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Mavacamten (MYK-461) is a small-molecule allosteric inhibitor of sarcomeric myosins being used in preclinical/clinical trials for hypertrophic cardiomyopathy treatment. A better understanding of its impact on force generation in intact or skinned striated muscle preparations, especially for human cardiac muscle, has been hindered by diffusional barriers. These limitations have been overcome by mechanical experiments using myofibrils subject to perturbations of the contractile environment by sudden solution changes. Here, we characterize the action of mavacamten in human ventricular myofibrils compared with fast skeletal myofibrils from rabbit psoas. Mavacamten had a fast, fully reversible, and dose-dependent negative effect on maximal Ca2+-activated isometric force at 15°C, which can be explained by a sudden decrease in the number of heads functionally available for interaction with actin. It also decreased the kinetics of force development in fast skeletal myofibrils, while it had no effect in human ventricular myofibrils. For both myofibril types, the effects of mavacamten were independent from phosphate in the low-concentration range. Mavacamten did not alter force relaxation of fast skeletal myofibrils, but it significantly accelerated the relaxation of human ventricular myofibrils. Lastly, mavacamten had no effect on resting tension but inhibited the ADP-stimulated force in the absence of Ca2+. Altogether, these effects outline a motor isoform-specific dependence of the inhibitory effect of mavacamten on force generation, which is mediated by a reduction in the availability of strongly actin-binding heads. Mavacamten may thus alter the interplay between thick and thin filament regulation mechanisms of contraction in association with the widely documented drug effect of stabilizing myosin motor heads into autoinhibited states.

Kraft’s work focuses on the mechanisms that regulate autophagy in response to nutrient availability.

Permeability of the cardiac cell membrane to choline ions was estimated by measuring radioactive choline influx and efflux in cat ventricular muscle. Maximum values for choline influx in 3.5 and 137 mM choline were respectively 0.56 and 9 pmoles/cm2·sec. In 3.5 mM choline the intracellular choline concentration was raised more than five times above the extracellular concentration after 2 hr of incubation. In 137 mM choline, choline influx corresponded to the combined loss of intracellular Na and K ions. Paper chromatography of muscle extracts indicated that choline was not metabolized to any important degree. The accumulation of intracellular choline rules out the existence of an efficient active pumping mechanism. By measuring simultaneously choline and sucrose exchange, choline efflux was analyzed in an extracellular phase, followed by two intracellular phases: a rapid and a slow one. Efflux corresponding to the rapid phase was estimated at 16–45 pmoles/cm2·sec in 137 mM choline and at 1.3–3.5 pmoles/cm2·sec in 3.5 mM choline; efflux in 3.5 mM choline was proportional to the intracellular choline concentration. The absolute figures for unidirectional efflux were much larger than the net influx values. The data are compared to Na and Li exchange in heart cells. Possible mechanisms for explaining the choline behavior in heart muscle are discussed.

1. In the dark adapted Mnemiopsis, mechanical stimulation causes luminescence along the eight rows of paddle plates. The tactile receptors for this reaction lie only in the paddle plate rows, and are connected only longitudinally along these rows. 2. The tactile receptors for ciliary and muscular movement are distributed generally over the surface and are connected by a nerve net. 3. Luminescence may occur at 3°C. provided the animal has been kept sufficiently long at that temperature. Ciliary action goes on at – 0.6°C. 4. Luminescent paper made by spreading the luminescent secretion of Mnemiopsis on filter paper, yields the following effects. The paper shows luminescence in solutions of K2SO4, KCl, MgSO4, SrCl2, CaCl2; no luminescence in NaCl, MgCl2. Changes in pH value of salt solutions between pH 6 and 8 do not affect the phenomenon. Illumination of the paper with strong light for longer time than necessary to suppress luminescence in the living animal has no effect on the subsequent luminescence of the paper. Hence in the animal, light affects luminescence through the photoreceptor system; the nervous system carries the impulse to the luminescent organs. 5. The power of luminescence of the animal is suppressed by sufficiently intense light, the relation between the intensity and the time requisite being expressed by the equation for the Bunsen-Roscoe photochemical law, namely, I · t = K. 6. It is suggested that the reaction scheme involved in luminescence is of the following form See PDF for Equation in which A is the luminescent substance in the resting, dark adapted animal, L is the light-giving decomposition product, and D is a product which does not yield light. 7. The luminescent substance receives double innervation and the character of the decomposition is determined by the type of nerve fiber stimulated.

