Cloning of the Zea mays controlling element Ac from the wx-m7 allele

Summary The cloning of the controlling element Ac from the wx-m7 allele of Zea mays is described. The cloned fragment carries a 4.3 kb insertion that by restriction analysis is indistinguishable from the Ac insertion in Ac wx-m9. It is located approximately 2.5 kb upstream of the Ac wx-m9 insertion. Offprint requests to: P. Starlinger     

Diverse post-translational modifications of the pannexin family of channel-forming proteins

The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions.     

Effects of solution polarity and viscosity on peptide deamidation.

Deamidation kinetics were measured for a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala, 0.02 mg/mL) in aqueous solutions containing glycerol (0-50% w/w) and poly(vinyl pyrrolidone) (PVP, 0-20% w/w) at 37 degrees C and pH 10 to determine the effects of solution polarity and viscosity on reactivity. The observed pseudo-first order deamidation rate constants, k(obs), decreased markedly when the viscosity increased from 0.7 to 13 cp, but showed no significant change at viscosities >13 cp. Values of k(obs) also increased with increasing dielectric constant and decreasing refractive index. Molecular dynamics simulations indicated that the free energy associated with Asn side-chain motion is insensitive to changes in dielectric constant, suggesting that the observed dielectric constant dependence is instead related primarily to the height of the transition state energy barrier. An empirical model was proposed to describe the effects of the viscosity, refractive index and dielectric constant on k(obs). Analysis of the regression coefficients suggested that both permanent and induced dipoles of the medium affect the deamidation rate constant, but that solution viscosity is relatively unimportant in the range studied.     

Failure to detect infection in fallow deer (Cervus dama) exposed to Theileria cervi from white-tailed deer

A frozen stabilate was produced from Theileria cervi sporozoites in salivary glands of adult Amblyomma americanum. The stabilate was inoculated into three fallow deer (Cervus dama) and two white-tailed deer (Odocoileus virginianus). Following inoculation, the white-tailed deer developed parasitemias as determined by blood smear examination at 11 and 13 days postexposure. Repeat examination of blood from the three fallow deer for 30 days postexposure failed to reveal observable piro-plasms. These findings indicate that fallow deer are not as susceptible to the Theileria cervi found in white-tailed deer from North America. Thus, there are some questions regarding the taxonomic position of this organism.     

Proposed Mechanism of Inheritance and Expression of the Human Fragile-X Syndrome of Mental Retardation

A mechanism is proposed for the inheritance and expression of the fragile-X-linked syndrome of mental retardation in humans. Two independent events are required for expression of the syndrome: the fragile-X mutation, and X chromosome inactivation in pre-oogonial cells. The fragile-X mutation at site Xq27 has little or no effect until the chromosome is inactivated in a female as part of the process of dosage compensation. At a stage where the inactivated X chromosome would normally be reactivated in preparation for oogenesis, the mutation results in a local block to the reactivation process. This block to reactivation leads to mental retardation in progeny by reducing the level of products from the unreactivated Xq27 region in male cells, and, for a heterozygous female, in somatic cells in which the normal X chromosome has been inactivated. Published data relevant to this proposed mechanism are discussed.     

Reconstitution of mitochondrial F0.F1-ATPase with phosphatidylcholine using the nonionic detergent, octylglucoside

A reconstitution procedure has been developed for the incorporation of the mitochondrial F0.F1-ATPase into the bilayer of egg phosphatidylcholine vesicles. The nonionic detergent, octylglucoside, egg phosphatidylcholine, and the lipid-deficient, oligomycin-sensitive F0.F1-ATPase (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) were combined in a 4770:320:1 detergent/phospholipid/protein molar ratio and then centrifuged on a discontinuous sucrose gradient to isolate the F0.F1-phosphatidylcholine complex. The specific activity of the reconstituted F0.F1-ATPase was as high as 14.5 mumol/min/mg protein, whereas with no added lipid the activity ranged between 1.4 and 2.2 mumol/min/mg protein. This reconstituted preparation exhibited greater than 90% oligomycin sensitivity which demonstrated the intactness of the multisubunit enzyme complex. The phosphatidylcholine/protein molar ratio of the reconstituted F0.F1 was 250:1 with less than 0.4% of the added octylglucoside remaining. Titrations with both phosphatidylcholine and octylglucoside demonstrated that the specific activity and oligomycin sensitivity were highly dependent on the concentrations of both phospholipid and detergent in the original reconstitution mixture. Analysis of the reconstituted ATPase by electron microscopy demonstrated that the catalytic portion of the enzyme complex projected from the phospholipid bilayer with an orientation similar to that observed with submitochondrial particles. The F0.F1-phosphatidylcholine complex was able to trap inulin, which suggests a vesicular structure impermeable to macromolecules. The electrophoretic mobility of the complex was identical to that for liposomes of egg phosphatidylcholine alone. The reconstitution conditions utilized give rise to an enzyme-phospholipid complex with very low ionic charge that demonstrates high oligomycin-sensitive ATPase activity.     

Ammonium release by zooplankton in suspensions of heat-killed algae and an evaluation of the flow-cell method

The maximum excretion rate of NH₄ (39 nmol mg dry wt^–1h^–1) was directly measured for Daphnia pulex by measuringNH₄ accumulation in bottles containing D. pulex and dense, satiatingsuspensions of heat-killed algae. Ammonium release rates inthe algal suspensions were compared to those of individual animalsremoved from the suspension and placed in flow cells. Ammoniumrelease rate, R (nmol mg dry wt^–1 h^–1). in the flowcell decreased very rapidly with time, t (min), after removalaccording to the relation R = 26 + 25e^–0.16t. Ammoniumexcretion obtained by the flow cell method after extrapolationto time zero was not significantly different from that obtainedin the bottles. The considerable experiment-to-experiment variationin NH₄ excretion was in large part correlated (r² = 0.73) withthe feeding rate on the algae.     

Congenital Cystic Malformation of the Bile Ducts: Report of a Case and Review of Related Literature

A 20-year-old woman had a cyst of the proximal part of the common bile duct and a cyst of the left hepatic duct; these lesions were diagnosed preoperatively by intravenous cholangiography and successfully operated upon. At the time of writing, she has been followed up for one year.Congenital defects in the biliary system are rare and, in a review of the literature, only two cases were found similar to this one. It is generally accepted that these lesions are congenital, but the exact pathogenesis is unknown.Alonso-Lej, Rever and Pessagno² reviewed the literature in 1959 and found 403 authentic congenital cysts of the hepatic ducts. The most common congenital defect is a single choledochal cyst of the lower end of the common bile duct. Pain, jaundice and tumour are the main symptoms.Until the advent of intravenous cholangiography, these lesions were seldom recognized preoperatively. Means of operative repair as well as complications and prognosis are reviewed.