Biosynthesis of the labelled rifamycin B in the presence of different 14C- and 3H-precursors

The results of studying the effectiveness of incorporation of the label from different 14C- and 3H-precursors into the molecule of rifamicin B during its biosynthesis are presented. The regularities of the label incorporation into the antibiotic composition as dependent on the time of the precursor addition were investigated. A radiochemically pure preparation of 14C-rifamicin B with specific radioactivity of 3 mcurie/mg was obtained.     

Matrix association of early- and late-replicating chromatin studied by single-cell electrophoresis

CHO-K1 cells were synchronized at the G(1)/S border by mitotic shake-off and aphidicolin incubation. Pulse-labeling with tritium was done at 30 min, 2 or 5 h into the S-phase, with chase incubations for different times in non-radioactive medium. The cells were subjected to neutral microelectrophoresis to extend the DNA into "comets," after which the label was visualized through autoradiography. At zero chase time, all label was positioned in the head. The displacement of label into the tails increased with time, reaching a maximum at about 5 h after the pulse. A lag phase of 2-3 h was observed for the early-labeled cells before the displacement started. Also, more label was released after overnight serum starvation, but this was reversed through a 3-h incubation at normal growth conditions. It was found that late-replicating chromatin is organized in larger domains than early-replicating chromatin, and DNA polymerase seems to be an important organizer. Early-replicating chromatin has other important attachments to the nuclear matrix, dependent on metabolic activity.     

Differential effects of cAMP and cGMP on in vitro epidermal cell growth.

Primary keratinocyte cultures free of dermal fibroblasts were used to investigate the effect of varying cyclic AMP (cAMP) concentrations on epidermal cell function. Addition of 10^?3, 10^?4 or 10^?5 M dibutyryl cAMP to plated cells (day 1) results by day 5 in a dose dependent increase of [³H]TdR incorporation into DNA as determined by increases in both the labeling index and incorporation of ³H label into an isolated DNA fraction. 8-Bromo cAMP, another cAMP analogue, likewise induced keratinocyte proliferation. The proliferative response was dose and time dependent, and 5- to 6-fold increases in ³H label incorporated into DNA were seen at day 6, 8 and up until day 15 of culture. Moreover, elevation of cellular cAMP by addition of cholera toxin, an irreversible stimulator of adenylate cyclase, also demonstrated a time dependent stimulation of [³H]TdR uptake into DNA and increased the labeling index. Specific histochemical staining for keratinaceous protein (Kreyberg technique) demonstrated that elevated cAMP levels also enhance the production of specialized (differentiated) epidermal cells. Determination of the level of cAMP and cyclic GMP (cGMP) by RIA of partially purified fractions of the cultures revealed that addition of 8-bromo cAMP or cholera toxin to the cultures increased the levels of cAMP but not of cGMP. Addition of 8-bromo cGMP to the keratinocytes on day 1 at concentrations of 10^?6, 10^?7 or 10^?8 M had no effect on culture proliferation on days 4, 6 and 8, although qualitative changes in the electron microscopic pattern of the culture stratification and specialization were observed. The results indicate (1) both large and moderate increases in cellular cAMP levels induce keratinocyte culture proliferation and specialization in the absence of fibroblasts or dermal influences, (2) the quantitative enhancement of keratinocyte growth and specialization occurs without apparent participation of cGMP, (3) cGMP may be a qualitative effector of epidermal cell differentiation.     

Photoaffinity labeling of the 1,25-dihydroxyvitamin D-3 receptor.

Underivatized 1,25-dihydroxy[26,27-3H]vitamin D-3 was successfully used to photoaffinity label the 1,25-dihydroxyvitamin D-3 receptor. The covalent incorporation of tritium into the receptor protein was induced by ultraviolet irradiation of the receptor-1,25-dihydroxy[26,27-3H]vitamin D-3 complex in crude pig intestinal nuclear extract. The amount of incorporated label increased with increasing time of irradiation and was dependent on light of wavelengths 220-280 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography were used to demonstrate that label was incorporated primarily into the 1,25-dihydroxyvitamin D-3 receptor. In addition, the label incorporation was eliminated by competition with a 100-fold excess nonradioactive 1,25-dihydroxyvitamin D-3, indicating that the label was specific for the steroid binding site. Since 1,25-(OH)2[26,27-3H]vitamin D-3 is readily available and requires no special precautions for its preparation and handling, it should be a useful photoaffinity label for future studies of the receptor.     