1. A process of extraction and assay, which combines the features of several existing methods, is described for the lytic materials which can be obtained from blood, plasma, serum, and tissues. At least two alcohol-soluble substances, one ether-soluble ("soap-like") and the other insoluble in ether in the cold ("lysolecithin-like"), can be obtained from preincubated blood, plasma, or serum. The hemolytic activity (or concentration) of the soap-like lysin obtained from blood is greater than that of the lysolecithin-like substance, but for plasma and serum the reverse is true, i.e. the red cells are involved in the production of the soap-like lysin, and probably supply some of it when acted upon by enzymes contained in plasma and serum. Preincubation of the blood or plasma increases the yield of lysin two- or threefold, and small quantities of both soap-like and lysolecithin-like lysins can be obtained from unpreincubated blood or plasma. 2. The soap-like lysins obtained from preincubated mouse liver are some 5 to 15 times as active as, or occur in some 5 to 15 times the concentration of, those obtained from blood or plasma. The lysolecithin-like lysins of preincubated liver are about twice as active as, or occur in about twice as great concentration of, those obtained from blood. Because of the shape of the time-dilution curve for these lysins, the relations between their activities, or concentrations, are often quite different from those which one would anticipate if one were to consider only the times required for the production of hemolysis. 3. Paper chromatography can be used to separate the soap-like and the lysolecithin-like lysins obtainable from small quantities of preincubated mouse liver homogenates or preincubated mouse blood. The presence of lysins is detected by their effect on the red cells of a suspension as it wets the paper. Various technical procedures for separating lytic components and for demonstrating that they move on the paper along with protein components are described. 4. Paper strip electrophoresis can be used to show that the supernatant fluid of a preincubated mouse liver homogenate contains at least two protein components and at least two lytic components, not very closely associated in their electrophoretic behavior. 5. Observations on the physical nature of the alcohol- and ether-soluble lysin point to its having a soap-like character. Its activity, as well as that of the lysolecithin-like lysin, is inhibited by cholesterol, by lecithin, and by various fractions of serum. Some of these effects have been studied quantitatively. The most inhibitory of the protein fractions are those which contain lipoproteins; i.e., II + III and IV + V.

When I(131)-albumin is given intravenously to rabbits, the radioactive breakdown products that are released into the plasma and urine can be extracted into acetone. Paper chromatography and paper electrophoresis show that about 80 per cent of these are I(131)-iodide and the remainder are organic I(131)-iodine compounds. When I(131)-iodide is given to rabbits taking iodide in their drinking water, the radioactivity is quantitatively excreted, without being accumulated in the tissues and without becoming attached to the plasma proteins. The rate of excretion can be defined by a first order rate process with a rate constant, a, ranging between 1 and 3day(-1). The organic I(131)-iodine compounds liberated during the metabolism of I(131)-albumin can be closely matched by a mixture of the organic I(131)-iodine compounds liberated during the metabolism of I(131)-monoiodotyrosine, I(131)-diiodotyrosine, and the amino acids released by digestion from I(131)-albumin. These organic I(131)-iodine compounds are not accumulated in the body and their radioactivity does not become attached to the plasma proteins. Their radioactivity is excreted as fast or faster than that of I(131)-iodide, and, to a satisfactory approximation, the same equations describing the excretion of I(131)-iodide with the same constants may be used for describing the excretion of the organic I(131)-iodine. These results permit improved estimates of the distribution and catabolism of I(131)-albumin.

Protection of bacteriophage T1 against x-rays was tested in the presence of concentrations of (NH4)2SO4 ranging from 10–6 M to saturation (4.26 M). Survival of T1 in concentrations of 10–6 to 10–3 M after irradiation did not differ significantly from survival in distilled water after irradiation. From 10–3 M to 10–1 M there was a steep rise in survival, with a leveling off as the concentration approached saturation, giving over-all a 2,000-fold increase in survival. The mechanism of salting out protection in these experiments is apparently due chiefly to dehydration, which protects the virus particles against the indirect effects of x-irradiation. Postirradiation effects, tested by the inactivation of phage added to irradiated media, approach in magnitude the effects obtained by irradiation of the phage particles themselves in the various solutions. Filter paper adsorption analyses indicate a close correlation between concentrations of (NH4)2SO4, ability of the filter paper to adsorb phage, and protection against x-rays, both during and after irradiation.

Wense has reported the isolation of crystalline adrenalin from larvae of the beetle Tenebrio molitor. An attempt to repeat his procedures failed to yield any evidence of adrenalin. Analysis of mealworm extracts by paper chromatography and colorimetric means also yielded no indication of adrenalin, though adrenalin added to mealworms can be detected by these procedures in amounts less than 1 mg. per 100 gm. Mealworm extracts reveal on the paper chromatogram the presence of two other orthodiphenols, neither of which appears to be dopa but which may be 3,4-dihydroxyphenyllactic acid and 3,4-dihydroxyphenylacetic acid, compounds recently isolated elsewhere from this organism.

Two unknown radioactive areas appeared after radioautography and two dimensional paper chromatography of culture medium in which Escherichia coli was grown. These materials were studied by paper chromatography and paper electrophoresis of several derivatives and identified as ethionine and ethionine sulfone, the latter an artifact. Chromatographic coincidence of the unknowns and their derivatives with authentic materials establishes the identification. Ethionine was found in cellular extracts and in the growth media of Escherichia coli, Bacillus megaterium, Pseudomonas aeruginosa, and Aerobacter aerogenes but not in Scenedesmus, Saccharomyces cerevisiae, or bovine lymphosarcoma cells. Ethionine was synthesized by resting E. coli cultures from radioactive sulfate and from radioactive methionine. Growing cells labeled ethionine within 1 minute after addition of radioactive sulfate to cultures. Levels of radioactivity in ethionine increased with time. No incorporation of this amino acid could be detected in the cellular proteins formed under the conditions of this study.

Data on barium-zinc filtrates of liver homogenates and of boiled liver indicate that the free glucose content of liver exceeds the blood glucose level. For instance, for boiled liver, the glucose level is 10.1, compared with a blood level of 5.4 mM/kg. Method of preparation of the tissue is important for the interpretation of the final results, as has been shown in appropriate control experiments. Various methods including paper chromatography were used to show that the reducing substance in liver is glucose. The relation of the high glucose content of liver to hexokinase activity, phosphate activity, and to glucose transport between liver cell and blood is discussed.

