Turnover of rat liver 28 S ribosomal RNA: nicking as the initial step of degradation.

The large ribosomal subunits of rat liver contain small amount of minor RNA components which migrate through polyacrylamide gels between the two major rRNAs, and some 28 S rRNA molecules contain hidden nicks which can be revealed by heat treatment. The minor RNA components were isolated, labeled in vitro with 125I, and hybridized with nucleolar DNA from rat liver. The hybridization-competition experiments with 28 S RNA established that the minor RNA components and 28 S rRNA have a common DNA origin. When RNA components were isolated from rats that had received [14C]orotate and [3H]orotate, 1 week and 16 h, respectively, before sacrifice, the 14C : 3H ratio was the highest in the minor components, followed by the nicked RNA, and the intact 28 S rRNA showed the lowest ratio. We also found that partial hepatectomy decreases and the actinomycin C-treatment increases the amount of the minor RNA components as well as the nicked 28 S RNA. From these observations, we conclude that the initial step of degradation of 28 S rRNA in vivo is the nick formation and that the nicked RNA subsequently gives rise to the minor components.


Semi-conservative and non-conservative replication of DNA in temperature-sensitive mouse L-cells.

The mode of DNA replication has been studied in wild-type mouse L-cells (WT-4) and in two subclones (TS A1S9 and ts C1 cells) which are temperature-sensitive in DNA synthesis. It has been demonstrated that DNA is replicated by the semi-conservative mechanism in WT-4 cells grown at 34 degrees C or at 38.5 degrees C throughout the logarithmic phase and into the stationary phase. Similar results were obtained with ts A1S9 and ts C1 cells grown at the permissive temperature (34 degrees C). When the latter cells were incubated at the non-permissive temperature (38.5 degrees C) inactivation of DNA synthesis appeared to proceed through three general stages. During the first 24 h after temperature upshift suppression of semi-conservative DNA replication occurred. During the second stage a very low level of semi-conservative synthesis was maintained. During the third stage, incorporation of dThd into DNA began to increase, often reaching 10-20% of control levels after 3-5 days. During this third stage DNA synthesis was effected by a non-conservative mechanism. Temperature-inactivated ts A1S9 cells and ts C1 cells were able to perform semi-conservative synthesis upon back-shift to 34 degrees C, using as template that DNA synthesized prior to temperature upshift.


The effects of thromboxane B2 and 6-ketoprostaglandin F1alpha on cultured fibroblasts.

The effects of thromboxane B2 and 6-ketoprostaglandin F1alpha on the synthesis of DNA, RNA and hexosamine-containing substances were studied. Thromboxane B2 (1 microgram/ml) added to cultures in the stationary phase caused the initiation of DNA synthesis and cell proliferation in a small proportion of cells. The same amount of thromboxane B2 also increased [3H]uridine incorporation into acid-insoluble fraction by 50% in 24 h, and stimulated the production of hexosamine-containing substances 2.5 fold over the control during the first 6 h. On the other hand, 6-ketoprostaglandin F1alpha was essentially inactive on these indexes except slight stimulation of DNA synthesis.


Change of ribonuclease H activity in developing and regenerating rat liver.

Ribonuclease H (RNAase H), which specifically degrades RNA in RNA-DNA hybrids, has been studied with proliferating normal liver. Partial hepatectomy results in approximately two-fold increase of RNAase H activity in 24 h after 16 h lag phase in liver after operation. The activity of RNAase H as well as DNA ligase in the liver of newborn rats is high and the activities of both enzymes gradually decrease with same ratio during differentiation to low levels in the adult.


An ATP-dependent inhibition of protein synthesis in ascites cell extracts by wheat germ protein.

A high speed supernatant fraction from wheat germ was shown to be a very effective inhibitor of protein synthesis in a cell-free protein synthesizing system from Ehrlich ascites cells. Low concentrations of the extract were equally effective in inhibiting the translation of EMC viral RNA, ascites cell mRNA (exogenous) and endogenous mRNA. The kinetics of inhibition in the presence and absence of pactamycin, as well as the observed inhibition of polyphenylalanine synthesis, indicate that the wheat germ inhibitor acts at the level of elongation. Preincubation of the ascites system with the wheat germ inhibitor in the presence and absence of ATP showed that ATP was required for the development of the inhibition. The inhibitor was partially purified and appears to be a basic protein with a molecular weight of 30 000--40 000. These results are discussed with respect to the hypothesis that this may be another example of a protein kinase-induced inhibition of protein synthesis.


Functional modification of liver form-B RNA polymerase activity by a protein fraction from rats accustomed to controlled feeding schedules.

A protein fraction extracted from the liver of rats accustomed to eating during the first 8 h of a daily 12 h dark period is able to increase homologous form-B RNA polymerase activity. When the protein factor is added to RNA polymerase extracted at the end of the daily fasted period (0.9:OOh) the sensitivity of enzyme to ionic strength is modified, and resembles that of the enzyme extracted during the eating period (15:00 h) either using liver deproteinized DNA or chromatin as template. The chromatin-bound RNA polymerase activity is also modified in the presence of the factor, showing a modification of enzyme sensitivity to ionic strength similar to the one induced by food intake.


Purification and properties of RNA polymerase I from Ehrlich ascites cells.

DNA-dependent RNA polymerase I (or A) (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) was purified from Ehrlich ascites cells after solubilization from isolated nuclei. The purification was accomplished by a procedure involving initial precipitation with ammonium sulfate, following by chromatographies on DEAE-Sephadex and phosphocellulose ion exchange resins and gel filtration on Sepharose 6B. A chromatographically homogeneous enzyme was obtained which was purified about 2300-fold relative to nuclear extracts. The specific activity of the most purified enzyme fraction was 230 nmol of [3H]UTP incorporated into RNA per mg of protein in 10 min at 37 degrees C, which is similar to those reported for the highly purified RNA polymerase I from mouse myeloma and calf thymus. The elution position on Sepharose 6B gel filtration indicated a molecular weight of approx. 580 000. Analysis of the purified enzyme by polyacrylamide gel electrophoresis under nondenaturing conditions revealed only one protein band. Certain heterogeneity in the RNA polymerase I fractions was found in the early chromatographic steps, but not in the most purified fractions.


Synthesis, separation and characterization of the mono- and diazide analogs of ethidium bromide.

Ethidium bromide is used to characterize nucleic acid secondary and tertiary structural properties and the biological consequences of drug interactions. The mono- and diazido analogs of ethidium have proven valuable as photoaffinity probes in chemical and biological studies on nucleic acids, since they render the ethidium-nucleic acid interaction covalent. Although both of these compounds have been synthesized previously, the published synthesis procedure for the monoazide is inadeqlate since a major portion of the product has been identified as the diazide analog. This lack of purity severely limits the usefulness for nucleic acid research. The procedure presented here for the synthesis, separation, purification and crystallization of these analogs should provide the quantities and quality of these important reagents needed to perform a variety of chemical and biological experiments.


Complex kinetics of human leukocyte and platelet pyruvate kinases.

In the presence of SH group protectors, human leukocyte and platelet pyruvate kinases demonstrate biphasic kinetics with respect to the phosphoenolpyruvate substrate. SH group oxidation by oxidized glutathione reveals positive cooperativity kinetics for purified preparations of leukocyte and platelet pyruvate kinases. Complete reversal of the phenomenon may be obtained by incubation for several hours in dithiothreitol. This communication illustrates the existing relationships between enzyme conformation, the redox state of the SH groups, and the observed kinetics.


Selective reactivity of rhodanese sulfhydryl groups with 5,5'-dithio-bis(2-nitrobenzoic acid).

The reactivity of the sulfhydryl groups of sulfur-containing and sulfur-free rhodanese (thiosulfate : cyanide sulfurtransferase, EC 2.8.1.1) with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) has been investigated. Only 0.6 sulfhydryl group of the sulfur-containing enzyme reacts with DTNB. After removal of sulfur from persulfide groups a further 0.6 sulfhydryl group (i.e. a total of 1.2) becomes accessible to the reagent. The resulting enzyme-thionitrobenzoate complex shows an absorption spectrum with a shoulder at 325 nm due to bound thionitrobenzoate. Both thiosulfate and cyanide remove thionitrobenzoate from the enzyme restoring its catalytic properties. The modified enzyme is protected from alkylation by iodoacetate until thionitrobenzoate is removed. The existence of a further sulfhydryl group close to the catalytic one is suggested.


RMI 12330 A, an inhibitor of adenylate cyclase in rat liver.

RMI 12330 A, (N,-(cis-2-phenylcyclopentyl) azacyclotridecan-2-imine hydrochloride), has been reported to inhibit choleratoxin-induced intestinal hypersecretion, presumably via an inhibition of mucosal adenylate cyclase (ATP:pyrophosphate-lyase (cyclizing), EC 4.6.1.1). We report here that the adenylate cyclase activity of a rat liver plasma membrane preparation was inhibited by concentrations of RMI 12330 A ranging from 10 muM to 5mM. Similar effects were observed when the adenylate cyclase preparation was assayed in the presence of 10 mM NaF, 0.1 muM glucagon or 1 muM (--)-epinephrine plus 10 muM GTP. The effect of RMI 12330 A was not due to the inhibition of the regenerating system present in the incubation medium, since the effect was preserved in its absence. The inhibition brought about by RMI 12330 A was due to a decrease in the maximal velocity of the reaction; the affinity of the enzyme for the substrate remained unmodified. The inhibition was immediate and irreversible, even after several washes of the membranes previously preincubated with the drug. Complete inhibition of cyclase was obtained at a concentration of 370 nmol of RMI 12330 A per mg of membrane protein. The drug acted with a similar dose-response curve upon intact as well as detergent-dispersed cyclase preparations.


Some properties of the membrane-bound and solubilised forms of the protein disulphide isomerase of rat liver microsomes.

1. An improved method of measurement of the protein disulphide isomerase (protein disulphide-isomerase, EC 5.3.4.1) activity of microsomal preparations is described. 2. This enzyme is shown to be released from the membranes into solution in the ultracentrifuge. 3. Some of the properties of the enzyme in the membrane bound and soluble forms are described. 4. The results lead to the suggestion that the enzyme present in rough membrane is different from the present in smooth membrane as well as being differently situated in the membrane. 5. Some of the effects of sucrose on the activity of the membrane bound enzyme are consistent with the view that the membranes may undergo irreversible conformational changes during preparation and storage.


External yeast beta-fructosidase: stereospecific labeling by [3H]conduritol-beta-epoxide and isolation of labeled peptides from the active site.

