The calcium signal and phosphatidylinositol breakdown in 2H3 cells

Phosphatidylinositol (PI) and its phosphorylated derivatives are rapidly broken down in 2H3 cells stimulated with antigen, with a time course which coincides with the generation of the Ca signal. Stimulated PI breakdown is absolutely dependent on Ca2+ in the medium with a concentration dependence similar to that of the Ca signal and histamine release described in the preceding paper. However, PI breakdown does not depend on the rise in free cytoplasmic Ca2+ concentration in stimulated cells over the range 100 nM to 1 microM. Thus, stimulation by the ionophore A23187 causes only a small increase in PI breakdown and the Ca signal stimulated by antigen can be selectively blocked with appropriate concentrations of Zn2+ (100 microM) or La3+ (10-100 microM) which have small or negligible effects on stimulated PI breakdown. Both PI breakdown and the Ca signal appear to depend on a common external Ca2+ site (or sites) with Km approximately equal to 0.4 mM, and the data are consistent with either independent activation of PI phosphodiesterase and the Ca signal after antigenic stimulation, or with PI breakdown as a component of the mechanism by which the Ca signal is generated.     

Gastrointestinal helminths of the northern bobwhite in Florida: 1968 and 1983

We collected 153 northern bobwhites (Colinus virginianus) over a 10-mo period from Tall Timbers Research Station near Tallahassee, Florida, USA. Five species of gastrointestinal helminths were encountered commonly (greater than 30% prevalence): Cyrnea colini, Raillietina cesticillus, R. colinia, Heterakis isolonche, and Trichostrongylus tenuis. Other helminths included Brachylaima sp., Rhabdometra odiosa, Mediorhynchus papillosis, Cheilospirura spinosa, Dispharynx nasuta, Gongylonema ingluvicola, and Tetrameres pattersoni. During the intervening 15 yr since the last year-round study of this population of birds, C. spinosa and T. pattersoni have declined markedly, and Strongyloides sp. probably has become extinct locally. Prevalence and intensity seem more likely to fluctuate in parasite species that have complex life cycles.     

Chromosomal localization of group-specific component by in situ hybridization

Group-specific component (GC), an alpha 2-globulin plasma protein synthesized primarily in the liver, is the major vitamin D-binding protein in plasma. It has two common phenotypes, GC1 and GC2, which appear in all human populations. Using the cDNA insert containing the entire coding sequence of GC2, the GC gene was mapped to human chromosomal bands 4q13----q21.1 by in situ hybridization.     

Cloning and expression of a cDNA encoding a maize glutathione-S-transferase in E. coli.

The isolation and characterization of a family of maize glutathione-S-transferases (GST's) has been described previously. These enzymes are designated GSTs I, II and III based on size, substrate specificity and responsiveness to safeners. GST III has been shown to act on the herbicide alachlor as well as the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Clones were isolated from a maize cDNA library in lambda gt10. Three clones contained the entire coding region for GST III. The sequences of these clones were consistent with the known amino terminal GST III protein sequence. Moreover, expression of one of these clones in E. coli resulted in a GST activity as measured with both CDNB and alachlor, proving that at least one of the clones encodes an active GST III species. With the enzyme expressed in E. coli it will become possible to study enzyme structure-function relationships ex planta. While a number of different GST proteins are present in maize tissue the GST III gene is present in single or low copy in the genome.     

Refolding of serine proteinases

Bovine trypsinogen and chymotrypsinogen were successfully refolded as the mixed disulfide of glutathione using cysteine as the disulfide interchange catalyst. The native structures were regenerated with yields of 40%-50% at pH 8.6 and 4 degrees C, and the half-time for the refolding was approximately 60-75 min. We then refolded threonine-neochymotrypsinogen, which is a two-chain structure held together by disulfide bonds and produced on cleavage of Tyr 146-Thr 147 in native chymotrypsinogen [Duda CT, Light A, J Biol Chem 257 9866-9871, 1982]. Neochymotrypsinogen was denatured and fully reduced, and the thiols were converted to the mixed disulfide of glutathione. The two polypeptide fragments, representing the amino- and carboxyl-terminal domains, were separated on Sephadex G-75. Mixtures of the polypeptide fragments varying in the ratio of their concentration from 1:5 to 5:1 were refolded with yields of 21-28%. The lack of dependence on the concentration of either fragment and the relatively high yields suggest independent folding of the amino- and carboxyl-terminal domains. When the globular structures of the domains formed, they then interacted with one another and produced the native intermolecular disulfide bridge and the proper geometry of the active site.     

