Solubility of hemoglobin S by sedminetation, equilibrium, and antisickling compounds.

The effect of the compounds guanidine, arginine, lysine, and aspartic acid and the salt arginine aspartate on the solubility of deoxyhemoglobin S (Hb S) was studied by sedimentation equilibrium at 20–22 °C. Guanidine and arginine were found to be most effective, whereas aspartic acid and lysine had only a small effect. The effectiveness of these compounds in solubilizing Hb S is relatively pH independent. It is unlikely that the small effect of lysine and aspartic acid on the solubility of Hb S can account for the antisickling properties of lysine and aspartic acid previously reported (Sophianopoulos, A. J., et al. (1974) Clin. Biochem.7, 112–118). The effect of guanidine and arginine is large enough to account for a large part of such antisickling properties (Sophianopoulos et al. (1974). The nonideality of concentrated hemoglobin solutions (up to 0.3 g cm^?3) has been studied in detail. By using the liganded as well as the unliganded forms of both Hb S and Hb A, it was found that the magnitude of the virial (nonideality) coefficients can change with varying solution conditions. A comparison of pure Hb S with hemolysates is made using viscosity and sedimentation velocity.     

Comparative chiasma analysis using a computerised optical digitiser

A new computerised technique has been devised for measuring the distribution of chiasmata along diplotene bivalents. The method involves the introduction into the field of view of the microscope, of a fine light spot which can be accurately manipulated along the chromosomes of each bivalent. The data recorded include (a) the positions of the chiasmata along the bivalent in terms of their relative distances from the centromere and (b) the individual bivalent and cellular chiasma frequencies. — The method has been applied to the analysis of chiasma distribution patterns in the two known species of the genus Caledia, C. species nova 1 and C. captiva and in two chromosomal races of the latter. Statistical tests indicate that within bivalents at least 40% of the comparative distribution patterns of chiasmata between races and species are significantly different. Similar comparisons between populations within races reveal only 18% significant differences. — The observed distribution patterns of chiasmata in this genus suggest that chiasma formation is sequential from centromere to telomere. — The variation in the frequency and distribution of chiasmata between races and species suggests that the interference distances between successive chiasmata are, at least partially, independent of chiasma frequency and position. — The interracial and interspecific differences in chromosome structure are correlated with changes in chiasma pattern.     

Absence of “void area” in freeze-etched vesicles of the Alnus crispa var. mollis Fern. root nodule endophyte

Nitrogen-fixing root nodules of Alnus crispa var. mollis Fern. were studied by transmission electron microscopy and by freeze-etching technique. Ultrathin sectioning of septate vesicles of the actinomycetal endophyte showed an electron transparent zone, the so-called void area, between the vesicle cell wall and its encapsulation material. This void area was not observed in the freeze-etching replicas of cryoprotected nodular tissue. It is suggested that the void area is the result of the coming-off of the vesicle cell wall from the capsule and that its formation reflects difficulty in fixing the voluminous mature vesicle of the root nodule endophyte.     

The origin of the Adams-Schuster difference spectrum of oxyhemoglobin.

The characteristic difference spectrum reported by Adams and Schuster (Biochem. Biophys. Res. Commun. 1974, $\text{58}$, 525) on the addition of inositol hexaphosphate to oxyhemoglobin is similar to the difference spectrum between (i) isolated α- and β-chains, (ii) α- and β-semihemoglobins, (iii) addition of inorganic phosphate to oxyhemoglobin, (v) change in temperature of a solution of oxyhemoglobin, (v) change in pH of carp carboxyhemoglobin and (vi) addition of inositol hexaphosphate to α-semihemoglobin. The spectrum may also be generated by differentiation of the spectra of oxyhemoglobin and carboxyhemoglobin, implying that the common feature of the results reported above is a shift in the position of the absorption bands. This shift may arise from several causes and so its interpretation is uncertain.     

Regulation of phosphatidylcholine synthesis in rat liver endoplasmic reticulum.

The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation.     

Human placental diamine oxidase. Improved purification and characterization of a copper- and manganese-containing amine oxidase with novel substrate specificity.