Angiotensin II acts through the angiotensin 1a receptor to upregulate pendrin

Pendrin is an anion exchanger expressed in the apical regions of B and non-A, non-B intercalated cells. Since angiotensin II increases pendrin-mediated Cl(-) absorption in vitro, we asked whether angiotensin II increases pendrin expression in vivo and whether angiotensin-induced hypertension is pendrin dependent. While blood pressure was similar in pendrin null and wild-type mice under basal conditions, following 2 wk of angiotensin II administration blood pressure was 31 mmHg lower in pendrin null than in wild-type mice. Thus pendrin null mice have a blunted pressor response to angiotensin II. Further experiments explored the effect of angiotensin on pendrin expression. Angiotensin II administration shifted pendrin label from the subapical space to the apical plasma membrane, independent of aldosterone. To explore the role of the angiotensin receptors in this response, pendrin abundance and subcellular distribution were examined in wild-type, angiotensin type 1a (Agtr1a) and type 2 receptor (Agtr2) null mice given 7 days of a NaCl-restricted diet (< 0.02% NaCl). Some mice received an Agtr1 inhibitor (candesartan) or vehicle. Both Agtr1a gene ablation and Agtr1 inhibitors shifted pendrin label from the apical plasma membrane to the subapical space, independent of the Agtr2 or nitric oxide (NO). However, Agtr1 ablation reduced pendrin protein abundance through the Agtr2 and NO. Thus angiotensin II-induced hypertension is pendrin dependent. Angiotensin II acts through the Agtr1a to shift pendrin from the subapical space to the apical plasma membrane. This Agtr1 action may be blunted by the Agtr2, which acts through NO to reduce pendrin protein abundance.     

The use of (3H) thymidine-adsorbed on activated charcoal for the study of non-premitotic DNA synthesis in newborn rat hepatocytes

DNA synthesis in newborn rat hepatocytes was studied in the first three days of life by means of repeated injections of (3H) thymidine. One group of animals was treated with the label adsorbed on activated charcoal (experimental group) and another group (controls) was given the label diluted in saline. The specific activity of DNA was higher in control group, but its increase was not linear with time; in the experimental group, the radioactivity was lower, but its increase with time was linear. The percentage of labeled nuclei was higher in the experimental animals than in the controls and increased linearly with time. The average number of grains/nucleus was considerably smaller in the experimental group than in the controls, in which also the percentage of labeled cells showed considerable variations during the first three days of life. It is concluded that activated charcoal adsorption increases label availability with time and, by keeping a lower label concentration in the pool, reduces the risk of radiation damage.     

A simple method for determination of rotational correlation times and separation of rotational and polarity effects from EPR spectra of spin-labeled biomolecules in a wide correlation time range

A method using nitroxide radical spin labels for determining both the isotropic rotational correlation time tau R and the environmental polarity of the label is described. By means of a least square fitting method, the values of an effective hyperfine tensor A' and of an effective g value tensor g' of randomly oriented spin labels are determined from X-band EPR spectra on the basis of an effective time-independent Hamiltonian. The traces of the tensors deliver the information about the environmental polarity of the label and are not dependent on the rotational correlation time tau R. A new averaging parameter S (tau R), calculated on the basis of the principal values of the tensor A', permits the evaluation of the rotational correlation time tau R in a very wide time range between 10(-10) and 10(-6) s. An application of this method to spin-labeled methemoglobin over a large temperature range and in environments of different polarity is discussed.     

Changes in axonal transport of phospholipids in the regenerating goldfish optic system

Changes in axonally transported phospholipids of regenerating goldfish optic nerve were studied by intraocular injection of [2-³H]glycerol 9 days and 16 days after nerve crush at 30°C. The four major glycerophospholipids all showed substantial increases in transported radioactivity above non-regenerating controls at both time points, these being maximal (15- to 35-fold) in the optic nerve-tract at 9 days and about half as great at 16 days. In the contralateral optic tectum transported label increased 6- to 13-fold at 9 days and 10- to 25-fold at 16 days in the various glycerophospholipids. While all glycerophospholipids showed absolute increases in both tissues, PS and PI increased relatively more, especially in the tectum. The regeneration-associated increases in transported label of all glycerophospholipids were larger than those previously demonstrated for gangliosides and glycoproteins in the same system. Special Issue dedicated to Dr. Eugene Kreps.     

