Regulation of rat liver pyruvate kinase. The effect of preincubation, pH, copper ions, fructose 1,6-diphosphate and dietary changes on enzyme activity

1. Preincubation of partially purified rat liver L-type pyruvate kinase at 25 degrees for 10min. causes a marked increase in co-operativity with respect to both the substrate, phosphoenolpyruvate, and the allosteric activator, fructose 1,6-diphosphate. 2. The results are consistent with the existence of two forms of liver L-type pyruvate kinase, designated forms L(A) and L(B). It is postulated that form L(A) has a low K(m) for phosphoenolpyruvate (about 0.1mm) and is not allosterically activated, whereas form L(B) is allosterically activated by fructose 1,6-diphosphate, exhibiting in the absence of the activator sigmoidal kinetics with half-maximal activity at about 1mm-phosphoenolpyruvate. In the presence of fructose 1,6-diphosphate, form L(B) gives Michaelis-Menten kinetics with K(m) less than 0.1mm. It is further postulated that preincubation converts form L(A) into form L(B). 3. The influence of pH on the preincubation effect was studied. 4. The inhibition of pyruvate kinase by Cu(2+) was studied in detail. Though phosphoenolpyruvate and fructose 1,6-diphosphate readily protect the enzyme against Cu(2+) inhibition, little evidence of significant reversal of the inhibition by these compounds could be found. 5. The effects of starvation, fructose feeding and preincubation on the pyruvate kinase activity of crude homogenates of various tissues of the rat were also studied.     

Oxyphenbutazone (Tandearil?) in Rhinoplasty—A Clinical Evaluation of Effectiveness

To evaluate the effectiveness of Oxyphenbutazone as an anti-inflammatory agent, a double-blind study of Oxyphenbutazone and a placebo in a group of 42 patients who had nasal cosmetic operations involving osteotomy was carried out. The observations included direct objective measurement of the width of the palpebral fissure after operation, grading of the severity of postoperative edema and ecchymosis from photographs, and observations by the patients regarding the clearing of the postoperative discoloration. It appeared from the results of these observations that Oxyphenbutazone is not effective in preventing postoperative edema in such operations or in promoting more rapid resolution of postoperative edema. It did appear to enhance the clearing of postoperative periorbital ecchymosis.     

Differential recovery of prostacyclin synthesis in cultured vascular endothelial vs. smooth muscle cells after inactivation of cyclooxygenase with aspirin

Conflicting findings from clinical trials on the use of aspirin in preventing myocardial infarction emphasize the importance of understanding the effects of aspirin on vascular cells. Cultured vascular endothelial cells and smooth muscle cells of human, rat and bovine origin synthesized prostacyclin, a key component in vascular homeostasis, when superfused with 14C arachidonic acid. Prostacyclin synthesis was inactivated following brief treatment with aspirin, which irreversibly acetylates cyclooxygenase. Marked differences were observed between endothelial and smooth muscle cells in the recovery of cyclooxygenase after aspirin treatment. Smooth muscle cells recovered within 3 hours by a process that required serum factors replaceable by epidermal growth factor (EGF) and TGF-beta. Recovery in both smooth muscle and endothelial cells was blocked by cycloheximide but not by actinomycin-D. Endothelial cell recovery occurred much more slowly, requiring up to 24 hours and was not dependent on serum factors or EGF. Furthermore, it was suppressed by growth inducing agents such as endothelial cell growth factor (ECGF) and was enhanced by conditions favoring growth arrest and cellular differentiation. Regulation of expression and recovery of cyclooxygenase following inactivation by aspirin thus differs considerably in the endothelial and smooth muscle compartments of the vasculature.     

Site-specific aflatoxin B1 adduction of sequence-positioned nucleosome core particles

The question of how the presence of nucleosomal packing of DNA modifies carcinogen interaction at specific sites cannot be answered by studies on whole chromatin or bulk nucleosomes because of the heterogeneity of DNA sequences in the particles. We have circumvented this problem by using nucleosomes that are homogenous in DNA sequence and hence in DNA-histone contact points. A cloned DNA fragment containing a sea urchin 5 S gene which precisely positions a histone octamer was employed. By using 32P end-labeled DNA and genotoxins that allow cleavage at sites of attack, the frequency of adduction at every susceptible nucleotide can be determined on sequencing gels. The small methylating agent dimethyl sulfate and the bulky alkylating agent aflatoxin B1-dichloride (AFB1-Cl2) were used to probe the influence of DNA-histone interactions on DNA alkylation patterns in the sequence-positioned core particle. We find dimethyl sulfate to bind with equal preference to naked or nucleosomal DNA. In contrast, AFB1-Cl2 binding is suppressed an average of 2.4-fold at guanyl sites within nucleosomes compared with AFB1-Cl2 affinity at the corresponding site in naked DNA. The DNA is more accessible in regions near the particle boundary. We observe no other histone-imposed localized changes in AFB1-Cl2 sequence specificity. Further, sites of DNase I cleavage or proposed DNA bending show neither enhanced nor reduced AFB1-Cl2 adduction to N7-guanine. Since AFB1-Cl2 binding sites lie in the major groove, nucleosomal DNA appears accessible to AFB1-Cl2 at all points of analysis but with an access which is uniformly restricted in the central 100 nucleotides of the core particle. The data available do not indicate further localized or site-specific perturbations in DNA interactions with the two carcinogens studied.     

Origin of the autoreactive anti-type II collagen response. II. Specificities, antibody isotypes and usage of V gene families of anti-type II collagen B cells

Autoantibodies play an important role in the pathogenesis of type II collagen-induced arthritis in mice. We have earlier reported a high frequency of cells producing anti-CII autoantibodies and a low frequency of cells producing multispecific antibodies, in regional lymph nodes 9 to 11 days after primary immunization with CII. It is shown here that anti-CII antibodies produced during primary immune response are IgG-antibodies mainly of IgG2a, IgG1 and IgG2b subclasses while IgM antibodies dominate primary responses elicited by OVA and denatured CII as analyzed with a large panel of hybridomas. Anti-CII antibodies generated during the primary response recognize at least five different epitopes on the CII molecule. The specificities of these antibodies for various epitopes result from combinational association of products encoded by genes derived from various VH and VK families and/or by the occurrence of somatic mutations. It is suggested that the primary anti-CII autoantibody response involves activation of memory B cells and is in this aspect different from the origin of "natural" autoantibodies.