A study of the metabolic pathways of H(3)-thymidine utilization in buds of Lilium longiflorum and root tips of Vicia faba was undertaken in order to obtain information that might explain the binding of H(3) from H(3)-thymidine in the cytoplasm of these plants. H(3)-thymidine was administered for various periods of time, the tissues were fixed and processed in the manner routinely used in preparation for sectioning and autoradiography, and the radioactivity removed in this way from the tissues was determined. It was found that the ethanol/acetic acid fixative contained the major portion of the radioactivity. Analysis of this extract by paper chromatography showed that the radioactivity was distributed among various degradation products of thymidine, principally beta-ureidoisobutyric acid and beta-aminoisobutyric acid. Time course experiments with Vicia showed that these degradation products rapidly appeared in the tissue during incubation with H(3)-thymidine, while H(3)-thymine appeared in the incubation medium. Preliminary studies indicated that Vicia root tips incubated with H(3)-dihydrothymine for 24 hours would bind a small amount of H(3) non-specifically in the cells. It seems unlikely that utilization of degradation products of H(3)-thymidine is sufficient to explain labeling which is concentrated in the cytoplasm.

Mature Arbacia eggs were extracted with cold dilute perchloric acid, the extract concentrated, and the concentrate digested in hot perchloric acid. Thymine was recovered from the digest by paper chromatography, and the amount per egg found to be about 5 times the amount per sperm. This was the amount expected from previous experiments and is believed to represent all or almost all of the thymine in the egg. The result supports previous observations that DNA is absent from the mature egg although present in the nucleus of the egg in the germinal vesicle stage. No thymine could be recovered from a similar extract of 5,000 times as many sperm of the same species. The observations are consistent with the theory that DNA and its derivatives act as metabolic antagonists of the corresponding ribose compounds.

The fine structure of integumental erythrophores and the intracellular location of pteridine and carotenoid pigments in adult goldfish, Carassius auratus, were studied by means of cytochemistry, paper and thin-layer chromatography, ionophoresis, density-gradient centrifugal fractionation, and electron microscopy. The ultrastructure of erythrophores is characterized by large numbers of somewhat ellipsoidal pigment granules and a well-developed system of tubules which resembles endoplasmic reticulum. The combined morphological and biochemical approaches show that pteridine pigments of erythrophores are located characteristically in pigment granules and are the primary yellow pigments of these organelles. Accordingly, this organelle is considered to be the "pterinosome" which was originally found in swordtail erythrophores. Major pteridines obtainable from goldfish pterinosomes are sepiapterin, 7-hydroxybiopterin, isoxanthopterin, and 6-carboxyisoxanthopterin. Density-gradient fractions indicate that carotenoids are mostly associated with the endoplasmic reticulum. Both tyrosinase and possibly a tyrosinase inhibitor containing sulfhydryl groups are present in the pterinosome. The possible existence of a tyrosinase inhibitor is suggested by the marked increase of tyrosinase activity upon the addition of iodoacetamide or p-chloromercuribenzoic acid. In the light of their fine structure, pigmentary composition, and enzymatic properties, the erythrophores and pterinosomes are discussed with respect to their probable functions and their relationship to melanophores.

Randomly dividing cultures of Saccharomyces cerevisiae were briefly exposed to radioactive adenine and then treated successively with dilute acid, ribonuclease, buffered formaldehyde, and NaOH. This treatment was shown to remove virtually all the radioactivity of the labelled cells other than that in DNA. Thus, in subsequent autoradiographs, only cells which had been synthesizing DNA during exposure to the precursor were labelled. The ages of these individuals within the cell cycle were estimated by measuring their sizes. This revealed that incorporation into DNA occurred almost exclusively during the first quarter of the cell cycle, starting with the initial appearance of the bud. This behaviour agreed closely with that of cells growing in artificially synchronized cultures.

C5-deficient mice differed from C5-sufficient mice both quantitatively and qualitatively in C5 protein, C5 mRNA, and the C5 gene. C5-deficient protein was present as decreased amounts of an unprocessed, single-chain precursor. C5-deficient mRNA was decreased in amount and present in two forms, the smaller of which was the same as the single form in normal cells. Nuclei from both normal and deficient cells contained the larger form of C5 mRNA, and C5-deficient DNA demonstrated differences from the normal pattern on Southern analysis for two restriction enzymes. These data suggest that the primary transcript of the C5-deficient gene is abnormal, retarding the processing of the C5 mRNA, and that the C5-deficient mRNA codes for an abnormal protein.

Monoclonal antibodies were prepared to identify molecular components specific to the mitotic apparatus of sea urchin eggs. The mitotic apparatus or asters induced within unfertilized eggs by taxol treatment were isolated from Strongylocentrotus purpuratus and used for immunization of mice. After fusion with spleen cells, the supernatant of hybridomas were screened in two stages by indirect immunofluorescence staining, first on isolated sea urchin mitotic spindles in 96-well microtiter plates to identify rapidly potential positive hybridomas, and second, on whole mitotic eggs on coverslips to distinguish between spindle-specific staining and adventitious contamination. Two hybridomas, SU4 and SU5, secreted antibodies reactive to microtubule-containing structures in eggs during the course of development. They preferentially stained the centrosphere both in isolated mitotic apparatus and in whole metaphase eggs, which was further confirmed by staining the isolated centrospheres with these antibodies. SU4 recognized a major 190-kD polypeptide on immunoblots as well as a species at 180 and 20 kD, whereas hybridoma SU5 stained a species at 50 kD. Thus, these polypeptides may be components of the centrosphere.

