Effects of phospholipids on the function of (Ca2(+)-Mg2+)-ATPase.

The ATPase activity for the (Ca2(+)-Mg2+)-ATPase purified from rabbit skeletal muscle sarcoplasmic reticulum is lower when reconstituted into bilayers of dimyristoleoylphosphatidylcholine [(C14:1)PC] than when it is reconstituted into dioleoylphosphatidylcholine [(C18:1)PC]. The rate of formation of phosphoenzyme on addition of ATP is slower for (C14:1)PC-ATPase than for the native ATPase or (C18:1)PC-ATPase. The reduction in rate of phosphoenzyme formation is attributed to a reduction in the rate of a conformational change on the ATPase following binding of ATP but before phosphorylation. The level of phosphoenzyme formed from Pi is also less for (C14:1)PC-ATPase than for (C18:1)PC-ATPase. At steady state at pH 6.0 in the presence of ATP Ca2+ is released from (C18:1)PC-ATPase into the medium, but not from (C14:1)PC-ATPase. These effects of (C14:1)PC on the ATPase are reversed by addition of androstenol to a 1:1 molar ratio with (C14:1)PC. The results are interpreted in terms of a kinetic model for the ATPase.     

Complementing amino acid substitutions within loop 6 of the alpha/beta-barrel active site influence the CO2/O2 specificity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase

Photosynthesis-deficient mutant 45-3B of the green alga Chlamydomonas reinhardtii contains a chloroplast mutation that causes valine-331 to be replaced by alanine within the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. This amino acid substitution occurs in loop 6 of the alpha/beta-barrel active site, three residues distant from catalytic lysine-334. The mutation reduces the specific activity of the enzyme and also reduces its CO2/O2 specificity factor by 42%, but the amount of holoenzyme is unaffected. In a previous study, an intragenic-suppressor mutation, named S40-9D, was selected that causes threonine-342 to be replaced by isoleucine, thereby increasing the CO2/O2 specificity of the mutant enzyme by 36%. To determine which other residues might be able to complement the original mutation, nine additional genetically independent revertants have now been analyzed. Another intragenic suppressor, represented by mutation S61-2J, causes glycine-344 to be replaced by serine. This change increases the CO2/O2 specificity of the mutant enzyme by 25%. Of the revertants recovered and analyzed, the mutant enzyme was improved only due to true reversion or by intragenic suppression mediated by substitutions at residues 342 or 344. Changes in the physical properties of the two pairs of complementing substitutions indicate that steric effects within loop 6 are responsible for the observed changes in the CO2/O2 specificity of the enzyme.     

The determination of the pKa of histidine residues in proteins by Raman difference spectroscopy

Sensitive Raman difference spectroscopy was used to monitor the protonation and deprotonation of histidine residues in apo-transferrin. We have shown previously that the behavior of small molecules and/or small molecular groups bound to proteins or other large macromolecules can be studied by Raman difference spectroscopy (Yue, K.T. et al. (1989) J. Raman Spectrosc. 20, 541-545). Using this method, we have measured the Raman difference spectra of human transferrin at different pH values with respect to pH 8.9, titrating its various histidine residues. About 12 +/- 2 of the 19 residues were titrated. The pH difference spectrum of transferrin obtained is very similar to that of histidine in solution, but with clear differences in the 1200-1400 cm-1 region. A titration curve with pKa of 6.08 +/- 0.01 fit the data of histidine in solution and a value of 6.56 +/- 0.02 was found for the average value of the 12 histidine residues inside transferrin. The technique has enough sensitivity at present to monitor a single histidine residue in a 130 kDa molecule and to determine the titration curve of one residue in a 40 kDa protein.     

Studies of fluids simulating blood-like rheological properties and applications in models of arterial branches

