Purification and structural analysis of interferon

Studies with crude or partly purified interferon have provided a significant amount of structural information. However, complete biochemical characterization required purification to homogeneity. Earlier work on fractionation has met with many difficulties because interferon was available only in minute quantities. A scale-up of production, adaptation of multi-step purification schemes, use of high-resolution separation techniques and highly sensitive analytical methods have yielded pure interferons and hence many structural data. Specific activities, amino-acid compositions, partial sequences and structural homologies of many interferons were determined. Finally, cloned copy DNA (cDNA) fragments derived from specific interferon mRNA, as well as isolated interferon genes, have been sequenced and the data were used to elucidate complete sequences of many interferons with a high degree of confidence.     

Regulation of H Excretion : EFFECTS OF OSMOTIC SHOCK

Osmotic shock, a 15-minute plasmolysis followed by a 15-minute rehydration in the cold, is a nondestructive technique which inhibits fusicoccin-stimulated H⁺ excretion from oat mesophyll cells (Avena sativa L.). Osmotic shock also causes a loss of intracellular solutes and stimulates H⁺ uptake, but osmoregulation can still occur, and enhanced H⁺ uptake is observed only at low external pH. It is concluded that osmotic shock interferes directly with the excretion of H⁺ rather than affecting only H⁺ or counter ion uptake.     

Stimulation of dopamine synthesis and release by morphine and D-Ala2-D-Leu5-enkephalin in the mouse striatum in vivo

The effects of opiates on dopamine (DA) release and synthesis were assessed in the mouse striatum $\text{in vivo}$ by simultaneously measuring 3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels after inhibition of aromatic amino acid decarboxylase. This method was developed to assess stimulus-coupled changes in DA synthesis and release. Peripheral injections of morphine and intraventrcular injections of D-Ala²-Leu⁵-enkephalin elevated DOPAC levels, indicating that “opiates” stimulated DA release. Concomitantly, the rate of DA synthesis was increased. The effects were dose-dependent, saturable and antagonized by naloxone. When morphine and the enkephalin analog were given together in saturating doses, the effects of the two agents were not additive. Thus, the involvement of different receptors in the mediation of the effects of morphine and enkephalins could not be demonstrated.     

Responses of Avena coleoptiles to suboptimal fusicoccin: kinetics and comparisons with indoleacetic Acid

Proton excretion induced by optimal concentrations of indoleacetic acid (IAA) and fusicoccin (FC) differs not only in maximum rate of acidification but also in the lag before onset of H⁺ excretion and in sensitivity to cycloheximide. Because these differences might simply be a consequence of the difference in rate of proton excretion, FC and IAA have now been compared using oat coleoptiles (cv. Victory) under conditions where the rates of acidification are more similar, i.e. suboptimal FC versus optimal IAA. As the concentration of FC is reduced, the rate of H⁺ excretion decreases, the final equilibrium pH increases, and the lag before detectable acidification increases up to 7-fold. This enhanced lag period is not primarily a consequence of wall buffering, inasmuch as it persists when a low concentration of FC is added to sections which were already excreting H⁺ in response to IAA. An extended lag also occurs, upon reduction of FC levels, in the hyperpolarization of the membrane potential, before enhancement of O₂ uptake and before the increased rate of Rb⁺ uptake. The presence or absence of a lag is not a distinguishing feature between FC and IAA actions on H⁺ excretion and cannot be used to discriminate between their sites of action. In contrast, the insensitivity of FC-induced H⁺ excretion to cycloheximide, as compared with the nearly complete inhibition of this auxin effect by cycloheximide, persists even at dilute concentrations of FC. This seems to be a basic difference in H⁺ excretion by IAA and FC.     

Possibility of analytical application of the partition in aqueous biphasic polymeric systems technique

The partition behaviour of a number of ionic and nonionic surface-active substances in the dextran-polyethylene glycol system was examined. The strictly linear dependence of the logarithm of the partition coefficient on the alkyl chain length for a homologous series of nonionic surfactants provides a measure of the difference in the relative hydrophobicity between the two phases of the system, in terms of the free energy of transfer of a CH₂ group from the bottom phase to the top phase of the system. This difference is found to be altered in the presence of NaCl or KCl depending on the salt concentration. It is concluded that the influence of the salt composition of the system on the distributed solutes' behaviour may be due to the effect of the ions on the hydrophobicity difference between the phases.The partition of ionic amphiphiles is found to be dependent on the relative hydrophobicity of the compounds as well as on their charge. It is shown that at salt concentrations up to about 0.1 M NaCl the charged solute partition is determined by its charge as well as its relative hydrophobicity, in the presence of 0.1–0.2 M NaCl the substance distribution is highly dependent on its charge and slightly on its lipophility. At the salt concentrations above 0.2 M the solute partition is determined just by its hydrophobic character and seems to be totally independent of its charge. It is concluded that the partition technique can be used for analytical purposes. The method seems to be unique in providing quantitative information on the amphiphilic surface properties of the solutes being partitioned.     

The synthesis of apoproteins of very low density lipoproteins isolated from the Golgi apparatus of rat liver.

