Natural cytotoxic cell-specific cytotoxic factor produced by IL-3-dependent basophilic/mast cells. Relationship to TNF

The murine IL-3-dependent mast cell line, PT18-A17, and the rat basophilic leukemia cell line, RBL-2H3, were found to mediate natural cytotoxic (NC) activity via the release of a soluble factor which specifically lysed NC-sensitive WEHI-164 but not NK-sensitive YAC-1 tumor cells. The release of this NC cell-specific cytotoxic factor was enhanced by triggering of both types of cells via IgE receptor bridging. This factor had activity on TNF-sensitive but not TNF-resistant cell lines and could be neutralized by two independently produced polyclonal anti-mouse TNF antisera. It was not neutralized by antibodies against mouse IFN-alpha/beta or IFN-gamma. Moreover, it was not neutralized by a monoclonal or a polyclonal anti-human TNF, demonstrating that the rodent TNF differed antigenically from human TNF. These results indicate that the cytotoxic factor released from a murine IL-3-dependent mast cell line and from a rat basophilic leukemia cell line is immunologically and functionally related to murine TNF.     

Dual regulation of resistance against Toxoplasma gondii infection by Lyt-2+ and Lyt-1+, L3T4+ T cells in mice

Studies were performed to attempt to define the T cell subset responsible for resistance to Toxoplasma gondii. A temperature-sensitive mutant (ts-4) strain of T. gondii was used for immunization because it causes infection but does not persist in the host. Immunization with this strain induced marked resistance against lethal challenge infection with virulent strains of T. gondii in mice. The resistance could be transferred to normal recipient mice by i.v. injection of spleen cells from ts-4-immunized mice. Marked inhibition of cyst formation in the recipient mice was also noted. The protective activity of immune spleen cells was removed by pretreatment of the spleen cells with anti-Thy-1.2 and C, indicating that T cells are responsible for the observed protection. Pretreatment of immune spleen cells with anti-Lyt-2.2 and C completely ablated their protective effect; pretreatment with anti-Lyt-1.2 or anti-L3T4 and C had lesser effects on their ability to transfer resistance. The effect of anti-Lyt-1.2 was the same as that obtained with anti-L3T4. This suggested that one T cell subset that is partially responsible for protection has both Lyt-1.2 and L3T4 markers on the cell surface. These results indicate that there are substantial roles for both the Lyt-2+ and Lyt-1+, L3T4 T cell subsets in dual regulation of resistance against toxoplasma infection and that Lyt-2+ T cells are the principal mediator of the resistance.     

Complex of N-phosphonacetyl-L-aspartate with aspartate carbamoyltransferase. X-ray refinement, analysis of conformational changes and catalytic and allosteric mechanisms

The allosteric enzyme aspartate carbamoyltransferase of Escherichia coli consists of six regulatory chains (R) and six catalytic chains (C) in D3 symmetry. The less active T conformation, complexed to the allosteric inhibitor CTP has been refined to 2.6 A (R-factor of 0.155). We now report refinement of the more active R conformation, complexed to the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) to 2.4 A (R-factor of 0.165, root-mean-square deviations from ideal bond distances and angles of 0.013 A and 2.2 degrees, respectively). The antiparallel beta-sheet in the revised segment 8-65 of the regulatory chain of the T conformation is confirmed in the R conformation, as is also the interchange of alanine 1 with the side-chain of asparagine 2 in the catalytic chain. The crystallographic asymmetric unit containing one-third of the molecule (C2R2) includes 925 sites for water molecules, and seven side-chains in alternative conformations. The gross conformational changes of the T to R transition are confirmed, including the elongation of the molecule along its threefold axis by 12 A, the relative reorientation of the catalytic trimers C3 by 10 degrees, and the rotation of the regulatory dimers R2 about the molecular twofold axis by 15 degrees. No changes occur in secondary structure. Essentially rigid-body transformations account for the movement of the four domains of each catalytic-regulatory unit; these include the allosteric effector domain, the equatorial (aspartate) domain, and the combination of the polar (carbamyl phosphate) and zinc domain, which moves as a rigid unit. However, interfaces change, for example the interface between the zinc domain of the R chain and the equatorial domain of the C chain, is nearly absent in the T state, but becomes extensive in the R state of the enzyme; also one catalytic-regulatory interface (C1-R4) of the T state disappears in the more active R state of the enzyme. Segments 50-55, 77-86 and 231-246 of the catalytic chain and segments 51-55, 67-72 and 150-153 of the regulatory chain show conformational changes that go beyond the rigid-body movement of their corresponding domains. The localized conformational changes in the catalytic chain all derive from the interactions of the enzyme with the inhibitor PALA; these changes may be important for the catalytic mechanism. The conformation changes in segments 67-72 and 150-153 of the regulatory chain may be important for the allosteric control of substrate binding. On the basis of the conformational differences of the T and R states of the enzyme, we present a plausible scheme for catalysis that assumes the ordered binding of substrates and the ordered release o     