During normal development, the neural cell adhesion molecule N-CAM changes at the cell-surface from a sialic acid-rich embryonic, or E form, to several adult, or A forms that have less sialic acid (E-to-A conversion). To investigate the cellular and molecular mechanisms that underlie these changes, we have established conditions under which E-to- A conversion occurs in cultured explants of central nervous system tissues. Mouse cerebellum, chick spinal cord, and chick retina that express the E form of N-CAM were dissected and cultured on collagen gels. After 3-6 d in culture, increased proportions of A forms were synthesized, as revealed by specific immunoprecipitation and immunoblotting. The rate of E-to-A conversion and the proportions of the different A forms synthesized in vitro were similar to those observed for the tissues in vivo at comparable times. In addition, the explants incorporated radioactive precursors of amino sugars into N- CAM, and the electrophoretic mobilities of the E and A forms of N-CAM were altered by treatment with neuraminidase in a way comparable to that found for N-CAM obtained directly from tissue. These results suggest that the post translational processing in vitro was similar to that in vivo. Logistic studies on cell division and death in the explants suggested that E-to-A conversion resulted mainly from a specific increase in synthesis of A forms in individual cells rather than as a consequence of differential birth or death within distinct cell populations. The data were consistent with the possibility that the increase in synthesis of A forms occurred either in cells that had previously synthesized E forms or in a distinct population of cells that already synthesized A forms. Cells dissociated from embryonic central nervous system tissues and cultured in vitro were also found to undergo E-to-A conversion at the same rate as the explant cultures, which suggests that if intercellular signals were responsible for initiation of the change in synthetic pattern, they had already occurred in vivo before the time of culture. In pulse-chase experiments, the E form of N-CAM that was synthesized during the first day after explantation persisted as E form for several days, at times when newly synthesized N-CAM was predominantly in A forms. These results indicate that in cultured neural tissue, the E form of N-CAM is not processed into A forms but is gradually degraded and replaced by newly synthesized A forms.(ABSTRACT TRUNCATED AT 400 WORDS)

We examined the interaction of complement component C3 with surface molecules on Trypanosoma cruzi. Five- to six-fold more C3 was bound to epimastigotes (Epi) than to metacyclic trypomastigotes (CMT) of strain M88. Epi and CMT were surface iodinated, then incubated in C8-deficient serum, and detergent lysates were applied to anti-C3 antibody that had been coupled to Sepharose. We found that 9.20-10.24% of applied 125I-Epi protein bound to anti-C3-sepharose, compared to 2.64% binding of 125I-CMT protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that C3 was attached to 125I-Epi protein by a covalent bond. Samples eluted from anti-C3-sepharose with hydroxylamine revealed a single, major, 72 kD band, suggesting that C3b attaches almost exclusively to the 72 kD glycoprotein of Epi by a hydroxylamine-susceptible ester bond. An antiserum was prepared from lysates of serum-treated Epi that had been affinity-purified on anti-C3-sepharose. This antiserum immunoprecipitated a single 72 kD component (gp72) from surface-iodinated Epi, and specifically recognized only gp72 from Epi in immunoblots. In contrast to the results with Epi, gp72 on CMT was not found to be an efficient acceptor molecule for C3 deposition. The results are the first to evaluate the acceptor site for C3 deposition on a parasite, and they show that gp72 on Epi, but not gp72 on CMT, serves as the preferential acceptor for C3 during antibody-independent alternative complement pathway activation.