Transmethylation of Sterols in Aerobically Adapting Saccharomyces cerevisiae

The transmethylation of methyl-(14)C-methionine and methyl-(14)C-adenosylmethionine into the nonsaponifiable lipids of anaerobically grown yeast during adaptation to aerobic conditions was investigated. The rate and extent of methyl transfer increased with aeration time and was dependent upon the presence of a fermentable carbon source and O(2). Methionine and adenosylmethionine uptake rates increased in adaptation buffer but did not seem to be the rate-limiting factor for transmethylation under the conditions studied. Thinlayer chromatography of the nonsaponifiable fraction after exposure to label showed the labeled product to be ergosterol. Samples taken after short-term exposure to label were composed of two labeled steroidal products, one with kinetics of an ergosterol precursor.     

Membrane lipid fluidity as rate limiting in the concanavalin A-mediated agglutination of pyBHK cells.

The initial rate of concanavalin A-mediated agglutination of polyoma transformed Baby Hamster Kidney (pyBHK) cells follows Arrhenius kinetics. There is a smooth decrease in the agglutination rate from 37 degrees C to 22 degrees C with an activation energy of 11.8 +/- 0.2 kcal/mol in this region. There is a sharp decrease in agglutination rate below 22 degrees C. The addition of 0.1 mM 1,3-di-tert-2-hydroxyl-5-methylbenzene, a lipid perturber, increases the agglutination rate by a factor of two and increases the membrane lipid fluidity as determined by the spin label method. The rotational correlation time of the spin label 2N14 (2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide) was measured. The sum of the enthalpy of activation of rotational diffusion and the enthalpy of activation of translational diffusion is very nearly equal to the enthalpy of activation of agglutination. This is consistent with the rate limiting step of agglutination being receptor diffusion, which is probably limited in pyBHK cells by membrane lipid fluidity.     

Changes in lipid and protein synthesis during spermatozoan development and thoracic tissue maturation inDrosophila hydei

Summary The synthesis of protein and lipid was studied in the testis and thoracic tissues ofDrosophila hydei by radioisotope incorporation. The formation and accumulation of late stage gametes in the testes during maturation was accompanied by more than 4-fold increases in both lipid and protein synthesis rates. Though there was an 8-fold increase in the rate of protein synthesis during thoracic tissue maturation, the rate of lipid synthesis little more than doubled. By comparison the rates of protein synthesis were high in mature testis and thoracic tissues, whereas the rate of lipid synthesis was high in the mature testis but low in both immature and mature thoracic tissues.Label incorporation from pyruvate and L-alanine into protein in thoracic tissue appeared to be highly dependent on Krebs cycle activity, whereas pyruvate and L-alanine label incorporation into protein in the testis was primarily from L-alanine and its derivatives. Both pyruvate and L-alanine were decarboxylated prior to label incorporation into lipid in the testis and thoracic tissue.This work was supported by National Science Foundation Grant GB-13393.     

Utilization of uridine in developing ovarioles of the kelp fly, Coelopa

The fate of ³H-uridine in ovaries of the kelp fly C. frigida after injection into the female is followed by autoradiography and by thin layer chromatography over a time period of 4 hr. Autoradiography demonstrates that the label is incorporated initially into nuclear material in the nurse cells and follicle cells, and is transported from the nuclei into the cytoplasm within the first hour after injection. Reduced incorporation into nuclear material, after the first 2 hr following injection, is interpreted as depletion of exogenous precursor. Only a very small amount of label is found over the nuclei after 4 hr when the nurse cell cytoplasm is densely labeled. This indicates that most of the label is retained in long-lived products and is not available for the salvage pool.Analysis of the relative distribution of radioactivity in derivatives over a 4 hr time span corroborates the autoradiographic observations. The amount of radioactivity present in uridine, cytidine, and sugar nucleotides increases rapidly, though with different velocity for each nucleotide. The pattern of utilization during the first 2 hr, particularly of UTP, suggests preferential utilization of the exogenous precursor. After depletion of the salvage pool, labeled precursors provide low levels of specific activity for the nucleotide pool. The macromolecules synthesized after this time period do not show radioactivity.     