Chromatin reconstitution on small DNA rings. I

Chromatin was reconstituted using the four core histones on 359 base-pair nicked and closed rings by salt dialysis and/or at physiological ionic strength by means of polyglutamic acid. The products, which consisted of mono- and dinucleosomes, were characterized by gel electrophoresis, sedimentation in sucrose gradients and high-resolution electron microscopy. The results were as follows. (1) The efficiency of the reconstitution was found first to increase with the negative linking difference of the closed rings relative to their relaxed configuration to reach a maximum for -2 turns, and then to decrease for the largest difference of -3 turns. Discrepancies between topoisomers were also observed with regard to differential formation of mono- and dinucleosomes. Topoisomer -1 reconstituted monomers easily but reconstituted dimers with difficulty, whilst this discrimination was virtually absent in the case of topoisomers -2 and -3. Moreover, mononucleosomes on the nicked ring were, with respect to their electrophoretic mobility, similar to mononucleosomes formed on topoisomer -1 but not to those on the other topoisomers, whose mobilities were greater. These features were interpreted in terms of the linking number change associated with the formation of a nucleosome monomer and dimer, approximately -1 and -2 turns, respectively. (2) Two dinucleosome subtypes were found to form in a sequential manner. Their different electrophoretic mobilities and sedimentation coefficients suggested that the early subtype is lighter, probably because of an incomplete histone complement in the second nucleosome of that subtype as a result of an impaired co-operativity in octamer assembly due to the small ring size. (3) An electron microscopic examination of the chromatin reconstituted on topoisomer -2 revealed that both mono- and dinucleosomes adopt two different, salt-dependent, morphologies each: in type I, entering and exiting DNAs do not cross, whilst they do in type II. Type I configuration is favoured in lower salt, whereas type II is favoured in higher salt. Such behaviour explains why nucleosomes in dimers were found to be always diametrically opposed on the rings rather than sometimes apposed, as would have been expected from a random deposition of the histone cores.     

Inhibition of gastric acid secretion by peptide YY is independent of gastric somatostatin release in the rat

The purpose of this study was to determine whether the inhibitory action of peptide YY (PYY) on gastric acid secretion is attributable to the release of gastric somatostatin in rats. Two groups of rats (six rats/group) were anesthetized with urethane and prepared with gastric fistulas and jugular catheters. Pentagastrin (18 micrograms/kg-h) was given intravenously for 150 min to stimulate gastric acid secretion. Intravenous PYY (130 micrograms/kg-h) inhibited pentagastrin-stimulated gastric acid secretion significantly (P less than 0.05). Administration of iv PYY resulted in a 41% reduction (P less than 0.05) in pentagastrin-stimulated gastric acid secretion. In another group of anesthetized rats, administration of PYY (10(-7), 10(-8) M) failed to stimulate a release of somatostatin from the isolated-perfused rat stomach. Our findings indicate that PYY can inhibit gastric acid secretion independently of release of gastric somatostatin in the rat.     

Enhancement of allergic lung sensitization in mice by ozone inhalation

Inhaled ozone was found to exert an enhancing effect for allergic lung sensitization when mice contracted an aerosolized allergen. The animals were exposed to ozone concentrations of 0.24, 0.16, 0.13, and 0.10 ppm. After 4 days of continuous ozone exposure, the mice had allergen contact from an aerosolized solution of ovalbumin. The animals were then maintained in ambient air for several days before the cycle of ozone and aerosolized allergen was repeated over four allergen contact cycles. Mice were rested in ambient air for a week after the last allergen contact, and they were then tested for allergic sensitization by the intravenous injection of 2 mg of ovalbumin to induce anaphylactic shock in allergic individuals. The control groups of mice were maintained in ambient air throughout the experiment, but they experienced identical allergen contact with the ozone-exposed mice. The phenomenon of allergic enhancement from ozone inhalation was detected at 0.24, 0.16, and 0.13 ppm of ozone. The enhancing effect disappeared at 0.10 ppm of ozone. The study indicated a potential for increasing the number of allergically sensitized individuals when various allergens are inhaled during periods of high ozone exposure with the consequent adverse changes on respiratory membranes. The significance to human health of the allergic enhancement phenomenon by ozone needs investigation.     

Our method of correcting cryptotia

Our technique for the correction of cryptotia using both Z-plasty and the advancement flap is described. The main advantages are the simple design of the skin incision and the possibility of its application to cryptotia other than severe cartilage deformity and extreme lack of skin.     

Prefabricated sensate myocutaneous and osteomyocutaneous free flaps: an experimental model. Preliminary report

Principles of neovascularization have been reported for the successful creation of a variety of muscle and bone free flaps. This study demonstrates a simple and effective technique for construction of prefabricated sensate myocutaneous and osteomyocutaneous free flaps in a rat model. These experiments were carried out in 20 Sprague-Dawley male rats. In half the animals, a sensate myocutaneous flap was constructed by sandwiching the superficial inferior epigastric vessels between a laterally based external abdominal oblique muscle flap and a laterally based skin flap served by an identified cutaneous nerve. A similar preparation included a piece of iliac crest bone. Two to three weeks later, now neovascularized by the sandwiched vessels, the flaps were harvested and transferred as free flaps with high reliability. An increased number of potential donor sites, the versatility of design, and the ability to customize flaps to the specific recipient-site needs are proffered.     

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana, was studied. [24-14C]-24-Dehydro-26-deoxy-5 beta-ranol (3 alpha,7 alpha,12 alpha-trihydroxy-27-nor-5 beta-cholestan-24-one) was chemically synthesized from [24-14C]cholic acid and incubated with bullfrog liver homogenate fortified with NADPH. 24-Dehydro-26-deoxy-5 beta-ranol was shown to be converted into both 26-deoxy-5 beta-ranol and 24-epi-26-deoxy-5 beta-ranol [(24S)- and (24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrols] in addition to 5 beta-ranol [(24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol], which is the major bile alcohol of the bullfrog. [24-3H]-26-Deoxy-5 beta-ranol and [24-3H]-24-epi-26-deoxy-5 beta-ranol were prepared from 24-dehydro-26-deoxy-5 beta-ranol by reduction with sodium [3H] borohydride and administered respectively to two each of four bullfrogs by intraperitoneal injection. After 24 h, labeled 5 beta-ranol was isolated from the bile of the bullfrogs that received [24-3H]-26-deoxy-5 beta-ranol. In contrast little if any radioactivity could be detected in 5 beta-ranol or its 24-epimer after administration of [24-3H]-24-epi-26-deoxy-5 beta-ranol.