When cholesterinized antigen is dropped into an excess of water, the rapid flocculation of cholesterin crystals is prevented by the fact that, as tiny aggregates form, they adsorb a protective surface of hydrophilic lecithin (i.e., antigen) which endows the particles with its own stable surface properties and thus prevents further aggregation. The colloidally dispersed antigen-cholesterin particles have approximately the same isoelectric point (pH 1.9), critical potential (1 to 5 millivolts) and coagulation value (0.75 M NaCl) as pure antigen particles of the same concentration, while the corresponding values for cholesterin are pH 2.1 to 3.4 (probably due to an associated impurity), >100 millivolts, and Presumably, this adsorption of antigen by the cholesterin nucleus is determined by the fact that the former has a lower surface tension against water. At any rate, many surface active substances (serum; alcoholic extract of milk, egg or any animal tissue; Na-oleate; Na-glycocholeate; Na-taurocholate) cause a similar stable dispersion of cholesterin; and conversely, many otherwise water-insoluble substances of the most diverse chemical structure can be made to form a stable colloidal suspension by adding antigen to their alcoholic solutions before dropping into water. The colloidal suspension formed by antigen alone is very finely dispersed: only a few of the particles exceed the limits of dark field visibility. Cholesterin causes a marked increase in the number of these particles, out of all proportion to its mass; thus, one part of cholesterin to five of antigen causes a ten-fold increase in such visible particles, at the expense of the submicroscopic micellae formed by antigen alone. At the same time, the suspension becomes much more turbid. The particles remain discrete until the cholesterin: antigen ratio exceeds 1:1, when slight microscopic aggregation is observed; microscopic flocculation is seen only when this ratio exceeds 5:1, when there is not sufficient antigen to act as an efficient protective colloid. Cholesterin therefore causes a coarsened dispersion of antigen by forming a relatively large nucleus upon which antigen is adsorbed. As shown in the text, the larger the antigen particle the greater is its avidity for reagin per unit surface or mass. Thus, the coarse sol formed by dropping water-into-antigen is about twice as efficient as a finely dispersed antigen-into-water sol of the same concentration. The coarsened dispersion caused by cholesterin completely explains the greater sensitivity of the cholesterinized antigen in complement fixation. The same factor obtains in the flocculation reactions. In addition, the coarsened dispersion acts as a preliminary quasi-aggregation, facilitating by just so much the subsequent formation of visible clumps (or sedimenting aggregates) upon the addition of syphilitic serum; moreover, there is less surface in a coarse sol, with more reagin per unit surface, and correspondingly more efficient flocculation. The foregoing would be of purely academic interest were it not for the following considerations. From several points of view cholesterin is unsatisfactory as a sensitizer for antigen. Its solubility in alcohol is small. Even the 0.6 per cent concentration used in the Kahn test is difficult to keep in solution. Yet, as our experiments show, its sensitizing action increases indefinitely with its concentration. If it were sufficiently soluble, even 3 per cent could be used to advantage, increasing the sensitivity of 1½ per cent antigen for complement fixation some 200 to 400 per cent, instead of about 50 per cent, as does 0.2 per cent cholesterin. Since, as we have shown, the sensitizing action of cholesterin upon antigen is due solely to the coarse dispersion it causes, and since it is quite inert during the actual combination of the lipoid particles with reagin, it can be replaced by any substance with similar physical properties. The problem in hand was therefore to find a water-insoluble substance, very soluble in alcohol, with so high an interfacial tension against water that, as in the case of cholesterin, microscopic particles would adsorb antigen when the alcoholic solution of the two is dropped into water. Given such a substance, it would be possible to obtain a more sensitive antigen for both complement fixation and flocculation, but particularly for the former. These theoretical expectations have been realized in a group of substances shortly to be reported: they make possible an antigen which is from 2 to 10 times as efficient in the Wassermann test as any now available.

Erythrocyte membranes incorporated labeled phosphate from γ-adenosine triphosphate (AT32P) into phosphatidic acid and the polyphosphoinositides. Inositol-3H and palmitate-14C were also incorporated into the phospholipids but α-glycerophosphate-32P was not. The incorporation of γ-AT32P into phospholipids was increased when the erythrocyte ghosts were incubated in hypotonic media which lysed the cells. Lysis had little or no effect on the incorporation of inositol-3H and palmitate-14C into the phospholipids. If erythrocyte membranes were prepared in 1 mM ethylenediaminetetraacetate (EDTA), instead of 1 mM MgCl2, then the tonicity of the incubating medium did not influence the incorporation of γ-AT32P into the phospholipids. Erythrocyte ghosts, prepared by lysis in water, EDTA, or 1 mM calcium, lead, mercury, zinc, or cadmium, failed to reconstitute when placed in isotonic medium, inasmuch as they did not retain potassium against a chemical gradient. Ghosts prepared by lysis in 1 mM magnesium, barium, or strontium could be reconstituted. Ghosts which failed to reconstitute incorporated more labeled phosphate from γ-AT32P into the phospholipids than did intact or reconstituted ghosts. The larger incorporation of labeled phosphate by leaky ghosts was not due to a greater entrance of γ-AT32P into those cells. Primaquine phosphate and digitonin, at concentrations which are known to cause cells to form smaller vesicles or to lyse cells by removing cholesterol, did not increase the incorporation of labeled phosphate into the phospholipids. It is suggested that the increased metabolism of phospholipids may be involved in a membrane repair mechanism.

Ultralow temperature radioautography, suitable for the quantitative localization of diffusible solutes, was used to study the permeability of the nuclear envelope in the intact amphibian oocyte Sucrose-3H solutions were injected into mature oocytes, in volumes of 0 016–0 14% of that of the cell, and the subsequent movement of the solute was recorded. The resultant radioautographs show diffusion gradients in the cytoplasm and nucleus, and concentration gradients across the nuclear envelope Analysis of these gradients discloses that the nuclear envelope is as permeable as a comparable structure composed of cytoplasm, and is about 108 times more permeable than the oocyte plasma membrane The diffusion coefficient of sucrose in cytoplasm is 2 x 10-6 cm2/sec, or about one-third its diffusivity in pure water. This reduction can probably be accounted for by an effective lengthening of the diffusional path because of obstruction by cytoplasmic inclusions. The nuclear: cytoplasmic sucrose concentration ratio at diffusional equilibrium is about 3 05, or 1.6 times as great as expected from the water content of the two compartments This asymmetry is attributed to an unavailability of 36% of the cytoplasmic water as solvent Finally, sucrose entry into oocytes from a bathing solution was monitored by whole cell analysis and radioautography. These and the microinjection results are consistent with a model in which sucrose entry into the cell is entirely limited by the permeability of the plasma membrane. The results are inconsistent with cell models that hypothesize a short-circuit transport route from the extracellular compartment to the nucleus, and with models in which cytoplasmic diffusion is viewed as limiting the rate of solute permeation.

