Proton N.M.R. study of the conformational dynamics of porcine pancreatic colipase. Titration of aromatic residues.

The low-field portion of the 360 MHz proton N.M.R. spectrum of native porcine pancreatic colipase has been studied as a function of pH over the pH range 2-12. Resonances associated with the 26 protons of the aromatic rings of the two histidines, two phenylalanines and three tyrosines have been identified and tentatively assigned to specific residues. Titrations of pH yielded apparent pKa's of 7.9, 6.9, 10.4, 10.3 and 11.3 for His I (His 30), His II (His 86), Tyr I (Tyr 56 or 57), Tyr II (Tyr 56 or 57) and Tyr III (Tyr 53) respectively (tentative assignments). The high pKa value of His 30 is attributed to the vicinity of Asp 31. The mobility of the aromatic ring of Tyr 53 is hindered and an upper bound of 500 s-1 on the rate of rotation can be estimated. The aromatic rings of the 2 other tyrosine residues and of the 2 phenylalanine residues can rotate freely on the N.M.R. time scale. The study of perturbations in titration profiles and chemical shift values reveals a specific interaction of His 86 with Tyr I and, to a lesser extent, Tyr II. The existence of this interaction indicates that the protein folding brings in close spatial vicinity two distant regions of the covalent structure to form a "hydrophobic-aromatic" site which might be involved in the binding of bile salt micelles to pancreatic colipase.     

The protective effect of dietary fish oil on murine lupus

Dietary marine lipids markedly reduce the severity of glomerulonephritis and its associated mortality in inbred strains of mice developing autoimmune disease, a model for human systemic lupus erythematosus. We report here the influence of varying the dose of menhaden oil and the timing of its administration on the mortality of female (NZB x NZW) F₁ mice. After ingesting 25 wt% menhaden oil (MO) for periods of 1.5 weeks to 12 months, there was a stable content of tissue n-3 fatty acids, with total n-3 fatty acids of 28% and 35% in spleen and liver, respectively. The extent of protection from mortality was dependent on the dose of MO with marked protection at doses of 11 to 25%, marginal protection at 5.5% and no protection at 2.5% MO. Delay in the institution of MO until ages 5 or 7 months still resulted in large reductions of mortality. Conversely, institution of a MO diet from 6 weeks until ages 5 to 7 months followed by a change to beef tallow resulted in little protection. Serum levels of 4 cyclooxygenase products were reduced ranging from 26 to 76% in mice fed MO diets, compared to mice fed beef tallow, based on radioimmunoassay. The degree of reduction of mortality on different doses of MO was correlated best with tissue levels of C22:5, and levels of C20:5 and C22:6 were similar at high and low doses of MO, suggesting that levels of 22:5 may be related to the protective effects of marine lipids on autoimmune disease.     

Potential generation in bilayer lipid membranes in the NADH-flavin mononucleotide-ubiquinone-6-O2 system

Membrane conductance and generation of transmembrane potential by the NADH oxidation reactions in the NADH-flavin mononucleotide-ubiquinone-6-O₂ system have been studied. It has been shown that in solutions of a relatively low buffer capacity at pH 5.8 in the presence of a proton carrier, a potential is generated, the value of which depends on the concentration of the reducer and amounts to 40–60 mV. In the absence of a proton carrier at pH 8, a potential arises, which suggests a transmembrane negative charge transfer. Bilayer lipid membranes have been shown to possess proton selectivity if the reaction is run at pH 3.7. At a pH higher than 5.8 the proton selectivity disappears. Schemes of potential generation in lipid bilayers in different conditions are suggested and discussed.     

Further studies on the effect of aldosterone on electrical resistance of toad bladder

The fall in transepithelial electrical resistance which accompanies aldosterone stimulation of short-circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) in toad urinary bladder has been studied further to evaluate the possible causal role of this response in hormonal stimulation of Na⁺ transport. A steady-state change in tissue conductance was found to depend upon both the simultaneous stimulation of transport by the steroid and the metabolic state of the tissue. Changes in metabolic state alone did not alter resistance. A sustained increase in Na⁺ transport, dependent on pretreatment with aldosterone and elicited by addition of glucose, could be obtained without a sustained decrease in resistance. Amiloride, an inhibitor of Na⁺ uptake, produced changes in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ that were linearly correlated with its effects on tissue conductance. On the basis of the $\text{conductance}\text{-I}{}_{\mathrm{<mtext>sc</mtext>}}$ relationship with amiloride, the $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ response to aldosterone was about two-fold higher than would be predicted from its effects on conductance alone. Despite the apparent lack of a simple quantitative dependence of the change in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ on the change in conductance when the response is fully developed, the results suggest that conductance changes may mediate the initial or early stage of the response.     