The effect of electrical stimulation and ouabain on the uptake and efflux of l-[U-14C]valine in chopped tissue from guinea-pig cerebral cortex

1. Chopped tissue from guinea-pig cerebral cortex carried out an energy-dependent accumulation of l-[(14)C]valine. 2. The uptake was dependent on the extracellular concentration of Na(+) and was markedly inhibited by ouabain (20mum). The extent of the inhibition of uptake by ouabain was also Na(+)-dependent. 3. The accumulation of labelled valine was not directly dependent on the ATP and creatine phosphate contents of the slices. 4. Electrical stimulation increased the rate of [(14)C]valine uptake at first but ultimately led to a net loss of the label so that the amount of label present in the tissue was lower than in the controls. 5. The rate of loss of label during prolonged stimulation was dependent on the extracellular concentration of Na(+). 6. The efflux of labelled valine from slices preloaded at 164mm-Na(+) was studied at 164, 80 and 40mm-Na(+) with and without electrical stimulation or ouabain. 7. Lowering the Na(+) concentration or adding ouabain increased the rate of efflux. 8. Electrical stimulation had little effect on the rate of efflux at first but ultimately led to a more complete loss of label from the tissue than occurred in the control. A kinetic analysis of the efflux curves was attempted.     

Rapid phytochrome-mediated changes in the uptake by bean roots of sodium acetate [1-14C] and their modification by cholinergic drugs

Summary 4 min of red light increases the uptake of sodium acetate[1-¹⁴C] by excised, etiolated secondary roots of Phaseolus aureus Roxb. 4 min of far-red light reveres this effect. AMO-1618, which inhibits acetylcholinesterase activity, enhances the red-light effect, while d-tubocurarine, which blocks the animal acetylcholine receptor, inhibits it. Red light also increases basipetal translocation of the label. When the metabolic fate of the label was determined in dark-held roots, 36% of the label remained as acetate, 48% evolved as [¹⁴C]CO₂, 3% partitioned with acetylcholine, and 3% effluxed from the roots. The rest of the label was associated with the coarse residue left after extraction. The major effect of red light was to increase the uptake of the label in the acetate fraction.We interpret these observations to mean that the phytochrome mechanism immediately causes an increase in uptake of the label during brief irradiation with red light. Because of our previous demonstration that both red light and acetylcholine increase respiration, it is probable that the increased absorption of the label is a process requiring respiratory energy. These data support the concept of phytochrome as a membrane-bound functional system that in bean roots is mediated by the acetylcholine mechanism.Abbreviations ACh Acetylcholine - AChE acetylcholinesterase - ATP adenosine triphosphate - AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride - TPB tetraphenyl boron - D darkness - FR far-red - R red     

Proton involvement with the light-induced hindrance of spin label motion in the lumen of spinach thylakoids

The light-induced hindrance of spin label motion increases linearly with light intensity. However, it has not been possible to unambiguously demonstrate light saturation due to the very high rates of spin label reduction at high light intensity. The light-induced hindrance of spin label motion may be mimicked in the dark by subjecting thylakoids to appropriately low pH regimes. Uncouplers such as gramicidin-D and methylamine reduce the light-induced hindrance to dark levels as does ethylenedinitrilotetraacetate (EDTA) treatment. Valinomycin plus KCl which destroys the electric potential is only partially effective in reducing the light-induced hindrance. These results indicate that protons in the aqueous lumen of the thylakoids are closely involved with the observed light-induced hindrance of spin label motion.     

Receptors for calcitonin gene-related peptide on the rat liver plasma membranes

Specific binding sites for calcitonin gene-related peptide (CGRP) were identified in the rat liver plasma membrane. The binding of 125I-[TyrO]rat CGRP to rat liver plasma membrane was time dependent, saturable and reversible. Scatchard analysis of the data revealed a single class of binding sites with apparent dissociation constant of 260.8 pM and a maximal binding capacity of 26.6 fmol/mg of protein. Rat, chick, and human CGRP and their synthetic analogues inhibited label binding in a dose-dependent manner with relative potencies as follows; chick greater than rat greater than human greater than [TyrO]rat CGRP. Salmon, human and [Asu1'7]eel calcitonin also inhibited label binding but only at higher concentrations. These results clearly indicate the presence of specific binding sites for CGRP in rat liver plasma membrane and suggest that CGRP has possible biological actions on the rat liver.     