We studied several non-Newtonian fluids to determine how closely they simulate the flow behavior of human blood. The viscous and viscoelastic properties of these fluids were compared with human blood samples in steady flow and transient flow Couette viscometers and in an oscillatory tube flow viscoelasticity analyzer. We examined: 1) A polyacrylamide suspension (Separan AP30 and AP45) to which we added 4% isopropanol and 0.01% magnesium chloride. 2) A suspension of 2% Dextran with 16% by weight biconcave disc-shaped particles simulating red blood cells. 3) 40% ghost cells prepared according to Dodge in Tri (hydroxymethyl) aminomethane. These ghost cells were used to simulate the two-phase flow behavior of blood. 4) A suspension of 5% Dextran (70,000) with 12% polystyrene particles (diameter of 1 micron) and 10 mMol calcium chloride. All these fluids closely approximate the flow behavior of blood and can be used in a variety of different experimental situations. To measure velocity distribution using a laser-Doppler-anemometer, we used fluids #1 and #3 in a rigid T-junction simulating the first septal branch of the left descending coronary artery. The measurements were done in steady and pulsatile flow experiments at different flow rate ratios. The fluids showed large differences in velocity profiles compared to Newtonian fluids.     

Effect of photobioreactor inclination on the biomass productivity of an outdoor algal culture

The profiles of photon flux density incidented on a tubularloop photobioreactor in the day could be altered by inclining the bioreactor at an angle with the horizontal. The photon flux density at noon decreased with increasing angle of inclination, whereas the photon flux density in the early morning and late afternoon increased with increasing angle of inclination. The overall photosynthetic radiance received by the bioreactor inclined at 0, 25, 45, and 80 degrees was 1:0.89:0.77:0.62. Regardless of the angle of bioreactor inclination, the overall biomass output rate of a fed-batch culture over an 8-h/day period was comparable (26-36 g-biomass m(-2) bioreactor surface area day(-1)). As a bioreactor inclined at an angle occupied smaller land area, and daily biomass output rate per land area of a bioreactor inclined at 80 degrees (130 g-biomass m(-2) land) was about six times of that obtainable at horizontal position (21-g biomass m(-2) land). The bioenergetics growth yield from the absorbed photosynthetic radiance was not a constant but an inverse function of the photon flux density. The quasi-steady state chlorophyll content of the Chlorella cells varied between 36 and 63 mg g(-1) cells. Photoinhibition of the maximum photosynthetic capacity was not observed in this study.     

吲(口朶)乙酸和苄基腺嘌呤对菊芋再生新皮组织分化的影响

根据前人的研究,植物激素对部分茎段剥皮后新皮再生有一定的影响。草本植物菊芋(Helianthus tuberosus L.)在正常情况下环剥后,其维管组织的分化过程与杜仲、茄子很不一样,对于一些外源激素的刺激反应可能也有差异,为此,有必要应用一些外源激素对菊芋环剥后再生新皮的组织分化进行试验研究。     

The stability of foreign protein production in genetically modified plant cells

Summary The stability of foreign protein production in genetically engineered plant cells was studied. A cultured tobacco cell line was transformed with a chimeric molecule carrying a bacterial gene, ß-glucuronidase (GUS), under plant regulatory sequences. The specific GUS activity was monitored for 294 days with ten independently transformed cell lines either in the presence or the absence of selectable antibiotics. Specific GUS activity was stably maintained in five lines. About a two-to four-fold increase in the GUS activity was observed from three cell lines. The remaining two cell lines lost the activity within the first 70 to 210 days. The presence of antibiotics did not significantly alter the stability of the foreign protein production in all cell lines examined.     

Suprachiasmatic nucleus and photic entrainment of circannual rhythms in ground squirrels.

The efficacy of photoperiod as a zeitgeber for entrainment of circannual body weight and estrous rhythms was tested in female golden-mantled ground squirrels maintained for 3 or more years in either a simulated natural photoperiod (SNP) or a fixed LD 14:10 photoperiod (FP). The role of the retinohypothalamic tract--suprachiasmatic nucleus (RHT-SCN) projection in photic entrainment was assessed in animals that sustained destruction of the SCN (SCNX). Circannual rhythms were lengthened by the SNP as compared to the FP. Mean periods (tau's) for neurologically intact animals in the third year of testing were 49.6 +/- 0.3 weeks and 43.1 +/- 1.2 weeks (p less than 0.001) for the SNP and FP groups, respectively; furthermore, 56% and 7% of animals in these groups had tau's not significantly different from 365 days (p less than 0.005), and within-group variability was lower for SNP than for FP squirrels (p less than 0.01). SCNX squirrels differed from animals with the SCN intact (SCNC), as evidenced by higher within-group variability (p less than 0.001); only 29% of SCNX squirrels had tau's not different from 365 days (p less than 0.03 compared to the SCNC group). The coupling between estrous and body weight rhythms that was evident in SCN-intact SNP and FP squirrels was disrupted in SCNX animals. The RHT-SCN pathway is implicated in entrainment and in maintenance of normal phase relations among the several circannual rhythms. In a second experiment, female squirrels were maintained for 2.5 years in an accelerated SNP that compressed two normal annual photocycles into each calendar year. Of 12 squirrels, 3 had tau's that did not differ significantly from 6 months; 6 had tau's equivalent to 12 months; and 3 had tau's significantly different from both 6 months and 12 months. The data suggest that photoperiod is a major zeitgeber for entrainment of golden-mantled ground squirrels circannual rhythms.     

