Identification of Endogenous Gibberellins in the Winter Annual Weed Thlaspi arvense L

Eleven endogenous gibberellins (GAs) were identified by combined gas chromatography-mass spectrometry in purified extracts from shoots of field pennycress (Thlaspi arvense L.): GA_{1,9,12,15,19,20,24,29,44,51,53}. Traces of GA₈ and GA₂₅ were tentatively indicated by combined gas chromatography-mass spectrometry-selected ion monitoring. Comparison of the total ion current traces indicated that GA₁₉ and GA₄₄ were most abundant, while GA_{12,15,20,24,29,53} occurred in lesser amounts. Only small amounts of GA_1,9,51 were present. The levels of GA₈ and GA₂₅ were barely detectable. Consideration of hydroxylation patterns of the ent-gibberellane ring structure indicates two families of GAs: one with a C-13 hydroxyl group (GA_{1,8,19,20,29,44,53}) and another whose members are either nonhydroxylated (GA_{9,12,15,24,25}) or lack a C-13 hydroxyl group (GA₅₁). This suggests that in field pennycress there are two parallel pathways for GA metabolism with an early branch point from GA₁₂: an early C-13 hydroxylation pathway, leading ultimately to GA₁ and GA₈ and a C-13 deoxy pathway culminating in the formation of GA₉ and GA₅₁.     

Dissociation of the receptor for immunoglobulin E in mild detergents

We previously showed that, in the absence of phospholipids, exposure of the tetrameric receptor for immunoglobulin E to mild detergents dissociates the intact beta chain and two gamma chains from the alpha chains. Having developed a practical method for assaying the dissociation, we have now explored a variety of different detergents, detergent concentrations, temperatures, times, salts, pHs, and other factors that influence the detergent-induced dissociation. Our findings should be useful for optimizing the stability of the receptor and for future studies on recombination of the subunits. The data suggest the following: (1) The critical perturbant is micellar detergent. (2) Unlike solubilization of membranes, where a molar ratio of micellar detergent:lipid of 2 is adequate, dissociation of the receptor is incomplete even at molar ratios of micellar detergent:receptor of greater than 10(5) and may be limited by a reversible component. (3) Detergents that are best for solubilizing membranes are also best for dissociating the receptors. (4) The latter observation and other data implicate bound lipid as stabilizing the receptor. Our findings may be applicable to the study of interactions between membrane proteins in general.     

Phase separation of the receptor for immunoglobulin E and its subunits in Triton X-114

Above its critical micelle concentration, Triton X-114 in solution forms two phases at room temperature: a lower phase containing supramicellar aggregates and an upper phase largely depleted of detergent. This property of the detergent is potentially useful for separating under mild conditions proteins that bind detergent from those that do not (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). We studied the distribution of the receptor for immunoglobulin E (IgE) and its subunits in the two phases. IgE and IgE complexed either with intact receptors or with the alpha chains of the receptor alone are principally partitioned into the upper phase, whereas the unliganded receptor as well as the isolated alpha, and especially the beta and gamma chains of the receptor, preferentially partition into the lower detergent phase. Chromatography of IgE and of the subunits of the receptor on a hydrophobic support showed that the beta and gamma chains have a considerably greater hydrophobic surface than the alpha chains or IgE. These results indicate that the distribution of a protein in the two phases of phase-separated Triton X-114 is not an all-or-none effect based upon whether it binds detergent or not. Rather, it reflects the overall balance between the hydrophobic and hydrophilic properties of the protein's surface.     

Role of intracellular pH in muscle fatigue

Intracellular pH of in vitro diaphragm preparations was determined following low- (5 Hz, 1.5 min) and high- (75 Hz, 1 min) frequency stimulation, using glass microelectrodes of the liquid membrane type (pHm). Results were compared with values obtained by the standard homogenate technique (pHh). High- and low-frequency stimulation reduced peak tetanic tension to 21 +/- 1 (SE) and 71 +/- 2% of initial values, respectively. Peak tetanic tension returned to resting values after 10- to 15-min recovery from high- or low-frequency stimulation. Resting pHm was 7.063 +/- 0.011 (n = 72), and after fatiguing stimulation declined to values as low as 6.33. During recovery pHm significantly increased and by 10 min had returned to prefatigue values. No difference was observed in the recovery of pHm between the low- and high-frequency stimulation groups (analysis of variance test, ANOVA), and in both groups pHm recovery was highly correlated to the recovery of peak tetanic tension (r = 0.94, P less than 0.001). Resting pHh was 7.219 +/- 0.023 (n = 13), which was significantly higher than the pHm value. In contrast to pHm, intracellular pHh was significantly higher during recovery from 75- vs. 5-Hz stimulation (P less than 0.05). For both groups pHh increased significantly with time and by 10 min returned to prestimulation values. The ANOVA test demonstrated that pHh values were significantly higher than pHm values during recovery from fatigue. The results from this study support our hypothesis that fatigue from both high- and low-frequency stimulation is at least partially due to the deleterious effects of intracellular acidosis on excitation-contraction coupling.     

