facilitate adsorption must meet these thermodynamic constraints. (+info)
Exposure of sperm head equatorin after acrosome reaction and its fate after fertilization in mice.
(38/631)
Equatorin is a sperm head equatorial protein, possibly involved in sperm-oocyte fusion (Toshimori et al., Biol Reprod 1998; 59:22-29). In the present work, we have shown that equatorin contained in the posterior acrosome is detectable only after spontaneous or induced acrosome reactions following fixation and permeabilization, but not in intact spermatozoa. The presence of protease inhibitors during sonication or ionophore treatments does not inhibit the exposure of the antigenic epitope. The zona-penetrated spermatozoa lying in the perivitelline space display equatorin, similar to those of the acrosome-reacted ones. After sperm-egg fusion during in vitro fertilization (IVF), the equatorin dissociates from the sperm head equatorial region and remains at the vicinity of the decondensing male pronuclei. During pronuclear apposition stage, it is pushed away from the pronuclei, possibly by the perinuclear microtubules. After first cleavage, equatorin is inherited by one of the proembryonic cells. The residual equatorin disappears after the second cleavage. Microinjected whole spermatozoa or sperm heads into the MII stage oocytes display equatorin similar to those of the perivitelline sperm. After activation, it dissociates from the sperm nuclei in a similar manner as during IVF. The mode of equatorin degeneration during fertilization is similar to those of the sperm tail components or mitochondria, but different from those of the membrane associated proteins. (+info)
Nature of the surviving plaque-forming unit of reovirus in water containing bromine.
(39/631)
The initial inactivation of reovirus in water containing 3 to 7 microns M bromine as HOBr was very rapid. Electron microscopy revealed extensive physical damage to the virions in as little as 1 min, but none were degraded beyond recognition. As treatment time continued, the reaction rate decreased toward a plateau of resistance, usually at about the 10-4 survival level; still no particles were lost. Progeny grown from these resistant plaque-forming units (PFU) were no more resistant to HOBr than the parent cultures. Small-number aggregation (adhering groups of two to ten virions counted by electron microscopy) had no detectable effect on the level of persistant PFU. Large aggregates seemed to be involved. Sonic treatment at 20 kHz after bromine exposure increased survival PFU titer 10- to 43-fold. Virus exposed to light centrifugation prior to bromine treatment did not show the plateau of resistance. Surviving PFU sedimented faster in a shallow sucrose gradient than single virions. Large aggregates were apparently too few to be counted by electron microscopy, but their penetration and inactivation must be achieved by any disinfectant chosen to rid water of reovirus. (+info)
Sonication of mouse sperm membranes reveals distinct protein domains.
(40/631)
Molecular interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized. To begin to characterize sperm components that are involved in sperm-ZP interactions, we isolated and density fractionated sperm membranes. The membrane fractions recovered from a density fractionation protocol were characterized, and sonication was compared with vortexing for preparation of sperm membranes by examining the distribution of proteins in the membrane fractions obtained from these 2 protocols. Biochemical and microscopic analyses were used to determine the composition of the sonicated membrane fractions, and immunoblotting was used to identify fractions containing some of the previously suggested ZP3 receptors. Transmission electron microscopy revealed that bands 1-3 contained membrane vesicles and band 4 contained axonemal and midpiece fragments. SDS-PAGE revealed that bands 1 and 2 shared many proteins, but band 3 contained a number of unique proteins. Surface labeling with 125I demonstrated that bands 1 and 2 contained the majority of the sperm surface protein markers, whereas band 3 contained minor amounts of surface markers. Lectin-binding characteristics of sperm membrane glycoproteins were used to compare the relative distribution of glycosylated proteins in vortexed or sonicated membrane preparations. These characterizations indicate that sonication enhanced the differential distribution of sperm membrane proteins among the density fractions and suggests that this method is preferable for preparation of membrane fractions to be used for identification of proteins that mediate sperm-egg interactions. (+info)