Acidic glycosaminoglycans in developing sterno-costal cartilage of the hydrocephalic (ch+/ch+) mouse

The sterno-costal cartilage of the hydrocephalic mouse carrying the autosomal recessive gene (ch+/ch+) has 40 ± 3% of the acidic glycosaminoglycan concentration of the normal control containing the satin marker (+sa/+sa). The acidic glycosaminoglycan concentration of the sterno-costal cartilage in the heterozygous mouse (ch+/+sa) is significantly higher (114 ± 8%) than the normal control. The distribution of the acidic glycosaminoglycans in the sterno-costal cartilage is similar in the normal, heterozygous and homozygous mice at all stages of development studied, (prenatal, newborn and postnatal) being 78 ± 4% chondroitin 4(6)-sulfate and 22% hyaluronic acid and/or keratan sulfate. The concentration of acidic glycosaminoglycans in the sterno-costal cartilage decreases as development progresses in all three gene types of mice. The reduced level of acidic glycosaminoglycans in the sterno-costal cartilage of the autosomal recessive mouse, ch+/ch+, is associated with a defect in the formation of the sternum. The higher than normal acidic glycosaminoglycan concentration in the sterno-costal cartilage of the heterozygous mouse ch+/+sa is associated with delayed calcification of the sternum. This study characterizes the molecular locus of a defect in the extra-cellular matrix of a mouse carrying a lethal gene and may help in understanding proteoglycan disorders (mucopolysaccharidosis) in the human.     

The effect of local application of homocysteine on neuronal activity in the central nervous system of the rat

Homocysteine, a monocarboxylic, sulfur-containing amino acid, produces convulsions in rats and mice when administered systemically. Convulsions and high serum concentrations of homocysteine are among the symptoms that characterize patients with homocystinuria, a hereditary disorder of amino acid metabolism. In order to evaluate the effects of homocysteine on the central nervous system directly, extracellular recordings were made from neurons in rat cerebral cortex, cerebellum and midbrain during local application of homocysteine by pressure ejection or iontophoresis. Both methods of drug delivery produced dose-dependent increases in the activity of neurons in every area tested. Activity was increased by D, L-homocysteine and L-glutamate in 67 percent of cells tested with both drugs. The doses required to produce equivalent excitations in this group of cells were similar, suggesting that homocysteine is at least as potent as glutamate. The excitatory effects of both homocysteine and glutamate were antagonized by local application of betaine, a biological methyl donor which blocks convulsions produced by systematic administration of pentylenetetrazol and electroshock as well as homocysteine. The effects of local application of homocysteine were also blocked by local application of the glutamate antagonist glutamate diethylester (GDEE). In 6 of 7 cells tested, GDEE appeared to preferentially affect homocysteine-induced excitations. These data indicate that homocysteine has an excitatory action on neurons, a finding which may account for some of the symptoms associated with certain disorders of amino acid metabolism.     

Diurnal variations of plasma testosterone in men

Plasma testosterone (T) levels were assayed by a Competitive Protein Binding (CPB) technique in a group of 31 healthy males. In 22 subjects a single blood sample was taken between 8:00 and 9:00 A.M. and the mean T concentration was 6.84 ± 2.11 ng/ml. In the other 9 normal men, blood samples were taken every 4 hours. The existence of temporal variations for testosterone was confirmed by finding the highest mean plasma levels at 4:00 A.M. (9.28 ± 1.17 ng/ml) and lowest mean levels at 8:00 P.M. (2.66 ± 0.52 ng/ml).     

The effect of catecholamines on ion transport in the toad bladder

Noradrenalin (8 · 10⁻⁶ M) and adrenalin (6 · 10⁻⁶ and 6 · 10⁻⁷ M) were found to cause marked stimulation of short-circuit current (S.C.C.) in isolated toad bladder, but isoprenalin (8 · 10⁻⁷ M) was found to be without effect. The percentage rise in S.C.C. due to noradrenalin was found to be inversely proportional to the initial S.C.C. or total conductance of the bladder. Again in the case of noradrenalin the rise in S.C.C. was almost completely abolished by α-adrenergic blockade but not by β-blockade. This rise in S.C.C. was found not to be significantly different from the rise in net Na⁺ flux. Bidirectional Cl⁻ fluxes were estimated using ⁸²Br as a companion radionuclide to ³⁶Cl. No significant net Cl⁻ flux was apparent, either before or after addition of any of the three catecholamines tested. However, in some cases the unidirectional Cl⁻ fluxes rose markedly following addition of noradrenalin or of adrenalin and this change was not reflected in a change in total conductance. This anomaly was noted to occur in bladders whose initial conductance was of the order of 0.5 kΩ⁻¹ · cm⁻² or greater. The evidence presented suggests that two actions of catecholamines on ion transport in toad bladder are (a) to increase Na⁺ transport via stimulation of α-adrenergic sites and (b) at the concentrations tested to cause an increase in passive Cl permeability in bladders whose initial conductance is high.     