A New Plating Medium for the Isolation of Enteric Pathogens: II. Comparison of Hektoen Enteric Agar with S S and E M B Agar

During this study, 2,855 stool specimens from patients at Cook County Hospital were cultured for enteric pathogens. Hektoen Enteric Agar (HE) was compared with E M B and S S Agars by replicate samplings with both direct and indirect methods. Shigella species were recovered more than twice as often on HE Agar as on S S Agar by both methods. With the direct method only, out of 98 Shigella isolated, 97 were isolated from HE Agar, 74 were recovered from E M B Agar, and 40 were found on S S Agar. In addition, HE yielded better isolation of Salmonella strains than did S S or E M B by either direct or indirect methods. The greater efficiency of HE medium is discussed with respect to colonial recognition of enteric pathogens.     

Crystallization and preliminary X-ray characterization of a soybean seed lipoxygenase

An isoenzyme of soybean (Glycine max L. Merrill cv. Provar) lipoxygenase (EC 1.13.11.12) has been crystallized using the vapor diffusion method. Crystals were grown from solutions of the protein (7 mg/ml) using 10 to 20% (w/v) polyethylene glycol 8000 in citrate/phosphate buffer (pH 5.7) containing 0.5% (w/v) n-octyl-beta-D-glucopyranoside. The crystals reached maximum dimensions of 0.3 mm x 0.2 mm x greater than 2 mm. The enzyme crystallized in space group C222(1) with unit cell dimensions a = 246 A, b = 193 A and c = 75 A. A calculated Vm value of 2.35 A3/dalton was obtained assuming two molecules per asymmetric unit. The density of the crystals was found to be 1.16 g/ml, which confirmed the presence of two molecules per asymmetric unit and indicated a solvent content of 47.5%.     

IgE receptor-mediated hydrolysis of phosphoinositides by cytoplasts from rat basophilic leukemia cells

Cytoplasts (plasma membrane sacs containing cytoplasm, endoplasmic reticulum, and few organelles) were prepared from rat basophilic leukemia cells by treatment with cytochalasin B and centrifugation at 33 degrees C through stepwise gradients of Ficoll. To compare the relative ability of cytoplasts and cells to generate second-messengers (inositol phosphates, Ca2+) in response to stimulation of the high affinity receptor for IgE, we normalized our results per recovered receptor by using the tightly bound IgE as a marker. This marker correlated well with other estimates of plasma membrane recovery. Furthermore, data normalized on this basis correlated well with data expressed as percentage of phosphoinositides hydrolyzed. The purest fraction of cytoplasts (containing about 6% of the receptors) was satisfactorily devoid of organelles and, at early times, generated about 50% as much inositol phosphates per receptor as did the intact, untreated cells. This response of the cytoplasts, like that of the cells, was totally dependent upon aggregation of the receptors. The response by the cytoplasts (in the 5-min time frame which we examined), unlike that of the cells, was not enhanced by the presence of extracellular Ca2+. Furthermore, unlike the cells, the cytoplasts failed to raise their intracellular free Ca2+ levels after addition of polyvalent Ag. This result suggests that aggregation of the receptors may be insufficient, by itself, to open the normal Ca2+ channels.     

Isolation and characterization of Y chromosome DNA probes.

A sorted, cloned Y chromosome phage library was screened for unique Y chromosome sequences. Of the thousands of plaques screened, 13 did not hybridize to radiolabeled 46,XX total chromosomal DNA. Three plaques were characterized further. Clone Y1 hybridized to multiple restriction enzyme fragments in both male and female DNA with more intense bands in male DNA. Clone Y2, also found in female and male DNA, is probably located in the pseudosutosomal region because extra copies of either the X or Y chromosomes increased Y2 restriction enzyme fragment intensity in total cellular DNA. Clone Y5 was male specific in three of four restriction enzyme digests although in the fourth a light hybridizing band was observed in both male and female DNA. Clone Y5 was sublocalized to band Yq 11.22 by hybridization to a panel of cellular DNA from patients with Y chromosome rearrangements. Clone Y5 can be used to test for retention of the proximally long arm Y suggested to cause gonadal cancer in carrier females. The long series of GA repeats in Y5, anticipated to be polymorphic, may provide a sensitive means to follow Y chromosome variation in human populations.     

Topography of intermediates in transcription initiation of E.coli

Three characteristic footprinting patterns resulted from probing the Escherichia coli RNA polymerase T7 A1 promoter complex by hydroxyl radicals in the temperature range between 4 degrees C and 37 degrees C. These were attributed to the closed complex, the intermediate complex and the open complex. In the closed complex, the RNA polymerase protects the DNA only at one side over five helical turns. In the intermediate complex, the range of the protected area is extended further downstream by two helical turns. This region of the DNA helix is fully protected, indicating that the RNA polymerase wraps around the DNA between base positions -13 and +20. In the open complex, a stretch between base positions -7 and +2, which was fully protected in the intermediate complex, becomes accessible towards hydroxyl radicals but only in the codogenic strand, indicating that the DNA strands are unwound. Our data suggest that only the DNA downstream of the promoter is involved in this unwinding process.