Gly-238-Ser substitution changes the substrate specificity of the SHV class A beta-lactamases

The SHV-type beta-lactamase SHV-2A is related to SHV-1 by a Gly-238-Ser replacement. Strains carrying SHV-2A are resistant to the third generation cephems cefotaxime and ceftizoxime, whereas those that carry SHV-1 are sensitive to these drugs. We present a kinetic analysis of a SHV-1 and SHV-2A enzymes, with the goal of gaining insight into the role of residue 238 in hydrolyzing cefotaxime and ceftizoxime. SHV-2A shows altered kinetic properties for a number of other cephems that also have heterocyclic side chains at the amino position of the 7-aminocephalosporanic acid nucleus (R1 side chain), including a significantly higher kcat/Km than does SHV-1 for cephaloridine, cephalothin, and cefotiam. Two cephems with straight chain R1 substitutions, cephalosporin C and cephacetrile, are not hydrolyzed more efficiently by SHV-2A. These results indicate that the Ser-238-Gly substitution increases the affinity toward cephems with a heterocyclic ring in the R1 side chain. In addition, the data for ampicillin and benzylpenicillin show that addition of a nitrogen to the second carbon of the R1 side chain of a penem results in a lower kcat/Km for SHV-2A relative to SHV-1. These data strongly suggest that the previously proposed hydrogen bond formation between Ser-238 and the second carbon nitrogen of cefotaxime is not an important factor in hydrolysis by SHV-2A. We propose that the Gly-238 to Ser-238 replacement in SHV-2A has altered the hydrophobic pocket so that it can better accommodate cephems with bulky R1 side chains.     

Rat pineal gland phosducin: cDNA isolation, nucleotide sequence, and chromosomal assignment in the mouse

The pineal gland contains a soluble phosphoprotein, phosducin, which is homologous to that of retinal photoreceptors. Phosducin has been shown to bind the beta, gamma subunits of the retinal G-protein transducin. Retinal phosducin has been cloned and now we report a rat pineal cDNA encoding phosducin. A 1217-nucleotide cDNA was isolated from a rat pineal library by DNA-DNA hybridization with a polymerase chain reaction-amplified cDNA of bovine retina mRNA for phosducin. Northern blot analysis demonstrates that the mRNA for phosducin is approximately 1.3 kb in both rat pineal and rat retina. The translated mRNA from rat pineal encodes a protein with 246 amino acids, compared to the 245 amino acids of bovine retina phosducin. The predicted molecular weight of rat pineal phosducin is 28,201. Immunoblot analysis with affinity-purified antibodies against bovine retina phosducin identify a single immunoreactive protein of approximately 33 kDa in both rat retina and rat pineal. The amino acid sequence of rat pineal phosducin is homologous to that of bovine retina phosducin, revealing 89% identity and another 5.7% similarity. Both rat pineal and bovine retina phosducins are acidic proteins with pIs of 4.3 and 4.5, respectively. The translated protein lacks hydrophobic domains that would suggest an integral membrane protein. Rat pineal phosducin has a single consensus phosphorylation domain for protein kinase A that is nearly identical to that of retinal phosducin, which is phosphorylated by protein kinase A in situ. Rat phosducin also contains three potential phosphorylation domains for protein kinase C and nine for casein kinase II as well as a predicted site for N-glycosylation. The cDNA encoding phosducin was used to localize the gene within a linkage group to a large segment of mouse chromosome 1 in a conserved region with the long arm of human chromosome 1 with a panel of DNA samples from an interspecific cross. In keeping with a proposed role of retinal phosducin in down-regulation of the photo-transduction cascade, a modulatory role in signal transduction is proposed for pineal phosducin.