1. External yeast beta-fructosidase (beta-D-fructofuranoside fructohydrolase EC 3.2.1.26) was labeled by [3H]conduritol-B-epoxide, an active-site directed inhibitor of this enzyme. During the inactivation 1--2 mol of inhibitor were bound covalently per mol of enzyme. The labeled enzyme was digested with pepsin and 3H-labeled peptides were isolated. 2. Conduritol-B-epoxide is a racemic mixture of 1-D-1,2-anhydro-myo-inositol and 1-L-1,2-anhydro-myo-inositol, but only the latter one was now shown to be the reactive component. 3. The label was released from the enzyme by milk alkaline treatment and identified by paper chromatography as 1-D- or L-chiro-inositol, which suggested that an ester linkage had been formed by reaction of the epoxide with a carboxylate group at the active site of the enzyme. Position and role of this carboxylate group are discussed.


A comparison of the alpha-L-fucosidase activities of human liver and serum.

Human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) has been separated into four components by chromatography on Sephadex G-150 or DEAE-cellulose. These components differ in their relative stability to heat and acid treatment, and their response to neuraminidase. The serum enzyme was devoid of high molecular weight activity and probably contained more sialic acid residues than the corresponding enzyme from liver. All the liver components tested were able to liberate fucose from 2'-fucosyllactose but not fucose from other oligosaccharides.


Natural plant enzyme inhibitors. V. A trypsin/chymotrypsin inhibitor from Alocasia macrorhiza tuber.

A trypsin/chymotrypsin inhibitor was isolated from the tubers of Alocasia macrorhiza by extraction at pH 7.6, heat treatment at 80 degrees C, ammonium sulphate precipitation and successive column chromatography on CM-cellulose, DEAE-Sephadex A-50 and Sephadex G-100. The inhibitor was pure by cellulose acetate electrophoresis. The molecular weight was approximately 32 000 as determined by gel filtration on Sephadex G-100. The inhibitor acted on bovine trypsin, human trypsin and bovine chymotrypsin. It had no action on human chymotrypsin, subtilisin BPN', pronase, Aspergillus oryzae protease, human and porcine pepsins. The binding sites for bovine trypsin and chymotrypsin are not mutually exclusive. The inhibitor was stable over a pH range of 1--10. The purified inhibitor was far more thermostable than the crude inhibitor. The purified inhibitor lost only 33% of activity on heat treatment at 95 degrees C for 2 h. Trinitrobenzene sulphonate treatment resulted in the loss of antichymotryptic activity faster than the antitrypsin activity of the inhibitor.


Purification of proenzymic and activated human C1s free ofC1r. Effect of calcium and ionic strength on activated C1s.

1. A rapid method for the purification of the proenzymic and activated forms of C1s is presented. In the case of proenzymic C1s, di-isopropyl phosphorofluoridate (0.5--5 mM) is added at all stages of the purification procedure, which includes euglobulin precipation followed by DEAE-cellulose chromatography and affinity chromatography on anti-C1r IgG-Sepharose 6B. The final step completely removes contaminant traces of C1r and/or C1r, ensuring that the final preparation of C1s is stable in the proenzyme form and suitable for activation studies. 2. The apparent molecular weight of C1s and C1s determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis is 85 000 +/- 2000. Reduction followed by alkylation of C1s gives two fragments of apparent molecular weights 57 000 and 28 000. Results of N-terminal amino acid determination and labelling with di-iso[3H]propyl phosphorofluoridate are consistent with previous reports. 3. The influence of calcium and ionic strength on the structure and activity of C1s has been investigated. Calcium leads to a shift of the sedimentation coefficient from 4.3 to 5.6 S, whereas variation in ionic strength has no effect on this parameter. The thermal inactivation curve is profoundly modified both by calcium and ionic strength. In contrast, the esterase activity is only slightly influenced as judged from the absence of gross modification of Km and V.


Bovine serum albumin as a catalyst. VI. Specificity of several nucleophilic groups in the protein for N-dansylaziridine.

The reaction of N-dansylaziridine with serum albumin (both bovine and human) results in incorporation of about 3 mol of covalently bound dansyl label per mol protein. This indicates that a number of nucleophilic groups in these proteins (in addition to the free sulfhydryl group) will react with this reagent. The reaction has been studied in detail for bovine serum albumin and the results suggest that one of the sites labelled by the reagent may be at the unusual "catalytic site" responsible for the enzyme-like activity of bovine serum albumin recently described (Taylor, R.P., Chau, V., Bryner, C. and Berga, S. (1975) J. Am. Chem. Soc. 97, 1934-1942). The reaction of N-dansylaziridine with a variety of other proteins indicates a pattern of labelling consistent with high specificity for the sulfhydryl group. The explanation for the unexpected excess reactivity of albumin with the "sulfhydryl specific" reagent N-dansylaziridine must be related to the three-dimensional structure in albumin which enables a number of specific residues to manifest unusually high degrees of nucleophilic reactivity.


Multiple forms of brain adenylate cyclase: stimulation by Mn2+.

Mn2+-stimulated adenylate cyclase (ATP pyrophosphate-lyase-(cyclizing), EC 4.6.1.1) activity in detergent solubilized preparations from mouse brain. While NaF-stimulated activity was decreased by both solubilization and storage at 0--4 degrees C, the ability of the enzyme to be stimulated by Mn2+ was maintained for up to one week. By including Mn2+ in the assay of adenylate cyclase in gel fractions after isoelectric focusing, two distinct peaks of enzyme activity (pI1 - 5.8, pI2 = 6.4) were detected, suggesting the existence of more than one type of catalytic subunit in mouse brain cell membranes.


Comparison of intrinsic stabilities of free and bound enzymes by graphical removal of diffusional effects.

The enhanced stability usually exhibited by enzymes after immobilization may be attributed either to a stabilization effect of the solid matrix on the bound enzyme molecule or to the influences of diffusional limitations on the observed activity. To allow the comparison of the intrinsic statilities of free and bound enzymes a simple graphical procedure for the removal of external diffusional effects of stability curves is described. It is based on the determination of substrate concentration differences between the enzyme micro- and macroenvironment. Application of the method to aspartate aminotransferase bound to collagen membranes indicates that diffusional limitations for oxaloacetate are partly responsible for the observed stability enhancement. Comparison of the graphically obtained intrinsic profile with the stability curve of the soluble enzyme further demonstrate that the binding itself greatly increases the stability of aspartate aminotransferase.


Regulation of human erythrocyte hexokinase. The influence of glycolytic intermediates and inorganic phosphate.

Human erythrocyte hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was inhibited competitively with respect to MgATP2- by glucose-6-P (Ki - 10.8 muM) and fructose-6-P (Ki = 160 muM). Low concentrations of inorganic phosphate were competitive with respect to glucose-6-P and fructose-6-P, although higher concentrations of Pi were not able to overcome completely the inhibition by the hexose phosphates. The results are consistent with a model in which hexokinase exists in equilibrium either as free or phosphate-associated enzyme, the latter having a reduced but still substantial affinity for hexose phosphate. An alternative explanation could be found in the presence of two different enzymes, one with a high affinity for glucose-6-P being sensitive to regulation by Pi, one with a lower affinity for glucose-6-P being insensitive to Pi. A similar but less pronounced effect of Pi, was found on the inhibition by 2,3-diphosphoglycerate (Ki = 4.0 mM). Pi in the absence of inhibitor was also a competitive inhibitor with respect to MgATP2- (Ki = 20 mM). Furthermore a competitive inhibition with respect to MgATP2- was found by fructose 1,6-diphosphate (Ki = 4.3 mM), glycerate-3-P (Ki = 3.8 mM), glycerate-2-P (Ki = 12.5 mM), MgADP- (Ki = 1.0 mM) and MgAMP (Ki = 1.7 mM).


Autophosphorylation of cardiac 3',5'-cyclic AMP-stimulated protein kinase. Kinetic evidence for the regulatory subunit directly acting at the active site in the R2C2 complex.

The mechanism of the autophosphorylation reaction of bovine heart cyclic AMP-dependent protein kinase (ATP: protein phosphotranferase, EC 2.7.1.31) has been further examined using a kinetic approach. The reaction is first order in both ATP and protein kinase when both are present at comparable concentrations. Dilution has no effect on the fraction of regulatory subunit phosphorylated over a given interval of time, and this finding is in accord with the autophosphorylation proceeding via an intramolecular (or, more appropriately in this case, by an intracomplex) reaction. The possibility of regulatory subunit phosphorylation by uncomplex catalytic subunit or another R2C2 complex (the protein kinase complex of two catalytic subunits and the regulatory dimer) was clearly eliminated. These results are compatible with a subunit geometry permitting the regulatory subunit to bind at the protein substrate region of the kinase's active site and to undergo subsequent phosphorylation.


Studies of the N-bromosuccinimide inactivation of the enzyme rhodanese.

The enzyme rhodanese (Thiosulfate: cyanide sulphurtransferase, EC 2.8.1.1) is rapidly inactivated by treatment with N-bromosuccinimide. Spectrophotometric titration and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that neither tryptophan oxidation nor polypeptide chain cleavage can account for the inactivation. Sulfhydryl group assays using the colormetric reagent 5,5'-dithiobis(2-nitrobenzoic acid) after destruction of excess N-bromosuccinmide, indicate that approximately 2 sulfhydryl groups per enzyme molecule are lost. Further, rhodanese inactivated by N-bromosuccinimide can be reactivated (approximately 95%) by incubation with the substrate thiosulfate. It is postulated that N-bromosuccinimide inactivates rhodanese by inducing the formation of a disulfide bond involving the active site sulfhydryl group of the enzyme and a second sulfhydryl group which can be brought close to the active site in the flexible native structure.


Age-dependent modulation of 3'-phosphoadenosine-5'-phosphosulfate-galactosylceramide sulfotransferase by lipids extracted from the microsomal membranes and artificial lipid mixtures.

The 3'-phosphoadenosine-5'-phosphosulfate-galactosylceramide-sulfotransferase (cerebroside sulfotransferase) is microsomal enzyme, which shows a definite developmental activity pattern. This report gives evidence that the enzyme activity of partially delipidated microsomes is modulated by the cholesterol:phospholipid ratio of the extracted microsomal lipids in an age-dependent manner. These findings suggest that in vivo the enzyme activity is modulated by the lipid surrounding.


Enzyme affinity in the acylation of lysophosphatidylcholine.