Characterization of the plasma membrane ATPase of Candida tropicalis

1) Plasma membrane vesicles from Candida tropicalis were isolated from protoplasts by differential centrifugation and purified in a continuous sucrose gradient. 2) The plasma membrane bound ATPase was characterized. It is highly specific for ATP and requires Mg2+. It is stimulated by K+, Na+ and NH4+. Lineweaver-Burk plots for ATPase activity are linear with a Vmax of 4.2 mumoles of ATP hydrolyzed min-1.mg-1 protein and a Km for ATP of 0.76 mM. The ATPase activity is inhibited competitively by ADP with a Ki of 1.7 mM and non competitively by vanadate with a Ki of 3 microM. The activity is unaffected by oligomycin or azide but is sensitive to DCCD.     

A radioimmunoassay of plasma estradiol-17beta with the use of polyethylene glycol to separate free and antibody-bound hormone

A radioimmunoassay for plasma estradiol-17β was developed using polyethylene glycol to separate free from antibody-bound hormone. Specificity for estradiol-17β was achieved by a modified celite microcolunm procedure in which estradiol was.separated from interfering estrogens, including estrone. Using trace ³H-estradiol to monitor procedural losses, the method was shown to be sensitive and accurate. Intra- and inter-assay coefficient of variation of the method was 8.7 and 10.6%, respectively. Polyethylene glycol used for antibody precipitation appears to be a generally applicable method for steroid hormone radioimmunoassays. The simplicity, precision and rapid analysis, coupled with its lack of time dependence and ease in automation, makes this a convenient and practical method.     

Protein dynamics by solid-state nuclear magnetic resonance spectroscopy: Peptide backbone of the coat protein in fd bacteriophage

¹³C and ¹⁵N chemical shift anisotropy and ¹⁵N¹H dipolar powder patterns from backbone sites of the coat protein in fd bacteriophage are not averaged by motion. This means that the polypeptide backbone of the protein has no large amplitude motions rapid compared to 10⁴ Hz. Relaxation studies on the ¹³C_α and ¹⁵N amide resonances indicate the presence of motions on the 10⁹ Hz timescale. These results are reconciled with a model where an otherwise rigid backbone undergoes small amplitude, rapid motions.     

Methanol-stabilized intermediates in the thermal unfolding of ribonuclease A: Characterization by 1H nuclear magnetic resonance

The thermal unfolding of ribonuclease A has been studied in solutions of 25, 35 and 50% methanol ($\text{v}\text{v}$), using 360 MHz proton magnetic resonance spectroscopy. Several observations indicate that the native structure of the protein in methanol cryosolvents is very similar to that in aqueous solution. A detailed analysis of the unfolding process has been made using the C-2 protons of the imidazole side-chains of the four histidine residues. As denaturation proceeds new resonances appear, whose chemical shifts correspond to neither native nor fully unfolded species. These have been assigned to particular His residues by selective deuteration studies. The thermal denaturation transitions reveal a multiphasic process in each of the solvents, and become less co-operative with increasing concentrations of methanol. The denaturation is fully reversible with no evidence of hysteresis.The new resonances that appear during the unfolding process are attributed to partially folded species, which are stabilized by the presence of the relatively hydrophobic methanol. Based on the temperature dependence of the chemical shifts and the relative areas of the various resonances, a detailed sequence of events has been proposed to describe the unfolding process. Key features include the initial general loosening of the two domains, the subsequent movement of the upper S-peptide region (residues 13 to 25) away from the main body of the protein, followed by partial separation of the sheet structure and full exposure of the N-terminal helix, leading to complete separation of the “winged domains”, and ultimately the loss of the residual sheet and helix structure.     