Physiology of F-Pilin Synthesis and Utilization

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to study the synthesis and turnover of F-pilin in membrane preparations of Escherichia coli K-12 under conditions which have been reported to affect the production of F-pili. Incorporation of [³⁵S]methionine into membrane F-pilin by cells in log phase was barely detectable at 25°C, but increased with temperature. The labeled pilin band was prominent in membranes from 37°C cultures and even more prominent if the growth temperature was raised to 42°C. The appearance of other tra products in the membranes was similarly temperature dependent. In cultures grown in glucose minimal medium at 37°C, the relative amount of membrane pilin and traT product synthesis remained unchanged from early log phase through early stationary phase; provision of glycerol or arabinose as a substitute carbon source had no obvious effect. Turnover of traT product and membrane F-pilin, as assessed in an Flac tra mutant strain which is incapable of elaborating pili, was not rapid. Both traT product and pilin subunits labeled in mid-log phase cells were still apparent in the membranes after growth of the cells to stationary phase. The relative amount of labeled pilin decreased with prolonged incubation in stationary phase, but the relative amount of traT product did not decrease even after the culture was incubated for 24 h. When wild-type Flac piliated cells were used, a similar result was obtained, but in this case, loss of F-pilin from the preparations could be acclerated by blending the cells. Although intermittent blending during culture growth caused a slow depletion of the labeled pilin pool, continuous blending resulted in the rapid disappearance of this pool from our preparations. Loss of other membrane polypeptides was not accelerated by our blending procedure, and blending did not affect the turnover of the pilin pool of the Flac tra mutant. Our data are consistent with a model in which pilin subunits are assembled transiently into pili, conserved by retraction, and made available for subsequent reassembly. Growth in 0.01% sodium dodecyl sulfate did not accelerate loss of pilin from the Flac strain compared with the Flac tra strain, and we suggest that in the presence of sodium dodecyl sulfate at this concentration, F-pili are not elaborated from cell surfaces.     

Evidence for androgen independence of male mounting behavior in white-crowned sparrows (Zonotrichia leucophrys gambelii)

Previous experiments have shown that expression of mounting behavior in sexually inexperienced, adult male white-crowned sparrows does not require elevated plasma levels of androgen; adult males maintained on nonstimulatory short days mount sexually receptive females. The experiments reported here demonstrate that (1) sexually inexperienced, prepubertal males maintained on nonstimulatory short days show very low mounting rates in response to female sexual displays; (2) these males exhibit high mounting rates when exposed to stimulatory long days but androgen treatment on short days is ineffective in stimulating high mounting rates; and (3) prepubertal castration has no effect on the expression of mounting behavior by photostimulated adult males. Thus, there is no evidence that mounting behavior of adult male white-crowned sparrows depends on androgen.     

Nuclear Overhauser experiments at 500 MHz on the downfield proton spectrum of a ribonuclease-resistant fragment of 5S ribonucleic acid

The downfield (9-15 ppm) proton NMR spectrum of a RNase A resistant fragment of E. coli 5S RNA has been studied by nuclear Overhauser methods. The fragment comprises bases 1-11 and 69-120 of the parent molecule [Douthwaite, S., Garrett, R.A., Wagner, R., & Feunteun, J. (1979) Nucleic Acids Res. 6, 2453-2470]. The nuclear Overhauser data identify two double helical structures in the fragment whose sequences are (GC)3(AU)(GC)3 and (GC)2(AU)(GU). These structures correspond exactly to the central portions of the terminal stem and procaryotic loop helices which should exist in the fragment sequences according to the Fox-Woese model [Fox, G.E., & Woese, C. R. (1975) Nature (London) 256, 505-506] of 5S RNA secondary structure. The significance of these and other nuclear Overhauser effects detected for the structure of 5S RNA and its fragment is discussed.