Analogues of 1-palmitoyl-sn-glycero-3-phosphorylcholine were used to ascertain the respective roles of the 1-palmitoyl-sn-glycero and choline groups in binding this substrate to the transferase catalyzing the acylation reaction. 1-Palmitoyl-sn-glycero-3-phosphorylhomocholine proved to be an effective competitive inhibitor whereas 1-palmitoyl-2-deoxyglycero-3-phosphorylcholine was totally ineffective. The data support the view that the 1-palmitoyl-sn-glycero group plays the major role in determining enzyme affinity whereas the choline group functions primarily in the subsequent steps of the acylation reaction.


Quantitative 250 MHz proton magnetic resonance study of hydrogen-deuterium exchange. Angiotensin II hormone in trifluoroethanol.

Hydrogen-deuterium exchange kinetics of (Asn1-Val5) angiotensin II has been investigated by proton magnetic resonance at 250 MHz in deuterated trifluoroethanol, as an approach to the "in situ" hormone conformation. An interactive program was specially developed to perform the data analysis on a computer similar to those used for spectroscopic data acquisition. Nine exchange sites are evidenced and characterized by their individual kinetic parameters. Three of them are assigned to peptide NH hydrogens, and the six remaining to slowly exchanging side chain protons. At 11 degrees C, more than three peptide hydrogens, sterically hindered or involved in hydrogen bonds, do not exchange. These results corroborate previous circular dichroism and infrared investigations performed in the same solvent, and suggest a family of well-folded conformations, stabilized in trifluoroethanol by internal hydrogen bonds, involving both the backbone and the side chain hydrogens.


Proteins from disassembled microtubules characterized by oligospecific antisera.

The immunochemical properties of in vitro reassembled microtubules were investigated by immunoelectrophoretic techniques. The tubulin dimer gave no measurable immunochemical response, but the tubulin oligomer, the tau-factor and an antigen of about 135 000 daltons all gave precipitating antibodies. Those four proteins were investigated in reassembled microtubules, in DEAE-cellulose purified tubulin, and after molecular sieve chromatography of disassembled and NaCl-dissociated microtubules. Reconstitution of tubulin oligomer from tubulin dimer and tau-factor was also performed. The presence of a unique antigenic structure on tubulin oligomer which was not found in the dissociated components and the role of this aggregate as a nucleation center or intermediate in the assembly of microtubules is discussed.


Stability of phage T4 lysozymes. I. Native properties and thermal stability of wild type and two mutant lysozymes.

Two mutants of phage T4 lysozyme were prepared and characterized. One mutation substituted a tyrosine residue for tryptophan at position 138. The other substituted tyrosines at all three tryptophan positions of the wild type molecule (126, 138, 158). Comparative studies of the physical properties (absorption, fluorescence, circular dichroism) of the three enzymes were performed as a function of pH. Also, the proteins were reversibly melted as a function of pH. Since the unfolding reaction appeared to be a two-state process for all these proteins, the data were analyzed by the van 't Hoff procedure. The changes in stability and activity produced by substitution of Trp 138 were especially significant. The other substitutions were neutral. See the end of the paper for a summary of conclusions. In the appendix the appropriate thermodynamic relations are developed for a constant deltaCp transition.


Purification and characterization of rabbit transcobalamin II.

Rabbit transcobalamin II has been purified by labile ligand affinity chromatography and G-200 Sephadex gel filtration. Structural studies indicate Stokes' radii of 2.7 nm and 3.0 nm for transocobalamin II saturated and unsaturated with cobalamin. The amino acid content of the protein is very similar to that of human transcobalamin II (Allen, R. H. (1975) Prog. Hematol. 9, 59-84). The aminoterminal sequence for transcobalamin II is reported for the first time: Glu-Ile-Cys-Gly-Val-Pro-Lys-Val-Asp-Ser-Glu-Leu-Val-Glu-Lys-Leu-Gly-Gln-Arg-Leu-Leu-Pro-(Trp)-Met-Thr).  The ultraviolet and circular dichroic spectra of aquo-, hydroxo-, azido- and cyanocobalamin bound to rabbit transcobalamin II are described. On complex formation the molar absorption of the cobalamins increases and the major bands shift to longer wavelengths. The spectra are little affected by change in the fifth ligand, which indicates that the electron density around the cobalt atom is kept fairly constant by the transcobalamin II molecule. This is in contrast to the observations for the same cobalamins attached to human intrinsic factor and human transcobalamin I (Nexo, E. and Olesen, H. (1976) Biochim. Biophys. Acta 446, 143-150).


Conformational changes induced by the addition of carbamyl phosphate to sickle cell hemoglobin.

The conformational changes upon anaerobic carbamyl phosphate addition to dilute hemoglobin solutions have been studied by means of circular dichroism. Freshly prepared carbamyl phosphate stabilizes the conformation of pure deoxyhemoglobin without detectable NH2-terminal carbamylation. Addition of preincubated (i.e. partially converted to cyanate) carbamyl phosphate, however, results in such carbamylation and in the formation of the conformationally different carbamyldeoxyhemoglobin which exhibits enhanced oxygen affinity. Fresh carbamyl phosphate carbamylates hemoglobin in deoxygenated hemolysates containing erythrocyte phosphatases. Therefore the reversal of SS erythrocyte sickling by carbamyl phosphate is attributable to carbamylation by the carbamyl phosphate hydrolysis product, cyanate.


The isolation and partial characterization of transferrin binding components of the rabbit reticulocyte plasma membrane.

An affinity chromatograpy method utilising transferrin liganded agarose has been developed for the partial purification of transferrin binding components from Triton X-100 solubilised rabbit reticulocyte plasma membranes. A protein of molecular weight 30-35 000, shown to be located at the reticulocyte extra-cellular surface by lactoperoxidase 125I labelling, was isolated by the affinity method. The protein appeared to form a dimer of molecular weight 65-70 000 in Triton X-100 solution and was shown to associate with both 125I-labelled and unlabelled rabbit transferrin to form a high molecular weight complex in the same solution. N-[14C]Ethylmaleimide appeared to disrupt this association with transferrin and inhibit the formation of the dimer in Triton X-100 by binding to the protein. The protein appeared as a broad band of molecular weight 40 000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis.


Structural and composition of the N2 neuraminidase of influenza virus. Effect of carbohydrate content on the validity of molecular weight estimations.

Neuraminidase was isolated by proteolysis of the X7-(F1) (HON2) strain of influenza virus, and purified by gel filtration. The molecule contained a total of 46% (w/w) carbohydrate. The Mr was estimated as 152 500 (sedimentation diffusion) and 147 000 (sedimentation equilibrium). In 6 M guanidine-HCl the molecular weight was halved to 66 000 (sedimentation equilibrium). After irreversible reduction and blocking of sulphydryl groups the molecular weight was halved again to 33 500 (sedimentation equilibrium). These results confirm the tetrameric model of neuraminidase structure. They also provide strong evidence that the tetramer is composed of two disulphide linked dimers, themselves associated by non-covalent linkages. Theoretical considerations based on this model predict that assembly of the molecule must be accompanied by allosteric conformational changes in the subunits. The high carbohydrate content was thought to explain the discrepancy between the molecular weight values for the neuraminidase polypeptide obtained by different methods, and also the exceptional resistance of the molecule to digestion by proteolytic enzymes.


Interaction of sodium dodecyl sulfate and of non-ionic detergents with S-carboxyamidomethyl-k-casein.

Sodium dodecyl sulfate binds to S-carboxyamidomethyl-k-casein in a highly cooperative manner at a concentration near the critical micelle concentration, showing a strong dependence on ionic strength. The maximum number of sodium dodecyl sulfate molecules bound is attained above the critical micelle concentration, and is very close to the micelle aggregation number in the absence of protein. The binding sites on the protein for sodium dodecyl sulfate are localized mainly on para-k-casein part, which is a hydrophobic fragment of k-casein produced by rennin attack. The mode of the action of sodium dodecyl sulfate on S-carboxyamidomethyl-k-casein resembles that of several integral membrane proteins, rather than of water soluble proteins. On considering possible situations, it is suggested that the unusual interaction of S-carboxyamidomethyl-k-casein with sodium dodecyl sulfate is responsible for an anomalous migration of reduced k-casein observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Further, the suggestion was made by the binding studies of sodium dodecyl sulfate and non-ionic detergents that the sites which were involved in self-association of S-carboxyamidomethyl-k-casein participated in the binding sites of detergents.


Cellular localization of alpha-ketoglutarate: glyoxylate carboligase in rat tissues.

alpha-Ketoglutarate: glyoxylate carboligase activity has been reported by other laboratories to be present in mitochondria and in the cytosol of mammalian tissues; the mitochondrial activity is associated with the alpha-ketoglutarate decarboxylase moiety of the alpha-ketoglutarate dehydrogenase complex. The cellular distribution of the carboligase has been re-examined here using marker enzymes of known localization in order to monitor the composition of subcellular fractions prepared by differential centrifugation. Carboligase activity paralleled the activity of the mitochondrial matrix enzyme citrate synthase in subcellular fractions prepared from rat liver, heart and brain as well as from rabbit liver. Whole rat liver mitochondria upon lysis released both carboligase and citrate synthase. The activity patterns of several other extramitochondrial marker enzymes differed significantly from that of carboligase in rat liver. In addition, the distribution pattern of carboligase was similar to that of alpha-ketoglutarate decarboxylase and of alpha-ketoglutarate dehydrogenase complex. The data indicate that alpha-ketoglutarate: glyoxylate carboligase activity is located exclusively within the mitochondria of the rat and rabbit tissues investigated. There is no evidence for a cytosolic form of the enzyme. Thus the report from other laboratory that the molecular etiology of the human genetic disorder hyperoxaluria type I is a deficiency of cytosolic carboligase must be questioned.


Plasma ceruloplasmin. Evidence for its presence in and uptake by heart and other organs of the rat.

Evidence for the presence of the plasma protein, ceruloplasmin, in heart and other tissues of the rat was sought using various techniques. With p-phenylenediamine, ceruloplasmin-like oxidase activity was detected in heart post-mitochondrial and 100 000 X g supernatants in amounts far exceeding those that could be accounted for by residual blood. Much lower levels were detected in kidney, brain and liver. Oxidase activity of heart purified on DEAE-cellulose in the same way as rat plasma ceruloplasmin and behaved identically also in disc gel electrophoresis. The presence of ceruloplasmin in heart extracts was confirmed immunologically by Ouchterlony diffusion, using rabbit antibody raised against pure rat ceruloplasmin. When pure [3H]leucine-labeled ceruloplasmin was infused intravenously into a copper-deficient rat, radioactivity was concentrated in the heart and brain within 2 h; radioactive counts per g attained 11 and 3 times those of plasma in the two organs, respectively. A lesser concentration occurred in the liver. the results suggest that circulating ceruloplasmin (made by the liver) finds its way into the cells of some organs, especially the heart, a phenomenon which may be related to the function of ceruloplasmin to provide copper to the cytochrome oxidase of various tissues.