Chlorophyll isolation,structure and function: major landmarks of the early history of research in the Russian Empire and the Soviet Union

This paper covers major events of the early history of chlorophyll research in the Russian Empire and the Soviet Union from 1771 until 1952, when the modern period of studies on photosynthesis began in full swing. Short biographical sketches of key scientists, reviews of their major research contributions and some selected photographs are included. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of cycloheximide on protein and DNA synthesis and on the yields of chromosomal aberrations induced by DEB and ENU in vicia faba

Incubation of root tips in cycloheximide (CHM) at concentrations of 0.3–50 μg/ml inhibits the incorporation of [¹⁴C]leucine by 40–100% within 2 h. A depression in the incorporation of [³C]thymidine was observed after a 2-h incubation in CHM solution at 1 μg/ml.In root tips exposed for 2 h to CHM at 1 μ/ml the mitotic activity of cells was severely depressed within 15 h of recovery. Metaphases appearing after 20 h carried infrequent aberrations of the chromatid type. CHM at this concentration had no effect on the yield of aberrations induced by the alkylating agents diepoxybutane (DEB) and N-ethyl-N-nitrosourea (ENU) when applied as post-treatment.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system

High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin A_δ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H₂O solutions but not in ²H₂O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites.This proton resonance in chymotrypsin A_δ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N²(N-acetylalanyl)-N¹benzoylcarbazoyl-chymotrypsin A_δ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, ?18 parts per million, to a new high pH end point of ?14.4 parts per million (versus ?15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen.We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin A_δ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.     

On the decay of lateral phase separations in biological membranes

A theory for the decay of a lateral phase separation in a biological membrane has been developed based upon the edge decay of a population of circular molecular domains of uniform size. The theory has been applied to the case of vesicle fusion at a presynaptic membrane. It is shown that the efficiency of fusion decays exponentially in time with a rate constant $\text{solπ}\text{2τ}\text{N}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ which decreases as the rate at which bonds are broken within each domain (τ^?1) decreases and as the number of molecules within each domain (N) increases. Moreover, it has been speculated that this mechanism may offer in part an explanation for the slow, exponential decay during post-tetanic potentiation where it is known that the efficiency of neurotransmitter release at the presynaptic membrane is rate-controlling and decays exponentially.     

The inhibitive effects of steroid analogues in the binding of tritiated 5alpha-dihydrotestosterone to receptor proteins from rat prostate tissue.

The relationship between molecular structure and the binding potential of steroids to receptor proteins was investigated. Twenty-four selected steroids were studied in minced incubations of rat prostate tissue. Measurements of the inhibitory effects of the steroids on the binding of tritiated 5alpha-dihydrotestosterone to the receptor proteins were obtained in the 100,000 times g dialysed supernatant and the purified nuclear component of the prostate cells. The steroids that achieved the highest degree of inhibition were those compounds that exhibited a generally planar geometric shape and were known to possess potent androgenic activity. Several of the compounds were shown to possess a higher degree of inhibition than that of testosterone or 5alpha-dihydrotestosterone. The data is further supportive of the two step theory that necessitates the complexing of the free steroids to the receptor proteins in the cytosol before transport to the nuclei. Evidence is also suggestive of the presence of 17-esterase activity. The inhibitory effect of the steroids apparently involves the binding to the intracellular receptors and is not related to the uptake of 5alpha-dihydrotestosterone into the cell.     

Synthesis and characterization of core‐shell magnetic molecularly imprinted polymers for selective extraction of allocryptopine from the wastewater of Macleaya cordata (Willd) R. Br.

A novel type of magnetic molecularly imprinted polymers (MMIP) as the solid‐phase extraction sorbent was prepared, which can extract effectively the allocryptopine from the waster of Macleaya cordata (Willd) R. Br. In this study, MMIP was synthesized by using Fe₃O₄@SiO₂, 4‐vinyl‐pyridine, ethylene glycol dimethacrylate, and allocryptopine, and these ingredients worked as magnetic core, functional monomer, cross‐linker, and template, respectively. Concluded by the calculation of Gaussian 09 software, different ratio models of 4‐vinyl‐pyridine and allocryptopine were simulated, and the optimal ratio was 1:5 and the energy was ?2205.34 kJ/mol. Transmission electron microscopy, vibration sample magnetometry, X‐ray diffraction, Fourier transform infrared spectroscopy, and thermogravimetric analysis were used to determine the morphology and structure of MMIP. Furthermore, the results of adsorption experiments indicated that MMIP had high selectivity, excellent recyclability, and good adsorption performance (9.86 mg/g, 298 K). The adsorption process was consistent with the Langmuir adsorption isotherm (R² > 0.98, 298 K) and pseudo‐second‐order kinetics model (R² > 0.99, 298 K). After six times adsorption‐desorption experiments, the adsorption amount of MMIP only reduced to 8.5%. In the experiments of selective adsorption, MMIP has better adsorption properties for allocryptopine (ALL, C₂₁H₂₃NO₅) than those having the same functional group. The limit of detection (LOD) was 0.4 μg/mL. The relative standard deviation ranged from 0.09% to 0.72%. The recovery of allocryptopine in samples ranged from 93.60% to 106.19%. In addition, the synthesized complex had a certain adsorption effect on allocryptopine separating from the wastewater of Macleaya cordata (Willd) R. Br.