Chromosome velocity has been studied in living Melanoplus differentialis spermatocytes by phase contrast cinemicrography. Melanoplus chromosomes (and bivalents) differ in length by as much as 1:3.5. As expected, no size-dependent velocity differences were detected in anaphase, and this is also shown to be true for the less predictable movements during prometaphase congression. The size of the X chromosome can change during observation following x-irradiation, but this is equally without influence on velocity. However, an effect of position on velocity is found in both prometaphase and in anaphase: the chromosomes furthest from the central interpolar axis move 25 per cent faster than more central chromosomes. A simple mechanical model relating frictional resistance and mitotic forces to chromosome velocity is discussed in detail. Calculations from the model suggest that a significant difference in the force acting on a large, as compared with a small chromosome is necessary to account for the observed similarity in velocity. Therefore, it is concluded that the mitotic forces are so organized or regulated that velocity is, within limits, independent of load. The implications of velocity-load independence in relation to the molecular origin of mitotic forces are discussed.

Mouse peritoneal macrophages take up I*-HSA from their medium during in vitro cultivation. Conditions which promote I*-HSA uptake are the same as those which stimulate formation of pinocytic vesicles. Autoradiography of cells pulsed with 125I-HSA showed that intracellular isotope is localized in perinuclear granules, or secondary lysosomes. Following a pulse of 125I-HSA, intracellular radioactivity decreases and the amount of TCA-soluble isotope in the medium increases correspondingly. About 50% of the intracellular isotope is lost in 5 hr. The release of isotope from pulsed cells is not inhibited by parafluorophenylalanine, 2,4-dinitrophenol or by a reduction of the serum concentration of the medium. However, the processing of ingested 125I-HSA is reversibly inhibited by reduced temperature. The TCA-soluble radioactive material excreted by pulsed macrophages was identified as monoiodotyrosine.

Under restricted culture conditions, the growth and division of Saccharomyces cerevisiae was inhibited by the antimitotic drug Colcemid; in contrast, the related drug colchicine had no effect. The difference in the sensitivity of yeast to these two agents was not dependent on their ability to permeate the cell but rather reflected an inherent difference in the affinity of the two drugs for a cellular-binding site. The binding moiety was characterized by gel filtration as a macromolecule of approximately 110,000 mol wt with an affinity constant for Colcemid of 0.5 x 104 liters per mole; in addition, this macromolecule was retained by diethylaminoethyl (DEAE) ion exchangers. On the basis of these properties, the Colcemid-binding substance in S. cerevisiae cells was provisionally identified as microtubule subunits.

The initial rate of thymidine-(3)H incorporation into the acid-soluble pool by cultured Novikoff rat hepatoma cells was investigated as a function of the thymidine concentration in the medium. Below, but not above 2 microM, thymidine incorporation followed normal Michaelis-Menten kinetics at 22 degrees , 27 degrees , 32 degrees , and 37 degrees C with an apparent K(m) of 0.5 microM, and the V(max) values increased with an average Q(10) of 1.8 with an increase in temperature. The intracellular acid-soluble (3)H was associated solely with thymine nucleotides (mainly deoxythymidine triphosphate [dTTP]). Between 2 and 200 microM, on the other hand, the initial rate of thymidine incorporation increased linearly with an increase in thymidine concentration in the medium and was about the same at all four temperatures. Pretreatment of the cells with 40 or 100 microMp-chloromercuribenzoate for 15 min or heat-shock (49.5 degrees C, 5 min) markedly reduced the saturable component of uptake without affecting the unsaturable component or the phosphorylation of thymidine. The effect of p-chloromercuribenzoate was readily reversed by incubating the cells in the presence of dithiothreitol. Persantin and uridine competitively inhibited thymidine incorporation into the acid-soluble pool without inhibiting thymidine phosphorylation. At concentrations below 2 microM, thymidine incorporation into DNA also followed normal Michaelis-Menten kinetics and was inhibited in an apparently competitive manner by Persantin and uridine. The apparent K(m) and K(i) values were about the same as those for thymidine incorporation into the nucleotide pool. The over-all results indicate that uptake is the rate-limiting step in the incorporation of thymidine into the nucleotide pool as well as into DNA. The cells possess an excess of thymidine kinase, and thymidine is phosphorylated as rapidly as it enters the cells and is thereby trapped. At low concentrations, thymidine is taken up mainly by a transport reaction, whereas at concentrations above 2 microM simple diffusion becomes the principal mode of uptake. Evidence is presented that indicates that uridine and thymidine are transported by different systems. Upon inhibition of DNA synthesis, net thymidine incorporation into the acid-soluble pool ceased rapidly. Results from pulse-chase experiments indicate that a rapid turnover of dTTP to thymidine may be involved in limiting the level of thymine nucleotides in the cell.

After the addition of insulin to monolayers of chick fibroblasts previously incubated in serum-free medium, the rates of protein and RNA synthesis increase continuously during the first 8–10 h. Little stimulation of DNA synthesis or mitosis results with the addition of insulin alone in contrast to the addition of fresh serum which stimulates both markedly. The stimulation in RNA synthesis does not result from expansion of the nucleotide pool but is correlated with increases in RNA polymerase activity. All major classes of RNA are stimulated; processing of preribosomal RNA to 28S and 18S and the association of this mature RNA with ribosomes appear to occur normally. The kinetics of stimulation of 5S RNA differ from those of the synthesis of 4S and of ribosomal RNA. Insulin and serum appear to affect the synthesis or stability of certain transcripts differentially.