Studies on the solubilized porcine ileal intrinsic factor receptor and on a 340 000-dalton component binding vitamin B-12.

A 340 000-dalton component "C-III" was found when Triton X-100-containing extracts of ileal mucosa were incubated with human or porcine intrinsic factor vitamin B-12 preparations. It was not formed when abnormal human intrinsic factor, unable to attach to the intrinsic factor receptor, was used. Prolonged storage promoted the trnasfer of vitamin B-12 to it from the vitamin B-12-intrinsic factor recptor species C-I and C-II. The component was also present in ileal extracts prepared with or without detergent and it bound vitamin B-12 directly. Immunologically and by electrofocusing it could be classified as a cobalophilin but its molecular dimensions were larger than described for cobalophilin. It thus represents a novel vitamin B-12 binding protein, possibly a macromolecular acceptor of vitamin B-12 which accepts vitamin B-12 bound via intrinsic factor to the ileal intrinsic factor recptor. In the presence of EDTA or at low pH, vitamin B-12-intrinsic factor did not bind to any of the receptor species and under the same conditions it could all be dissociated from the receptor complexes but not from C-III. The dissociated receptor was able to recombine with vitamin B-12-intrinsic factor and it appeared to bind free and vitamin B-12-bound intrinsic factor in vivo.


Nephritogenoside, the receptor glycoprotein for concanavalin A in rat glomerular basement membrane. Demonstration of alpha-D-glucopyranosyl unit at the non-reducing terminus.

Rat glomerular basement membrane was extracted for 3 h with trypsin, pH 8.0. The supernatant solution was treated with trichloroacetic acid and the supernatant thus obtained was applied to Bio-Gel P200. The first of the two glycoprotein peaks was applied onto Sepharose derivatives of concanavalin A (Con A). Examination of the material retained by the unsolubilized Con A and subsequently eluted with methyl alpha-D-mannopyranoside reveals that the principal high affinity receptor for Con A is the renal glycoprotein, having antigenic activity that induces nephrotoxic antibody. This glycoprotein has also nephritogenicity (the activity capable of inducing glomerulonephritis in homologous animals by a single foot pad injection with Freund's incomplete adjuvant). Evidence  is given to show that this binding is specific. The remainder of the renal glycoprotein is unretarded and is revealed to contain none of the activities described above. When fluorescein isothiocyanate-labelled Con A is, conversely, injected into rats through the renal artery, the specific binding of Con A to the glomerular capillary loop is proved. The results demonstrated appear to, indicate that the receptor for Con A present in normal rat glomerular basement membrane can be identified as the well-established chemical substance, the nephritogenoside, having the alpha-D-glucopyranosyl unit at the non-reducing terminus which is facing the endothelial aspects of the glomerular capillary loop.


The supression of milk fat globule secretion by clochicine: an effect coupled to inhibition of exocytosis.

The effects of colchicine on release of milk lipids from mammary tissue were evaluated by biochemical analysis of milk and morphological study of the tissue following intramammary infusions of the alkaloid into lactating goats. Colchicine produces a reversible drop in milk yield. As the flow of milk resumes, 36--48 h after infusion, the fat content of the milk increases, phospholipid per g of total globule lipid falls, mean size of milk fat globules increases and diameters of fat droplets (presecretory milk fat globules) within lactating cells approximately double. These observations are consistent with the conclusion that colchicine suppresses milk fat globule secretion but that globules continue to grow in size wihtin cells during the suppression period. These findings indicate that secretion of milk fat globules and the skim milk phase are coupled.


[Nature of the electron-excited state in redox reactions of pigments. I. Photooxidation of chlorophyll a by n-benzoquine].

A method for studying the nature of electronic excited state under photooxidation is proposed. It is shown by an example of the oxidation of chlorophyll a with p-benzoquinone that the formation of cation-radicals of pigments proceeds only through the triplet state. On the basis of experimental data the values of the rate constants of formation of chlorophyll cation-radicals through the singlet-excited state (K2(1)--10(7) M-1 s-1) and triplet state (Kr=10(9) M-1 s-1) of the pigment are evaluated.


[Quenching of triplet states by Fe(II)- and Fe(III)-porphyrins].

The quenching of triplet states of metalloporphyrin molecules differing in the position of the lowest triplet state (from 9.000 to 15.000 cm-1) by ion complexes of octaethylporphin (OEP) has been investigated. High-spin ferric complex FeCl-OEP in dimethylformamide, toluene and ethanol, low-spin ferric complex FeCl(imidazole)2-OEP in toluene and diamagnetic ferrous complex Fe(piperidine)2-OEP in pyridine-piperidine (25:1) were used as quenchers. It has been found that all iron complexes investigated are effective quenchers of triplet states. The values of bimolecular quenching constants fall within the limits of (1 divided by 4) X 10(9) M-1 sec-1. Energy transfer to low-lying excited levels of iron porphyrins which are due to the presence of 3d electrons of ferric (ferrous) ion has been shown to play an essential role in the quenching process.


[Study of the interaction of water with cholesterol in organic media by the methods of dielectric and NMR spectroscopy].

Interaction between water and cholesterine in organic media was studied by means of dielectric and NMR-spectroscopy. It is shown that cholestine binds water in an equimolar ratio with the formation of strong hydrogen bonds between the hydroxil groups of cholesterine and water molecules. It is assumed that an ordered structure of cholesterine in the form of micellae is being formed around the water molecules. The conformation and size of the micellaes depend on cholesterine concentration and water content in dispersion.


[Determination of the molecular weight and form of the molecule histidine decarboxylase by the method of small-angle x-ray scattering].

Shape and molecular weight of histidine-decarboxylase from Micrococus sp. n. were studied by the method of X-ray small-angle scattering. The inertion radius of the molecule: Rg-2,93 nm. The shape of the molecule is adequately approximated by rotation ellipsoids of two possible variants: the elongated and flattened ones. The eccentricity in both cases is 1.6. The volume of the enzyme molecule V=190 nm3. The molecular weight of histidine-dexarboxilase obtained from the X-ray experiment M=102 000 c.u.


[Heat denaturation of hemocytocardiotoxin from the venom of the Central Asian cobra].

It has been stated that boiled for three hours haemocytocardiotoxin (HT) from cobra poison loses "direct" hemolytic activity and is unable to potentiate a haemolytic effect of phospholipase A. Surface activity of HT does not change. It is shown that in the course of heat denaturation the aggregation of toxin molecules to dimers and trimers takes place and electrophoretic mobility is decreased. The fluorescence of HT tyroxin residues supported the fact of its irreversible heat denaturation.


[The cell as an electrical circuit. I. Theoretical part].

The study deals with a theoretical analysis of the interaction between alternating signal and the model presenting the cells as a spatially closed formation with distributed parameters. Particular attention is paid to the distribution of the potential induced by an external alternating signal on the membrane surface; a significant influence of intracellular content on this distribution is proved. It is shown that at some values of electric parameters of the cell and signal frequency definite sites exist on the membrane surface where the conditions are realized at which the potential equals zero.


[Study of the microstructure of the water-protein layer of spin-labeled preparations of lysozyme by the magnetic relaxation method].

Possible applications of magnetic relaxation to the studies of microstructures of water-protein layer of spin-labelled proteins and enzymes are analyzed on lyzozymes taken as an example. It is shown that the effective time of correlation of dipole-dipole interaction of the complex spin label--water proton (Tauc), as well as thermodynamic parameters Eeff. and delta Seff. reflect the local state of solvatic surrounding of lyzozyme in case of long labels. Conformation transitions of lyzozyme are revealed at the change of temperature (5--80 degrees C) and as a result of binding of a specific inhibitor NAG. It is concluded that local physico-chemical properties of water-protein matrix of lyzozyme spin-labelled preparations depend on protein conformational state.


[Mechanism of oxidative phosphorylation. IV. Role of the closed state of mitochondrial membranes].

The clousness of membrane of rat brain mitochondria isolated in Krebs-Ringer's solution was broken by suspending the mitochondria in distilled water. Function characteristics of lyzed organells did not decrease but were somewhat higher than those of the organells suspended in Krebs-Ringer's solution. The results were discussed in relation to the question of the role of clousness of the mitochondrial membrane in the mechanism of oxidative phosphorylation.


[Polarity of cell permeability and its control by phytochrome].

Growing chloronema and rhizoid cells of the intact protonema of Funaria hygrometrica absorb luminescent stains selectively. The cation stains--acridine orange and neutral red, at pH 5.0 are absorbed by growing tips of the apical cells. The anion stain (uranine) is absorbed by the basal end of the apical cells and by intercalary cells of the protonema filaments. Red light inactivates the cell permeability to the cation and anion stains. Far-red light partly reversed the action of the red light solely on the initial stages of the irradiation. During the red light induced branching of apical cells the cation stains enter the cells through a new originated growing point.


[Chemiluminescence of plant tissues preserved in aldehydes and exposed to the salts of heavy metals].

It was established that plant tissues killed by various aldehydes in low concentrations, which evoked general enzyme inactivation for a long time retain permeability and lipoprotein bonds in the cell membranes. Such preparations from Elodea Canadensis leaves are used for: Cu, Zn, Pb, Hg toxical effect investigation. It was stated by chemoluminescence methods that these ions induce slow developed oxidative radical chain reaction in lipid cell membranes and break the lipoprotein bonds.


[Influence of chlorpromazine and temperature on glucose transport in human erythrocyte ghosts].

Glucose transport was determined by oxygen adsorption in solution when glucose was oxidized with glucoseoxidase, injected into erythrocyte ghosts. Temperature dependence of glucose transport velocity shows a break at 20 degrees C, which was described in literature. Small concentrations of chlorpromazine (about 2-10(-5) M) somewhat activates the transport but does not changes its temperature dependence. Higher concentrations of anaesthetics (beginning with 1-10(-4) M) inhibit the transport, decrease the temperature of break and increase the activation energyies of the process both lower and higher of the transition temperature. The change of working velocity of glucose carrier under the action of anaesthetic and temperature is accounted for by structural reconstructions in lipids, which are heterogeneously distributed along the erythrocyte membrane plane.