1. Isoelectric focusing studies of human placental diamine oxidase showed the pI value of the active enzyme to be 6.5. This information was used in modifying the enzyme purification by incorporating column chromatography on DEAE-Sephadex with ionic strength and pH gradient elution and this, together with affinity chromatography on concanavalin A--Sepharose, gave a highly purified preparation, with a specific activity of 7.0 units/mg. 2. The enzyme gave the expected stoicheiometry with p-dimethylaminomethylbenzylamine as substrate (Keq. 2700) and also oxidized [8-arginine]vasopressin, [8-lysine]vasopressin, collagen and tropocollagen. Polyacrylamide gel slices showed identical migration of diamine-oxidizing and [8-lysine]vasopressin-oxidizing activity. 3. The molecular weight, determined by ultracentrifugation, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, variable polyacrylamide-gel electrophoresis and Sephadex G-200 column chromatography, was estimated to be approx. 70000. 4. E.s.r. spectroscopy showed that copper and manganese were present in the purified enzyme. This result was confirmed by atomic absorption spectroscopy, which indicated a stoicheiometry for copper and manganese of approx. 1.0 and 1.2g-atom respectively/70000mol.wt. unit. 5. The e.s.r. spectral intensity did not decrease nor did the spectral line shape change when excess of p-dimethylaminomethylbenzylamine was added to the enzyme. 6. Addition of K13CN to the enzyme eliminated the copper e.s.r. signal without affecting the manganese signal. 7. The placental enzyme therefore appears to differ from other amine oxidases in terms of its metal cofactor requirement, molecular weight and substrate specificity, and possible roles in vivo for this enzyme are discussed.     

Kinetics of cell fusion induced by a syncytia-producing mutant of herpes simplex virus type I.

We have isolated a number of plaque-morphology mutants from a strain of herpes simplex virus type I which, unlike the wild type, cause extensive cell fusion during a productive viral infection. After the onset of fusion, there is an exponential decrease in the number of single cells as a function of time after infection. At a multiplicity of infection (MOI) of 3.8 plaque-forming units per cell, fusion begins 5.3 h after infection with the number of single cells decreasing to 10% of the original number 10.2 h after infection. As the MOI is gradually increased from 0.4 to 8, the onset of fusion occurs earlier during infection. However, when the MOI is increased from 8 to 86, the onset of fusion does not occur any earlier. The rate of fusion is independent of the MOI for an MOI greater than 1. The rate of fusion varies linearly with initial cell density up to 3.5 X 10(4) cells/cm2 and is independent of initial cell density at higher cell concentrations. To assay cell fusion we have developed a smiple quantitative assay using a Coulter counter to measure the number of single cells as a function of time after infection. Data obtained using a Coulter counter are similar to those obtained with a microscope assay.     

Regulation of resynthesis of the CO2-acceptor in photosynthesis. Feedback inhibition of transketolase

The presence of ribulose-5-phosphate epimerase (EC 5.1.3.1, epimerase) in samples of ribose-5-phosphate isomerase (EC 5.3.1.6, isomerase) obtained from spinach ( Spinacea aleracea L. cv. Bloomsdale Long Standing) was determined using (i) a sampling procedure which measured the quantity of xylulose-5-phosphate formed in the reaction mixture and (ii) a coupled enzyme assay in which the rate of oxidation of NADH was measured after establishing steady-state concentrations of xylulose-5-phosphate, dihydroxacetonephosphate and glyceraldehyde-3-phosphate by the action of epimerase, transketolase (EC 2.2.1.1), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). In preparations where the ratio of isomerase to epimerase activities was less than 100, both assay procedures yielded valid indications of epimerase activity. The steady-state assay system was found, however, to seriously underestimate epimerase activity in enzyme preparations which were enriched in isomerase. Cross plots of epimerase activity determined by the sampling and steady-state procedures demonstrated that an inhibitor of the coupling enzyme mixture was formed in the presence of high relative concentrations of the isomerase. The inhibited coupling enzyme mixture was fully active with glycer-aldehyde-3-phosphate. Inhibition of the coupling enzyme mixture was attributed to transketolase. Feedback inhibition of transketolase is proposed to be of physiological significance in the photosynthesis cycle, operating to restrict resynthesis of CO₂-acceptor under conditions where high steady-state concentrations of the intermediates of the photosynthesis cycle are maintained.     

A fatty acid synthase blockade induces tumor cell-cycle arrest by down-regulating Skp2

In eukaryotes, fatty acid synthase (FAS) is the enzyme responsible for synthesis of palmitate, the precursor of long-chain nonessential fatty acids. FAS is up-regulated in a wide range of cancers and has been suggested as a relevant drug target. Here, two independent approaches are taken toward knocking down FAS and then probing its connection to tumor cell proliferation. In one approach, Orlistat, a drug approved for treating obesity, is used as a potent inhibitor of the thioesterase function of FAS. In a separate strategy, the expression of FAS is suppressed by targeted knock-down with small interfering RNA. In both circumstances, the ablation of FAS activity causes a dramatic down-regulation of Skp2, a component of the E3 ubiquitin ligase that controls the turnover of p27Kip1. These effects ultimately tie into the retinoblastoma protein pathway and lead to a cell-cycle arrest at the G1/S boundary. Altogether, the findings of the study reveal unappreciated links between fatty acid synthase and ubiquitin-dependent proteolysis of cell-cycle regulatory proteins.     

Sequencing of the chicken non-erythroid spectrin cDNA reveals an internal repetitive structure homologous to the human erythrocyte spectrin.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.