The cationic antibacterial proteins of rabbit PMN lysosomes have been resolved into at least five subfractions. Each of these showed substantial selectivity in its antibacterial action against several pathogenic bacteria, including two smooth and two rough Escherichia coli strains, three Staphylococcus aureus strains, one S. albus, three proteus species and four different cultures of streptococcus. Each of the subfractions possesses a different electrophoretic mobility. Amino acid analyses of the three most cationic components revealed high contents of arginine consistent with their relative electrophoretic mobilities and very high arginine to lysine ratios. Aromatic amino acids were present in very low concentrations in these proteins and their light absorption at 2800 A was correspondingly weak. The evidence of antibacterial specificity, along with marked differences in the arginine-lysine ratios, shows that the cationic antibacterial components of rabbit PMN lysosomes are biologically and chemically heterogeneous.

40 min after injecting tritiated thymidine into an animal, 20–30% of the total plasma radioactivity is nonvolatile. This fraction decreases to about 6% 10 hr after the injection and 3% 24 hr after the injection. There appears to be material in this nonvolatile fraction that can label mastocytoma cells in culture. The labeling indices decrease with time after injection in the same way as the nonvolatile fraction. The 40 min plasma sample contains sufficient material to allow accurate assessment of the fraction of cells in S in culture after a 6 wk exposure. The circulating material is not apparently available for incorporation into those cells in cycle in the donor animal. The material appears to be related to the G0 cell-specific pool that has been described elsewhere. The trichloroacetic acid-soluble or ethanol-soluble nonvolatile activity appears to contain thymine, and some thymidine-phosphorylated compounds.

In this study, alanine was shown to be the N-terminal amino acid of a basic protein of low molecular weight that was isolated from either human or guinea pig brain. Antibodies prepared against the guinea pig protein were labeled with either fluorescein or ferritin. Studies with the labeled antibodies showed that an immunohistochemically similar protein is found in the myelin sheaths of central and peripheral nervous tissues of chicken and frog and a variety of mammalian species. Loss of integrity of the myelin during processing was shown to enhance markedly the antigen-antibody reaction.

Cultured human synovial cells secrete hyaluronic acid (HA) into the culture medium. Glucosamine-6-3H was shown to be a direct and relatively specific precursor of HA-3H by the following observations: the susceptibility of nondialyzable radioactivity in the medium to hyaluronidase, its migration with hexuronic acid on zone electrophoresis in polyvinyl chloride, its exclusion from Sephadex G-200, and the localization of radioactivity to glucosamine after hydrolysis of the labeled polysaccharide. The presence of intracellular HA-3H was established by sequential extraction of labeled cells and by radioautography of synovial cell cultures digested with hyaluronidase in situ. When cells were exposed to medium lacking glucose, glucosamine-3H-uptake was enhanced; and this made possible electron microscopic radioautographic studies. These studies demonstrate the early and continued presence of HA-3H within the Golgi apparatus.

Synthesis of collagen on polyribosomes has been demonstrated in vitro in chick embryo corium by radioisotope incorporation, zone centrifugation through sucrose gradients, and analytical ultracentrifugation. Collagen synthesis was associated with polyribosomes ranging in size, as reflected by their sedimentation constants, from about 180S to approximately 1600S. Most of the newly formed collagen, hydroxyproline, was present on the largest polyribosome aggregates ( approximately 350-1600S), but small polyribosomes ( approximately 180-200S) also contained collagen. On the basis of the proline-(14)C/hydroxyproline-(14)C ratios and the disrupting effect of collagenase, the proposal is made that the 350-1600S polyribosomes from this tissue are involved predominantly in collagen synthesis. The large polyribosomes are disrupted extensively by collagenase but only partially by ribonuclease and trypsin. Therefore, it appears that they are stabilized by the interaction of newly forming collagen chains. Evidence is presented consistent with the hypothesis that these large polyribosomes are formed by the aggregation of small polyribosomes (180-200S) through the interaction of collagen polypeptides. It is suggested that these small polyribosomes might be involved in the synthesis of subunits of the collagen alpha chain.

Cases of "distributive c-mitosis" (the term does not mean that colchicine has been used) in plant endosperm are described, in which the chromosomes of metaphase type (two-chromatid chromosomes) are distributed at random because of phragmoplast activity in a process similar to non-disjunction. There is some evidence that chromosmal fibres can be formed within the phragmoplast under special circumstances; during "distributive c-mitosis" some kinetochores show active movements due to cooperation with chromosomal fibres formed in the phragmoplast; while other chromosomes, as indicated by their arrangements and shape, are moved without any activity of kinetochores. Some components of the phragmoplast have the fastest movements occurring in mitosis. Some cases are described in which the phragmoplast divides telophase and interphase nuclei into two or more groups and moves the pieces a considerable distance apart. In a similar way, the phragmoplast may divide newly formed restitution nuclei. This phenomenon leads to a reduction of chromosome numbers, and the course of the process itself is reminiscent of amitosis.

It has been determined that cortisol and a few other steroids are transported outward from certain mammalian cells growing in vitro. The extrusion process is temperature dependent, glucose dependent, saturable, and operates for only a few selected steroids. Many, but not all, steroids are able to block the extrusion process but are not themselves transported. The outward transport process for steroids has been found in mouse fibroblasts, mouse lymphoma cells, and functional mouse adrenal gland tumor cells growing in vitro. The transport process is not present in two varieties of cells cultured from human sources—HeLa or diploid fibroblasts, WI-38.