[Model of an excitable medium in which may exist several stable impulses of different duration].

It is shown how with the help of minor changes of the type of non-linear function (corresponding for example to the current voltage characteristics of the active biological membrane) a non-monotonous relationship between the excitation pattern velocity and the value of the slow variable is obtained in a model of the excitable medium. In such a model several steady impulses may exist which propagate with equal velocity, but having different duration.


[Autoactivation in cell populations].

A mathematical model of cell population with cooperatively controlled mitotic activity is analysed. The mitotic activity of stem cells is activated by the cells themselves and is inhibited by differentiated cells. Analysis of the model shows that steady-state oscillations in the mitotic activity are possible in such populations. The appearance of self oscillations is due to a delay in the negative feedback loop. The oscillation period, determined to a linear approximation, is greater than a quadruplicate delay.


[Patterns in the reaction of rabbit visual cortex neurons to constant illumination].

A number of informational estimates for the reactions of excitation, inhibition and of A-type (according to Young) of rabbits' visual cortex neurons to constant light was calculated by means of electronic computer -6. It has been found that entropy as well as redundancy are sometimes the characteristics of reactions intensity: the correlation of their values is regularly connected with the type of reactions. Possibility of optimal (for exposing the reactions) quantization of records with respect to time was shown. The regularities of "thin" statistical reactions' structure consisting in the change in "force" and "distance" of intercennection action were found. Possible informational value of the obtained results is discussed.


[Model system of binaural hearing. III. Binaural processing of information about short sounds].

A model of neuronal structures of the medial superior olivary nuclei and inferior colliculus performing the determination of the direction on the source of the short (shorter than 10 ms) sound signals is presented. In the model the difference between the moments of the arrival of the two informational messages formed on the stage of monaural information processing is calculated. The result of this calculation is the firing probability of the primary detector (the neuron of the medial superior olivary nucleus). Because of the internal noise the curve of this probability as a function of the direction on the source is smoothly sloped. The estimation of the direction is the result of the statistical processing of the responses of the primary detectors ensemble. The direction on the source of sound is coded by a position of the secondary detector (the neuron of the inferior colliculus) on the direction "scale".


[Calculation of potential accuracy in measuring the angular coordinates of targets by the echolocator of bats using the equal-signal zone method].

Potential accuracy in measuring the course to a target is calculated. This accuracy is physically achievable it the hypothesis that an accurate measurement of angular coordinates of the target of bat's echolocator is realized by the method similar to that of equally signaling zone in radiolocation is true. Possible application of such a method is based on the "pulsation" of direction diagram in the course of radiation of the probing impulse. In this case crossing of partial diagrams of radiation corresponding to high- and low-frequancy regions of the impulse form the equally signaling zine. If the target is in this direction the amplitudes of autocorrelation functions formed by corresponding regions of reflected and probing impulses will be equal. The minimal error of the method is limited by the optimum duration of each of the compared correlation functions, which forms the basis for derivation of the formula evaluating this error. Numerical calculation of the accuracy of measurements of the angle achievable for the echolocator of Myotis blythi is performed by this formula proceeding from average experimental values of the echolocator characteristics. The model under consideration is shown to agree with a number of experimental data.


[Relationship between the amplitude of myocardial contractions in frogs and the frequency of electrical stimulation. Role of external and intracellular calcium in the coupling of excitation and contraction].

Ionic currents were studied on the frog atrial trabeculae (Rana ridibunda) at 20 degrees C using a double sucrose gap voltage clamp arrangement. The net inward current peaks did not change in the course of repetitive stimulation (0,5/s) in contrast to the increase of the contraction amplitude (isometric tension) in the similar conditions (Bowdich staircase). The slow component of the net inward current revealed under the action of TTX (2-10(-8) g/ml) was increased upon the increase of external Ca concentration but was blocked when D-600 was introduced into the solution. The inhibitory action of D-600 on the contraction amplitude was frequency independent (in the ranges: 0,1--0,7/s). The decrease of external Na+ (isoosmotic replacement of 70% NaCl by sucrose) or the increase (5-fold) of the external Ca2+ significantly enhanced the myocardial contraction depressed with D-600. However these contractions fall in the course of rhythmical stimulation, and the effect being strongly dependent on the rate of stimulation. The results confirm the assumption (see: Biophysics, 6, 1024, 1976), that intracellular Ca stores (sarcoplasmic reticulum, internal surface of the cellular membrane) are involved in the control of the contractility in the amphibian myocardial cells. Many peculiarities of the excitation-contraction coupling in the frog myocardial cells can be explaned if one assumes that: 1) there is no space separation of primary uptake and release of Ca ion sites in the frog myocardium; 2) the system of "resting Ca chanels" in the frog myocardial cells is not so well developed as in the mammalian myocardial cells.


[Model of representation of the phage structure of a 2-tone stimulus in the impulses of the neurons of the cochlear nucleus].

A model of the representation of information about the phase structure of oscillations in bitonal stimuli at the level of the neurons in cochlear neucleus is described. It is shown that for an agreement with the experimental results the degree of excitation of the final neuron of the model has to be proportional to the absolute value of the difference between the excitations coming to it from different groups of auditory nerve fibres. It is supposed that the sensitivity of neurons' response to the phase relations between the oscillations from which the stimulus consists could be due to possible dependence of the degree of receptor excitation either on bending or the hear cells or on the change of the sound pressure level in endolimphe.


[Photoluminescence of singlet oxygen in solutions of chlorophylls and pheophytins].

With the use of mechanical phosphoroscopes the photoinduced luminescence of singlet oxygen (1270 nm) have been found in air saturated solutions of chlorophyll a, bacteriochlorophyll a, protochlorophyll and pheophytins in CCl4, CS2, and freon 112. The excitation spectra of the luminescence coincide with the absorption spectra of the pigments. The relative quantum yields of the luminescence are determined, the data are used for calculation the probablities of intersystem crossing in pigment molecules. All the pigments are shown to quench 1O2, the rate constants of the quenching are measured. The quenching efficiency of the chlorophylls increases with reduction of the semiisolated double bonds, reduction of these bonds in molecules of the pheophytins decreases the constants of the quenching.


[Kinetics of dissociation of cyanide peroxidase complex].

The kinetics of dissociation ofHRP--CN- complex after reduction of the enzyme with ditionite was studied. The process goes in two consequent stages with different rate constants. The first stage can be related to the removal of CN- from Fe2+ and formation of out-of-equilibrium state of high-spin enzyme. The second stage possibly represents conformational relaxation of this state of the enzyme molecule.


[Problem of choosing radiation for x-ray analysis of biological objects in their native form (for instance collagen treated within the substance of the dermis)].

Some authors report that collagen in derma after industrial treating by the salts of chrom imply rather diffuse X-ray patterns. At the same time our X-ray photographs of these samples using Cr Ka-irradiation rather then Cu Ka-irradiation are all of fine quality. Therefore the collagen in derma is not distroed after salt treatment. In fact Ka-seria of Cu (using in all other investigations) cause transitions in Cr-atoms implying as diffuse phone on the film.


[Relationship between the modulus of elasticity in the surface of a membrane and the magnitude of its deformity].

The elasticity modulus E parallel in the membrane plane is frequency-independent in the range of 5--10 c.p.s. At room temperature E parallel 6-10(8) dn/cm2, at 4 degrees C E parallel depends strongly upon the degree of deformation-relative membrane surface change delta S/S, E parallel falls from 2-10(9) dn/cm2 if delta S/S = 0.1% down to 6-10(8) dn/cm2 if delta S/S = 1%. This property of E parallel explains the ability of mechanoreceptor membranes to produce a reaction if small stimulii are applied and to oppose the destruction if the latter are large.


[Role of lipid composition in the kinetics of free radical lipid oxidation in photoreceptor membranes].

The initial rate of lipid peroxidation (LPO) in photoreceptor membranes (PRM) of walleye pollock is 1.8--2.3 times higher than in frog PRM. Rhodopsin bleaching leads to an increase of LPO initial rate in PRM while having no effect on LPO in walleye pollack PRM. The content of unsaturated fatty acids in walleye pollock PRM is 1.4 times greater than in frog PRM. It is suggested that differences in LPO kinetics in PRM of walleye pollack and frog are caused by the differences in the lipid composition of the membranes (primarily by a high level of phospholipid molecular species with two unsaturated fatty acid residues in walleye pollock retina rod outer segments).


[Alteration of lipid peroxidation of rat liver endoplasmic reticulum in the presence of steroid hormones].

Effect of steroid hormones on peroxidation of lipids of rats' liver endoplasmic reticulum was studied. Accumulation of peroxidation products was measured by the change in chemoluminescence intensity and by the rate of malone dealdehydeformation. It was shown that typical antioxidants--estrogenes were the inhibitors of NADH-H and ascorbate-dependent systems of lipid peroxidation in rat liver microsomes, the enzymatic system being the most sensitive one.


[Thermal properties of DNA and polydeoxyribonucleotides in a wide range of ionic concentration of neutral salts and a polymer].

deltaHm, deltaT, Tm of melting of polydesoxyribonucletides polydAdT,, polyd(A--T)d(A--T), polyd(A--C)d(T--G) and DNA with different basic composition in a wide range of (10-2--4.0 M) ions (C2H5)4N+, Cs+ and Na+ were determined by the method microcalorimetry. It was established that the difference in melting heats of deltaHpolydAdT--deltaHpolyd(A--T)d(A--T) being approximately 0.6 kcal/mol b. p. represents that part of the energy which is caused by the heterogeneity of Stacking of interaction between (formula: see text) pairs. It is shown that the narrowing of deltaTm with increasing DNA concentration is connected with the decrease of the difference of polyGC and poly AT melting temperatures, what is with the parameter TGC--TAT which characterizes the melting of a free macromolecule.


[Mitochondrial ribosomes].

Some present-day conceptions on the structure and physiochemical and functional properties of mitochondrial ribosomes of higher and lower eukaryotes are reviewed. Mitochondrial ribosomes are compared to the ribosomes of prokaryotic and eukaryotic types and plastid ribosomes; biogenesis and functions of mitochondrial ribosomes are also discussed.


[Activity of chloroplast RNase under drought].

It is found, that the protein content in chloroplasts remained constant in conditions of drought, while RNase activity in chloroplasts was decreased in 2-2.5 times as compared with total leaves and hyaloplasma. No reliable differences in base composition of DNA and RNA were found in cotton variety "Tashkent 2" under the lack of water.