Spin label method reveals barnase-barstar interaction: a temperature and viscosity dependence approach

Spin labeling was used to study the protein-protein interaction between the enzyme barnase (Bn) and its inhibitor barstar (Bs). A mutant of barstar (C40A), which contains only one cysteine, C82, located near the Bn-Bs contact region, was selectively modified by two spin labels having different lengths and structures of the flexible tether. The formation of a strong protein complex resulted in significant restriction of spin label mobility at the C82 residue of barstar, as indicated by notable changes in the recorded EPR spectra. The dependence of the separation between broad outer peaks of the EPR spectra on viscosity at constant temperature was used to evaluate the order parameter S and the rotational correlation time tau (a temperature-viscosity dependence approach). The order parameter S, which characterizes fast reorientation of a spin label relative to the protein molecule, sharply increases and approaches unity when Bs binds to Bn. In addition, formation of a Bs-Bs complex was observed; it is also accompanied by restriction of spin label mobility. At the same time, the rotational correlation times tau of spin-labeled Bs, its complex with Bn, and the Bs dimer in solution agree well with their molecular masses. This indicates that barstar, its complex with barnase, and barstar dimer are rigid protein entities. The described approach is suitable for studying any spin-labeled macromolecular complexes.     

SWATH enables precise label‐free quantification on proteome scale

MS‐based proteomics has emerged as a powerful tool in biological studies. The shotgun proteomics strategy, in which proteolytic peptides are analyzed in data‐dependent mode, enables a detection of the most comprehensive proteome (>10 000 proteins from whole‐cell lysate). The quantitative proteomics uses stable isotopes or label‐free method to measure relative protein abundance. The isotope labeling strategies are more precise and accurate compared to label‐free methods, but labeling procedures are complicated and expensive, and the sample number and types are also limited. Sequential window acquisition of all theoretical mass spectra (SWATH) is a recently developed technique, in which data‐independent acquisition is coupled with peptide spectral library match. In principle SWATH method is able to do label‐free quantification in an MRM‐like manner, which has higher quantification accuracy and precision. Previous data have demonstrated that SWATH can be used to quantify less complex systems, such as spiked‐in peptide mixture or protein complex. Our study first time assessed the quantification performance of SWATH method on proteome scale using a complex mouse‐cell lysate sample. In total 3600 proteins got identified and quantified without sample prefractionation. The SWATH method shows outstanding quantification precision, whereas the quantification accuracy becomes less perfect when protein abundances differ greatly. However, this inaccuracy does not prevent discovering biological correlates, because the measured signal intensities had linear relationship to the sample loading amounts; thus the SWATH method can predict precisely the significance of a protein. Our results prove that SWATH can provide precise label‐free quantification on proteome scale.     

Preparation and properties of spin-labeled lecithin-cholesterol liposomes

Summary Lecithin-cholesterol vesicles of various compositions containing membrane-bound spin-labeled cholestane can be prepared by appropriate choice of initial concentrations of components during sonication. Increasing incorporation of spin label increases incorporation of cholesterol and decreases incorporation of lecithin, with the result that liposomes with cholesterol-lecithin molar ratios larger than 2 can be obtained. Besides associating with cholesterol-lecithin complexes in the liposome, the spin label seems to associate with cholesterol. Changes of the paramagnetic resonance spectrum of the liposome-bound spin label due to changes in liposomal cholesterol and spin label mole fractions — assessed by three parameters — can be used in cell-liposome interaction studies.     

Effect of retinoic acid on cAMP dependent protein phosphorylation in psoriatic fibroblasts

Retinoic acid treatment of psoriatic fibroblasts increases the activity of cyclic AMP dependent protein kinase. In this study we report that retinoic acid treatment of cultured psoriatic fibroblasts modifies their subsequent cAMP dependent protein phosphorylation. In the soluble fraction of normal fibroblasts cAMP clearly enhances the in vitro phosphorylation of proteins of MW 37,49,54,56,68,83 kD while retinoic acid treatment of the same cells results in a decrease of the cAMP dependent phosphorylation of the first five of the same proteins. In contrast, in psoriatic fibroblasts from psoriatic patients retinoic acid either has no effect or increases the cAMP dependent phosphorylation of some of these proteins. Moreover the phosphorylation of a protein of MW 54 kD, undetectable in untreated psoriatic cells, is more phosphorylated in the presence of cAMP after retinoic acid treatment. The appearance of this phosphorylated proteins is time dependent and dose dependent upon the addition of retinoic acid. These in vitro phosphorylation results suggest that retinoic acid treatment of psoriatic fibroblasts change the level of cAMP dependent phosphorylation of some cytosolic proteins. These specific phosphorylations could be implicated in a variation of cell functions.