The incorporation of [3H]leucine in vivo into very low density lipoproteins (VLDL) from the rat hepatic Golgi apparatus and serum was studied. A Golgi-rich fraction isolated on a discontinuous sucrose gradient between 0.5 and 1.1 M was found to contain VLDL having common antigenic determinants with serum VLDL. The incorporation of the [3H]leucine into the Golgi VLDL and serum VLDL suggested a precursor-product relationship. Analysis of the apoproteins of the Golgi VLDL by polacrylamide gel electrophoresis revealed protein bands with similar mobility to those of serum VLDL, except that the former contained virtually no rapidly migrating peptides with the mobility of serum apo-C-II and apo-C-III. The pattern of incorporation of the [3H]leucine into the apoproteins was similar in VLDL from Golgi apparatus and serum, except for the absence of radioactivity in the area of the gel of Golgi apo-VLDL corresponding to apo-C-II and apo-C-III. The radioactive amino acid was incorporated predominantly into the Golgi apo-VLDL bands with similar mobility to apo-B and an apoprotein or group of apoproteins containing the arginine-rich peptide of serum VLDL. In vitro incubation of the Golgi VLDL with [3H]leucine-labeled HDL resulted in the acquisition of a number of proteins, including the rapidly migrating proteins. Administration of colchicine prior to the injection of [3H]leucine resulted in the appearance of gel bands and radioactivity in the apo-C-II and apo-C-III areas of Golgi apo-VLDL, suggesting that these can be acquired if secretion of VLDL is slowed or inhibited. The hepatic Golgi apparatus was then divided into fractions of predominantly forming face (GF3) or secretory granules (GF1). After polyacrylamide gel electrophoresis of the apo-VLDL from GF, no visible bands or incorporation of [3H]leucine was found in the region of apo-C-II or apo-C-III. However VLDL from GF1, showed visible and radioactive bands in the apo-C-II and apo-C-III area although they represented a much smaller proportion of the total apoprotein than was found in the corresponding serum apo-VLDL. In the isolated perfused liver the percentage incorporation of [3H]leucine into the rapidly migrating apoproteins of Golgi VLDL was considerably less than that found in the corresponding apoproteins of perfusate VLDL, where circulating C lipoproteins are virtually absent. The data indicate that nascent VLDL begins to acquire the C-II and C-III apoproteins during its passage through the Golgi apparatus but that the main acquisition occurs during or after secretion into the space of Disse.     

Synthesis of myosin heavy and light chains in muscle cultures

The weight ratio of myosin/actin, the myosin heavy chain content as the percentage of total protein (wt/wt), and the kinds of myosin light chains were determined in (a) standard muscle cultures, (b) pure myotube cultures, and (c) fibroblast cultures. Cells for these cultures were obtained from the breast of 11-day chick embryos. Standard cultures contain, in addition to myotubes, large numbers of replicating mononucleated cells. By killing these replicating cells with cytosine arabinoside, pure myotube cultures were obtained. The myosin/actin ratio (wt/wt) for pure myotube, standard muscle, and fibroblast cultures average 3.1, 1.9, and 1.1 respectively. By day 7, myosin in myotube cultures represents a minimum of 7% of the total protein, but about 3% in standard cultures and less than 1.5% in fibroblasts cultures. Myosin from standard cultures contains light chain LC1, LC2, and LC3, with a relative stoichiometry of the molarity of 1.0:1.9:0.5 and mol wt of 25,000, 18,000 and 16,000 daltons, identical to those in adult fast muscle. Myosin from pure myotubes exhibits light chains LC1 and LC2, with a molar ratio of 1.5:1.6. Myosin from fibroblast cultures possesses two light chains with a stoichiometry of 1.8:1.8 and mol wt of 20,000 and 16,000 daltons. Clearly, the faster migrating light chain, LC3, found in standard cultures is synthesized not by the myotubes but ty the mononucleated cells. In myotubes, both the assembly of the sarcomeres and the interaction between thick and thin filaments required for spontaneous contraction occur in the absence of light chain LC3. One set of structural genes for the myosin light and heavy chains appears to be active in mononucleated cells, whereas another set appears to be active in multinucleated myotubes.     

The myofibroblast: Paradigm for a mechanically active cell

Tissues lose mechanical integrity when our body is injured. To rapidly restore mechanical stability a multitude of cell types can jump into action by acquiring a reparative phenotype—the myofibroblast. Here, I review the known biomechanics of myofibroblast differentiation and action and speculate on underlying mechanisms. Hallmarks of the myofibroblast are secretion of extracellular matrix, development of adhesion structures with the substrate, and formation of contractile bundles composed of actin and myosin. These cytoskeletal features not only enable the myofibroblast to remodel and contract the extracellular matrix but to adapt its activity to changes in the mechanical microenvironment. Rapid repair comes at the cost of tissue contracture due to the inability of the myofibroblast to regenerate tissue. If contracture and ECM remodeling become progressive and manifests as organ fibrosis, the outcome of myofibroblast activity will have more severe consequences than the initial damage. Whereas the pathological consequences of myofibroblast occurrence are of great interest for physicians, their mechano-responsive features render them attractive for physicists and bioengineers. Their well developed cytoskeleton and responsiveness to a plethora of cytokines fascinate cell biologists and biochemists. Finally, the question of the myofibroblast origin intrigues stem cell biologists and developmental biologists—what else can you ask from a truly interdisciplinary cell?     

Mitochondrial haplogroup N1a phylogeography,with implication to the origin of European farmers

Background  

Tracing the genetic origin of central European farmer N1a lineages can provide a unique opportunity to assess the patterns of the farming technology spread into central Europe in the human prehistory. Here, we have chosen twelve N1a samples from modern populations which are most similar with the farmer N1a types and performed the complete mitochondrial DNA genome sequencing analysis. To assess the genetic and phylogeographic relationship, we performed a detailed survey of modern published N1a types from Eurasian and African populations.