[Changes in the properties of hexokinase from hyaloplasm on binding with mitochondria].

It has been found that adenylic nucleotides at physiological concentrations solubilize mitochondrial hexokinase from muscles. The solubilizing effect of ATP is reversible in the presence of Mg2+ ions. It is shown that Mg2+ and glucose induce the transition of isoenzymes I and II of hexokinase from the free state to the bound one. Kinetic parameters of the free and bound forms of hexokinase have been determined. The stability of hexokinase has been found to vary, depending on its localization in the cell. Possible action mechanisms of reagents, exerting solubilizing and binding effects on the enzyme and the physiological role of hexokinase ability to exist in free and bound states are discussed.


[Conformational changes of erythrocyte membranes under the influence of biologically active substances].

As a result of studying rabbit erythrocyte membranes by using infrared spectroscopy method the absorption bands 1635 cm-1, 1645 cm-1, 1685 cm-1, 1695 cm-1 that probably belong to beta-structure of protein membranes have been discovered. The increase of the intensity of these bands under the influence of carbochol or eserine on membranes corresponds to increasing the lobe of beta-structure. The increase of theintensity of absorbtion bands corresponding to beta-structure and the change of intensity of absorption band of amide-1 in the field 1650-1662 cm-1 relating to alpha-helical and unordered structure are due to alpha leads to beta-transition in contractile protein. The change of a half of width of absorption band of amides A 3310 cm-1 under the influence of carbochol and other substances on cholinereactive systems has been found. This change correlates with the intensity of bands corresponding to beta-structure.


[Content of 5-methylcytosine in various families of repeating sequences of DNA in some higher plants].

Three families of repeating DNA sequences from 6 higher plants species are isolated. It is found that DNAs of plants examined at the same developmental stages differ considerably in the content of fractions with different repetition frequency of nucleotide sequences. The content of non-repeating sequences in the genome of the plants varies from 23.8 to 41.4%, the differences in the content of highly repeating fractions are less expressed (from 20.3 to 29.4% of the genome). The ratio of fractions changes considerably during ontogenesis: DNA of seeds contains more highly repeating sequences than DNA of seedlings. Fractions of nucleotide sequences with different repetition frequency, isolated from the same DNA, are little distinguished in the GC-pair content, and are characterized by a relatively high content of 5-methylcytosine: the methylation degree of cytosine is at least 17%. However, the degree of cytosine methylation is increased in fractions with high content of copies per genome in seedlings and leaves DNA of all the plants studied. The content of 5-methylcytosine in highly repeating fractions is 12% higher than in non-repeating sequences. Seed DNAs have some other distribution of 5-methylcytosine in different fractions: the degree of cytosine methylation in non-repeating and highly repeating sequences is practically the same.


[Characterization of protein composition of guinea pig skin melanosomes by polyacrylamide gel electrophoresis].

Protein composition and molecular weights of guinea pig skin melanosoma proteins were determined by polyacrylamide gel electrophoresis. Treatment of melanosomas extracts with sodium dodecyl sulfate, Triton X-100 and 1 N NaOH followed by electrophoresis revealed 26, 15 and 8-10 protein zones, respectively. It was found that melanosomas contain 4 main protein groups with respective molecular weights of 10 000-13 000, 21 000-42 000-94 000 and over 100 000.


[Metabolism of N-methyl-N-nitrosourea in various mouse tissues under inhibition of protein and nucleic acid synthesis].

The contribution of C1-pool and antimetabolite utilization to modification (labeling) of macromolecules of various mouse tissues after a single injection of 1-14C-methylnitrosourea was studied, using inhibitors of protein and nucleic acid synthesis. It was shown that about 50% of radioactivity found in the proteins, lipoproteins and DNA (but not in RNA) might be supplied by the C1-pool and/or through utilization of radioactive metabolites in the first 3-6 hours following the injection of 1-14C-methylnitrosourea. The data obtained allow to evaluete the degree of alkylation of macromolecules in various organs and tissues.


[Purification and comparative study of superoxide dismutase from animal tissues].

Cuprozinc superoxide dismutases were isolated from bovine liver, kidney and adrenal medulla. It was found that the enzyme from these sources exists in the form of three isoenzymes, the main izoenzyme being the most basic form. Physico-chemical properties of superoxide dismutases isolated from different tissues were found similar. Copper atoms in active sites of all superoxide dismutases investigated are bound with 4 nitrogen atoms.


[Specificity of fungal alpha-glucosidases].

Studies of substrate and cosubstrate specificities of mould alpha-glucosidases suggest that the binding site of the active center of mould alpha-glucosidase consits of two subsites--glucone and aglucone ones. The glucone site is capable to bind glucose and mannose, whereas the aglucone one- some compounds whose affinity for the enzyme may be expressed as follows: glucose greater than galactose greater than paranitrophenol greater than or equal to glycerol greater than ethanol approximately equal to methanol. Upon interaction of enzyme with alpha-D-glucoside the formation of a productive enzyme-substrate complex occurs when the glucosyl residue located at the non-reducible end of the substrate molecule occupies the glucone subsite and aglucone of the substrate occupies the aglucone subsite of the enzyme. After removal of the first product from the aglucone subsite the substrate is bound at this subsite. It is assumed that under cosubstrate excess it is capable to bind at the aglucone subsite prior to the removal of the first product and the formation of the substituted form enzyme--glycosyl. Under these conditions the cosubstrate removes the substrate from the aglucone subsite resulting in a formation of a non-productive tertiary complex enzyme--substrate--cosubstrate. The anomeric configuration of glucose produced under the action of alpha-glucosidase on maltose and starch was determined using a kinetic method.


[The nature of peptides firmly associated with the protein part of membrane lipoproteins in chloroplast biogenesis].

The biosynthesis of membrane lipoproteins in maize plastids at different stages of differentiation of the chloroplast lamellar system was studied. Prolamellar and lamellar system preparations were isolated from maize plastids and been or kidney bean chloroplasts, disintegrated by osmotic shock. Chloroplast lipoproteins are membraneous proteins of chloroplast lamellar systems. Peptids, lipids and carbohydrates "firmly" associated with the protein part of membrane lipoproteins were found at all stages of chloroplast ultrastructure formation. Incorporation of C14 amino acid increased with the time of incubation in "firmly" lipoprotein--bound peptids and in the polypeptid part of lipoproteins. Puromycin but not actidion D inhibited amino acid incorporation into the peptids and polypeptids of chloroplasts in vitro experiments. The maximal level of C14-peptids "firmly" associated with the protein part of membrane lipoproteins was observed in the membrane fraction of proplastids in vivo. It is pointed out that biogenesis process leads to the decrease of content of "firmly" lipoprotein--bound peptids in the chloroplast lamellae fraction.


[Mechanisms of stabilizing effect of vitamin E against lipid peroxidation in biological membranes].

Mechanisms of the inhibitory effect of vitamin E on the process of lipid peroxidation (LPO) in biological membranes are studied. Both alpha-tocopherol and its derivatives (a-tocopherylacetate, o- and p-tocopherylquinones possess no radical scavenging activity) inhibit non-enzymatic (Fe2+ + ascorbate)-induced LPO and prevent LPO-dependent inhibition of Ca2+ transport in sarcoplasmic reticulum membranes from skeletal muscles. The protective effect of alpha-tocopherylacetate, tocopherilquinones and partially of alpha-tocopherol is due to a stabilizing effect of these compounds on sarcoplasmic reticulum membranes, registered by a decrease of fluidity of membrane lipid bilayer (probed by nitroxile radical TEMPO) and by a decrease of its passive permeability for Ca2+. Under the enzymatic NADPH-dependent LPO induction in rat liver microsomal fraction a strong inhibitory effect of tocopherylquinones is similar to the effect of other electron acceptors (methylnaphtoquinone, TEMPO) and is due to their ability to compete with LPO reaction for reducing equivalents in NADPH-dependent electron carriers wich results in the formation of hydroxy-derivatives having pronounced radical scavenging activity.


[Isolation of NAG-vibrio neuraminidase by affinity chromatography].

Three molecular forms of non-agglutinating vibrio neuraminidase were isolated by affinity chromatography. All the three forms were found to be immunochemically related and had molecular weights of 90.000, 58.000 and 52.000, respectively.


[Applicability of quantitative kinetic spectrophotometric method for glucose determination].

A simple kinetic method for quantitative determination of glucose has been developed. The method is based on two enzyme reactions, catalyzed by glucose oxidase (E. C. 1.1.3.4) and peroxidase (E. C. 1.11.1.7). The advantages of the method are high sensitivity (up to 2 X 10(-5) M glucose in a sample) and rapidity (3-4 min per analysis). The method gives reliable results for determination of small glucose concentrations in the presence of cellobiose. The conditions have been found when the reproducibility of the method does not depend on the glucose oxidase activity.


[Proteolytic activity of lysosomes from dog kidney under the effect of amphotericin B in vitro].

Amphotericin B at concentration of 0.1-1 mcg/ml is found to increase the yield of proteases from isolated dog kidney lysosomes. Further increase of amphotericin B concentration results in the decrease of proteolytic activity in the incubation medium, which is due to a considerable inactivation of proteases by high concentrations of antibiotic. Like amphotericin B, sodium deoxycholate, which is a part of many drugs, sharply inhibits the activity of enzyme, but, in contrast to the antibiotic, it almost does not release proteases from lysosomes. It is suggested that early described degradation of proteins of plasmic membranes and chromatin in dog kidney cells under the injection of amphotericin B is a result of the damage of lysosome membranes by the antibiotic.


[Some properties of cytoplasmic thymidine kinase and nucleoside phosphotransferase from rat liver].

The activities of two deoxythymidine-phosphorylating enzymes--thymidine kinase and nucleoside phosphotransferase--were found in the cytoplasmic fraction of normal and regenerating rat liver. The specific activity of nucleoside phosphotransferase appeared to be by 50% higher than that of thymidine kinase. Nucleoside phosphotransferase has a broad specificity for the phosphate donor. This enzyme is more stable to heating and prolonged dialysis as compared to thymidine kinase. The enzymes respond differently to the addition of d-TTP, d-CTP and sturins A and B: thymidine kinase is strongly inhibited by these agents whereas nucleoside phosphotransferase is insensitive to d-TTP and d-CTP and is only slightly inhibited by sturins. On the other hand the activity of nucleoside phosphotransferase is considerably decreased after addition of ATP. Changes in the activities of both enzymes during 50 hrs following partial hepatectomy were studied. Two activity maxima were observed at 20-22 and 40-46 hrs of regeneration. Using polyacrylamide gel electrophoresis, three isoforms of both enzymes were found. The ratio between the isoenzyme content of the two enzymes from the cytoplasmic fraction of regenerating liver varied as compared to normal.


[Isolation and characterization of pigment-protein complexes from green serobacteria Chlorobium limicola forma thiosulfatophilum].

A subchromatophore fraction containing the reaction center P-840 was isolated from the chromatophores of green serobacteria Chlorobium limicola forma thiosulfatophilum by ultracentrifugation and its protein composition was characterized. After treatment of the chromatophores by Triton X-100 and subsequent polyacrylamide gel electrophoresis two pigment-protein complex containing bacterioviridin. The proteins of the pigment-protein complex containing bacterioviridin. The proteins of the pigment-protein complexes were obtained. The first complex contained mainly bacteriochlorophyll alpha with bacterioviridin contaminations and the second one-only bacterioviridin. The low-temperature absorption spectrum of the bacteriochlorophyll alpha-containing pigment-protein complex is identical to the absorption spectrum of a water-soluble pigment-protein complex isolated and characterized by Olson and contains proteins with molecular weights of 34.800, 33.100, 27.500 and 23.200. A protein with molecular weight of 32.700 was found in the pigment-protein complexes were compared to the proteins of the photochemically active subchromatophore fraction and chromatophores.


[Antigenic activity of angiotensin II and its fragments].

The antigenic activity of angiotensin and its seven fragments has been studied in cross-reaction with specific antibodies, elicited to angiotensin and its fragments: C-terminal hexapeptide and middle tetrapeptide. It has been found that all the fragments studied possess certain affinity for antibodies elicited to angiotensin, C-terminal hexapeptide and middle tetrapeptide. The middle tetrapeptide was identified to be the immunologically active centre of the angiotensin molecule.


Use of partial mammectomy to produce malnutrition in the rat.

Partial mammectomy in the Sprague-Dawley rat was studied as a method for producing malnutrition without accompanying differences in litter size. Throughout the litter period, experimental litters weighed less than controls. No difference in the rate of development of various anatomical landmarks was noted with this degree of malnutrition. Maternal behavior in the nursing dam was measured with respect to nursing, nesting, and retrieval behavior. Mammectomized subjects built and maintained better nests and spent more time in a nursing posture, but were less likely to retrieve their litter than were control animals. At weaning the intact mammae of the experimental animals were larger than those of control animals.


Effect of chronic maternal methadone exposure on perinatal development.

The effect of perinatal exposure to methadone on body growth and brain development was studied in rat. Female Sprague-Dawley albino rats received daily intraperitoneal injections of 5 mg/kg dl-methadone-HCl during gestation and lactation. Body weights were reduced in drug-treated mothers during gestation and the first 2 weeks of lactation. No differences in gestation time, litter size, or infant mortality were recorded, however methadone-treated offspring grew more slowly than controls. Weight deficits persisted in rats observed 5-1/2 weeks after cessationof drug exposure (group 1) and in animals continuing to receive daily interperitoneal injections of 5 mg/kg (group 2). From birth to day 21, brain weight and length, cerebral width and cerebellar weight and width were generally smaller in methadone-exposed rats. Brain measurements of group 1 and group 2 animals on day 60 revealed a reduction in brain and cerebellar weights and cerebral and cerebellar widths from control values. Brain:body weight and cerebellum:brain weight ratios were similar to controls. Analysis of these results indicates that maternal methadone treatment retards the growth of young rats and affects brain development.


Fetal exposure to 1:8 dihydroxyanthraquinone.

A survey of 160 consecutive maternity cases showed that a high percentage of mothers had exposed their fetus to laxatives of the anthraquinone type at some stage during the pregnancy. Administration of 1:8 dihydroxyanthraquinone at the time of induction of labour with estimation of the substance in maternal urine, amniotic fluid and the first urine obtained from the baby, indicated that the substance is absorbed from the maternal gut, crosses the placenta and is excreted via the fetal kidney into the liquor. In mothers and babies most of the drug appeared as glucuronide. Fetal catharsis did not appear to occur and the reasons for this are discussed.


Iodoprotein and iodoamino acid distribution in pig thyroid tissues at different periods of development.

A biochemical study of the thyroid tissue in pigs at various stages of development was undertaken. The iodine and protein concentrations, and the iodoamino acid content of thyroglobulin (TG) were determined. Expressed as mu 127I/100 mg wet tissue, the mean iodine content of pig fetus is 66.4, 89.2 in the piglet and 168 in the adult pig. TG accounts for 95% of total proteins; its content increases with age. Mean iodine content of TG is 0.70 mug 127I/100 microgram in the fetus, 0.78 in the piglet and 0.97 in the adult pig. The average number of molecules of thyroxine residues is 1.6 in the fetus, 2.2 in the piglet and 3.5 in the adult. Average triiodothyronine residues are lower around birth than in the adult. At all stages of development, synthesis of thyroxine is less effective in the pig than in the human, relative to the iodine content of TG. In contrast to the human, iodinated compounds are not found at a very low level near birth, but TG and iodine content regularly increase.


Plasma renin activity and its postnatal development in preterm infants. Preliminary report.

Immunoassayable plasma renin activity (PRA) was measured in 16 healthy preterm infants of different gestational ages. A total of 52 measurements have been performed between the second week of life and the date of the expected term. PRA was found markedly increased in preterm infants and a highly significative negative correlation with postmenstrual age was proved.


Amino acid nutrition of the fetal lamb.

With three exceptions amino acids were retained by the fetal lamb from the maternal circulation in proportions appropriate for the synthesis of whole blody protein. Only glutamate, aspartate and serine, amino acids involved in the synthesis of nucleic acids, seemed to be produced by the fetus itself.


Metabolic response to fasting in experimental intrauterine growth retardation induced by surgical and nonsurgical maternal stress.

Experimental intrauterine growth retardation was produced by two groups of neonatal rats by maternal sham surgery or by maternal obligatory exercise during the last 5 days of gestation (forced swim). Both experimental groups of neonates had lower plasma glucose and higher plasma insulin levels than controls after a 4-hour fast. Fetal stunting may be produced by a variety of maternal stresses in the rat and may be associated with alterations in the hormonal and glycemic response to fasting. Studies of the metabolic effects of experimental intrauterine growth retardation must be interpreted with caution if control animals have been subjected to intrauterine stress.


Uptake and metabolism of 14C-palmitate by fetal rabbit tissues.

The uptake and esterification of 14C-palmitate into lipid classes in placenta, fetal brown adipose tissue (BAT) and liver of rabbits were investigated in vitro. Fetal BAT showed a high rate of fatty acid uptake, 8.5 mumol-a-1 tissue-h-1. From 5 min onwards, the majority of incorporated label was in the triglyceride fraction. The placenta and fetal liver also incorporated I-[14C]-palmitate into both FFA and esterified lipid fractions, although at much lower rates than observed for BAT. In the liver, triglycerides, but in the placenta phospholipids, contained the majority of the label after 1 h incubation. BAT from both fetal and newborn rabbits released 14CO2 and the production of 14 CO2 was greater in the presence of noradrenaline. The specific activity of the CO2 was the same in stimulated and unstimulated tissue. It is concluded that BAT as well as the liver are important sites of free fatty acid removal from the fetal circulation. The potential for fatty acid oxidation is present in BAT of the 28-day rabbit fetus.


Measurement of placental blood flow in the pig and its relation to placental and fetal weight.

In 12 pregnant sows (Sus scrofa domestica), containing 144 live fetuses, placental blood flow was measured by the indicator fractionation technique using radioactive microspheres. In 8 of these litters, which contained 96 live fetuses, placental blood flow as obtained in absolute units. Significant positive correlations were observed between placental blood flow and placental weight, between placental blood flow and fetal weight, and between placental weight and fetal weight. It is clear that the runt, by comparison with his littermates, has a low birthweight in association with a small placenta and a low placental blood flow.


Baroreflex activity in conscious fetal and newborn lambs.

The immediate transient baroreceptor sensitivity was measured in 9 conscious fetal and 7 conscious newborn lambs for periods of at least 35 days following bolus injections of phenylephrine (20-50 mug/kg). Mean sensitivities were unchanged throughout gestation from 105 days at 6.7 +/- 0.4 msec/cm H2O (n = 45) and were insignificantly different from those in the newborn period, 5.9 +/- 0.4 msec/cm H2O (n = 78). In contrast, baroreflex sensitivities were less in 2 fetuses and 2 newborn lambs when pressures were increased by chronically implanted thoracic aortic balloon cuff; they were 3.03 +/- 0.11 (n = 127) and 0.91 +/- 0.11 msec/cm H2O (n =61), respectively. "Steady-state" heart period-arterial pressure curves indicate that the baroreflex operates down to levels of 40 cm H2O in the fetus which is lower than that achieved in the adult of other species, rabbit and man.


Confidence intervals and test of hypotheses concerning dose response relations inferred from animal carcinogenicity data.

Confidence intervals and hypothesis tests are developed for dose-response relations based on dichotomous data from animal carcinogenicity experiments. The functional form of the dose-response curve comes from the Armitage-Doll multistage carcinogenesis model and involves a polynomial in the dose-rate, with non-negative coefficients. Asymptotic distributions of the maximum likelihood estimators of these coefficients are used to construct confidence bounds on risk at a given dose and on the dose corresponding to a given risk. Likelihood ratio tests are developed for the presence of a positive dose-related effect and for the existence of a positive slope to the dose-response curve at zero dose. The latter test is of practical importance since a positive slope of the dose-response curve at zero dose rules out any "threshold-like" behavior and would often mean that any concentration low enough to insure a negligibly low cancer risk (e.g., 10(-6)) would be too low to be economically useful for applications such as food additives. Simulation experiments are performed to provide guidelines for applying the theory.


An extension of the sign test for replicated measurements.

An extension of the sign test is proposed for the case where there are paired groups or sets of replicated measurements; the individual measurements are unpaired between groups. A class of methods is investigated where the values of the groups of replicates are replaced by summary statistics (exceedances) whose distribution is independent, under the null hypothesis, of the underlying parametric structure. Possible adjustment for discontinuity of the underlying distribution (ties) is discussed and a normal approximation is presented. The proposed sign text extension, appropriate for small samples and large numbers of replicates, is used to compare the relative strengths of aqueous and lyophilized solutions of ragweed pollen at three dilutions for two types of reactions (erythematous and wheal area). Because correlations for individual reactions at two separate measurement times could not be estimated under the non-parametric procedures the analyses were performed for the cases where the responses at two measurement times were presumed to be (1) perfectly correlated, and (2) uncorrelated. Comparison of exact results with the normal approximation showed good correspondence for both cases, e.g., where n = 4 (n = number of subjects) and r = 8 (r = replicates) and n = 8 and r = 4. The analysis showed that the lyophilized product was superior to the aqueous at all solution strengths and for both measures of reactivity.


On the treatment of grouped observations in life studies.

Assuming a model of proportional failure rates, Cox (1972) presents a systematic study of the use of covariates in the analysis of life time. The treatment of tied observations is a particularly troublesome point in both theory and application. It appears that grouping rather than discrete time is the right way to handle ties. This paper studies methodology for grouped observations. A logistic model, which makes explicit use of Cox's earlier binary data methods, is introduced and illustrated with a numerical example. The model leads back to Cox's proportional failure rates when the lengths of the grouping intervals approach zero. This limiting process provides some enlightenment on controversial issues such as ignoring intervals in which no failures occur, determining whether the covariates may be functions of time, and treating ties.


Estimation for discrete time branching processes with application to epidemics.

Certain estimators for the mean of the offspring distribution of a Galton-Watson process are considered. The asymptotic behaviour of each of these estimators is studied when the true underlying model is in fact a multitype branching process or a branching process with a random environment. It is revealed which of the estimators remain consistent indicators of whether or not the process is subcritical, under these alternative underlying models. It is then indicated how this "robustness" result might influence the choice of an estimator by considering the problem of estimating the level of immunity required in a community in order to prevent major epidemics. The application is illustrated with references to smallpox using data from an outbreak in São Paulo, Brazil.


Assumptions for different ascertainment models in human genetics.

Some aspects of sampling family data in human genetics are discussed. Two well-investigated ascertainment models, the complete ascertainment model and the single ascertainment model, which have been derived using restrictive sampling assumptions, are shown to hold under four more general sets of assumptions.


Estimation of relative risk from matched pairs in epidemiologic research.

The matched pairs design is often used in epidemiologic research, both in prospective and retrospective studies. The Kraus estimator of relative risk (1958) has been derived in a number of ways. The development presented here employs an unconditional likelihood function. The estimator is shown to be valid only when disease incidence is low and relative risk is constant over the levels of the covariate. Asymptotic variances are derived.


An example on the risk dependence and additivity of intensities in the theory of competing risks.

Let vk(x) be the force of mortality when only risk Rk is acting in a population, and let muk(x) be the force of mortality due to Rk when all of the risks of death are acting, k = 1, ---, c. On the assumption of the independence of the risks of death, the sum of intensitives vk(x) equals the total force of mortality. This example shows that the equality can also hold when the risks of death are dependent and the forces of mortality, muk(x) and vk(x), differ.


Circadian rhythm analysis when output is collected at intervals.

It has long been recognized that many physiological and biochemical parameters show a repeating pattern of variation over 24 hours, i.e., a circadian rhythm. Halberg, Tong and Johnson (1965) used the sinusoidal model with 24-hour cycle to describe this variation. Tong (1976) described the polar coordinate transformation by which the sinusoidal regression problem can be treated as a linear regression problem. When the output of a system following diurnal variation, e.g. the human kidney, is collected at regular intervals and assayed, the expected quantity of substance present corresponds to the integral of the underlying output function. This paper shows that the polar coordinate transformation also linearizes this regression problem. More importantly, the covariance structure of Halberg et al. does not include interindividual variation. An alternative and more general model is proposed here based upon Rao's (1959) growth curve analyses. The latter method allows testing for adequacy of the sinusoidal model and leads to inferences about population parameters. An example is given.


Effects of serum-free culture media on human liver and fibroblastic cells.

The morphological aspects and amino acid variations of human fibroblast and liver cell monolayer cultures were studied in serum free media. Under these conditions, the behaviour of the two cell types differed greatly. The morphological changes for the liver cells, as compared with the fibroblasts, appear more quickly and some of these changes are particular. Furthermore, extracellular amino acid variations are of lesser importance for the liver cells, during the 10 days following the suppression of serum, except for serine.


Monodomain samples of dipalmitoyl phosphatidylcholine with varying concentrations of water and other ingredients.

Methods are presented for the preparation of large monodomain phospholipid bilayer arrays containing variable amounts of water approaching the two-phase limit. The optical birefringence of these lamellar phases of dipalmitoyl phosphatidylcholine (DDPC) is measured over a range of temperature and water content, and phase transitions are observed. The techniques employed for pure DPPC and water are extended in order to produce macroscopically aligned samples containing varying concentrations of cholesterol, inorganic salts, antibiotics, and chlorophyll a. Polarization studies of the 670-nm band of chlorophyll a indicate macroscopic orientational order in the chromophore under the same conditions.


An extrapolation method for reducing equilibration times in sedimentation equilibrium experiments.

We present a detailed investigation of the use of an extrapolation technique to decrease running times of sedimentation equilibrium experiments. If concentration profiles are available at time delta tau, 2delta tau, 3delta tau,...., cn(r) = c(r, n delta tau), then the Aitken transformation replaces the cn(r) + ĉn(r) = [cn + 1(r) cn - 1(r) - c2n(r)]/[cn + 1(r) + cn - 1(r) - 2cn(r)]. We show that the ĉn(r) converge to the equilibrium values c infinity (r) much more quickly than the cn(r). Savings in time are shown to range from a factor of approximately 2 for meniscus depletion experiments to factors of between 4 and 8 for lower speeds or smaller molecular weights. It is also shown that the technique is quite sensitive to noise, so that an accurate optical system is required to allow its optimal use.


Hydration of phosphatidylocholine. Adsorption isotherm and proton nuclear magnetic resonance studies.

Adsorption-desorption isotherms were obtained for water binding by 1,2-dimyristoylphosphatidylcholine in the temperature range 15 degrees-35 degrees C. The isotherms were analyzed by Brunauer et al.'s (BET) theory and also a polarization theory, the latter being more successful in fitting the data. There was some evidence for a change in the surface field of the lipid bilayer around 25 degrees C. Proton T1 and T2 measurements were used to obtain a log-normal molecular correlation time distribution for water protons in these systems. This distribution was compared with the isotherm data to effect a description of several classes of water molecules.


Human red cell hemolysis rates in the subsecond to seconds range. An analysis.

Hypoosmotic shock kinetics of the normal human red cell (25 degrees C) were investigated by means of a rapid kinetics apparatus, with a resolving time of about 50 ms. The results are compared with some current models for hemolysis. The fast hemolysis plots are not true symmetric sigmoids, in contrast to results from less stressful conditions, nor can they be simply fitted to an "all or none" process. In the most severe conditions, mixing with neat water, the velocities with which red cells start to hemolyze depend on the rate at which the cell is converted to a swollen sphere (lag phase). Under such conditions, the mean time to rupture and start of leaking is about 0.6 s. The rate of osmotically driven solvent flow is probably the principal controlling factor in the discocyte to sphere transformation. The overall course of hemolysis can be described in terms of two rate processes and a distribution of cell fragilities. The fragilities probably depend on the age of individual cells in the samples. In the low-salt region, the effect of hypotonicity as well as hypoosmolality is discerned. The surface charge on the red cell provided no driving force for rupture above salt concentration 0.10M, but at 0.05 M salt and below, electrostatic effects may contribute.


Physics of chemoreception.

Statistical fluctuations limit the precision with which a microorganism can, in a given time T, determine the concentration of a chemoattractant in the surrounding medium. The best a cell can do is to monitor continually the state of occupation of receptors distributed over its surface. For nearly optimum performance only a small fraction of the surface need be specifically adsorbing. The probability that a molecule that has collided with the cell will find a receptor is Ns/(Ns + pi a), if N receptors, each with a binding site of radius s, are evenly distributed over a cell of radius a. There is ample room for many indenpendent systems of specific receptors. The adsorption rate for molecules of moderate size cannot be significantly enhanced by motion of the cell or by stirring of the medium by the cell. The least fractional error attainable in the determination of a concentration c is approximately (TcaD) - 1/2, where D is diffusion constant of the attractant. The number of specific receptors needed to attain such precision is about a/s. Data on bacteriophage absorption, bacterial chemotaxis, and chemotaxis in a cellular slime mold are evaluated. The chemotactic sensitivity of Escherichia coli approaches that of the cell of optimum design.


Side-chain mobility and the calculation of tyrosyl circular dichroism of proteins. Implications of a test with insulin and des-B1-phenylalanine insulin.

Previous calculations using crystal structure coordinates (Strickland and Mercola [1976], Biochemistry. 15: 3857) have predicted that about 40 percent of the calculated tyrosyl circular dichroism of hexameric insulin is due to one of the four tyrosine residues: viz. the A14-tyrosine interacting with the nearby B1-phenylalanine ring group. We have tested this prediction by measuring the tyrosyl circular dichroism of an isomorphous analogue of insulin, des-B1-phenylalanine-insulin. Contrary to expectation, the resulting circular dichroism was the same as that of insulin. It is concluded that the B1-phenylalanine residue does not in fact make a large contribution to the circular dichroism of A14-tyrosine. This result is probably due to the thermal motion of the B1 and A14 ring groups not taken into account by the calculations. An example of the effects of thermal motion on the calculated circular dichroism is given and improvements that do take into account thermal motion are discussed.


Thermal-stimulated pressure and current studies of bound water in lysozyme.

The state of bound water in crystalized lysozyme was studied by four techniques: electret thermal depolarization currents, thermal-stimulated pressure, isothermal polization decay, and thermogravimetry. Hydration levels ranged from 0 to 40 mg water/g protein. Desorption of bound water dipoles was found to be the main process responsible for electrical depolarization. Two different binding sites for water were identified with long relaxation times at room temperature (order 10(2)s) and activation energies of 0.34 plus or minus 0.02 eV and 0.55 plus or minus 0.04 eV.


The representation of membrane admittance.

An integral representation for the membrane admittance in terms of its known current response to a voltage step function is presented. It is demonstrated that the frequency-dependent terms in the contribution to the membrane admittance by the ion-selective conductance of the nerve membrane are proportional to the static conductances. The additional information contained in the real and imaginary parts of the membrane admittance should allow the parameters of the ion conductance to be determined. Eventually, these measurements should also give information about the electric dipole displacement currents of the conductance systems themselves, and about the metabolically supported active ion transport currents that maintain the ion concentration gradients.