A large quantity of paraffin oil, sucrose solution, or sea water was injected into the eggs of the heart urchin Clypeaster japonicus shortly before the onset of the first cleavage. The injected oil became spherical, pushing the mitotic apparatus aside. The sucrose solution mixed with the protoplasm and caused disintegration of the mitotic apparatus, and the sea water formed a vacuole at the center of the cell. In all these cases, cleavage may take place almost normally in spite of the absence of the mitotic apparatus or its displacement within the cell. In some eggs, furrowing may take place when more than fifty per cent of the endoplasm has been replaced with sea water before onset of cleavage.

This commentary is on the paper by Angelini et al. Here, we set the original paper in the context of triggered arrhythmias, particularly early after depolarizations (EADs), emphasizing the importance of pharmacologically inhibiting late Ca2+ current to prevent EADs without affecting myocardial contractility.

In this issue of JEM, Bergman et al. challenge the data published in a previous JEM paper on the preventive effect of tolerogenic vaccination with a strong agonist insulin mimetope in type 1 diabetes. Daniel et al. provide a response to these data., In this issue of JEM, Bergman et al. (https://doi.org/10.1084/jem.20160471) challenge the data published in our previous JEM paper on the preventive effect of tolerogenic vaccination with a strong agonist insulin mimetope in type 1 diabetes. Here, we provide a response to these data and suggest that appropriate subimmunogenic conditions are required to induce Foxp3+ regulatory T cell conversion.

Arf6 positively regulates autophagosome membrane biogenesis by inducing PIP2 generation and PLD activation, which together may influence endocytic uptake of plasma membrane into autophagosome precursors., Macroautophagy (in this paper referred to as autophagy) and the ubiquitin–proteasome system are the two major catabolic systems in cells. Autophagy involves sequestration of cytosolic contents in double membrane–bounded vesicles called autophagosomes. The membrane source for autophagosomes has received much attention, and diverse sources, such as the plasma membrane, Golgi, endoplasmic reticulum, and mitochondria, have been implicated. These may not be mutually exclusive, but the exact sources and mechanism involved in the formation of autophagosomes are still unclear. In this paper, we identify a positive role for the small G protein Arf6 in autophagosome formation. The effect of Arf6 on autophagy is mediated by its role in the generation of phosphatidylinositol 4,5-bisphosphate (PIP2) and in inducing phospholipase D (PLD) activity. PIP2 and PLD may themselves promote autophagosome biogenesis by influencing endocytic uptake of plasma membrane into autophagosome precursors. However, Arf6 may also influence autophagy by indirect effects, such as either by regulating membrane flow from other compartments or by modulating PLD activity independently of the mammalian target of rapamycin.

Excessive cell growth in Drosophila intestinal stem cells lacking TSC blocks further cell division., Intestinal stem cells (ISCs) in the adult Drosophila melanogaster midgut can respond to damage and support repair. We demonstrate in this paper that the tuberous sclerosis complex (TSC) plays a critical role in balancing ISC growth and division. Previous studies have shown that imaginal disc cells that are mutant for TSC have increased rates of growth and division. However, we report in this paper that loss of TSC in the adult Drosophila midgut results in the formation of much larger ISCs that have halted cell division. These mutant ISCs expressed proper stem cell markers, did not differentiate, and had defects in multiple steps of the cell cycle. Slowing the growth by feeding rapamycin or reducing Myc was sufficient to rescue the division defect. The TSC mutant guts had a thinner epithelial structure than wild-type tissues, and the mutant flies were more susceptible to tissue damage. Therefore, we have uncovered a context-dependent phenotype of TSC mutants in adult ISCs, such that the excessive growth leads to inhibition of division.

[Jung and Lee][1] have responded in this issue to our recent paper ([Yu et al., 2014b][2]) in which we concluded that calmodulin (CaM) does not alter anion permeability of the mouse ANO1/TMEM16A Ca2+-activated Cl− channel. In our paper, we suspected that the Ca2+-CaM (abbreviated as CaM) effect

GD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3+ GM2− CD1−) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of ∼1:2,000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-γ followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1− tumors.

The role of vesicles in cargo transport through the Golgi apparatus has been controversial. Large forms of cargo such as protein aggregates are thought to progress through the Golgi stack by a process of cisternal maturation, balanced by a return flow of Golgi resident proteins in COPI-coated vesicles. However, whether this is the primary role of vesicles, or whether they also serve to transport small cargo molecules in a forward direction has been debated. Two papers (Martínez-Menárguez et al., 2001; Mironov et al., 2001, this issue) use sophisticated light and electron microscopy to provide evidence that the vesicular stomatitis virus membrane glycoprotein (VSV G)**Abbreviation used in this paper: VSV G, vesicular stomatitis virus membrane glycoprotein. is largely excluded from vesicles in vivo, and does not move between cisternae, whereas resident Golgi enzymes freely enter vesicles as predicted by the cisternal maturation model. Both papers conclude that vesicles are likely to play only a minor role in the anterograde transport of cargo through the Golgi apparatus in mammalian tissue culture cells.

Every so often, a paper comes along that brings clarity to an issue. Clarity is not necessarily a final resolution, but rather a conceptual framework for productively addressing that issue. Such a paper could be based on a breakthrough discovery, an intriguing observation, a flash of intuition, or a

One widely expressed K+ channel, often called the “BK” channel for its “big” single channel conductance, is regulated by intracellular Ca2+ and voltage: at constant voltage, the open probability (Po) increases with [Ca2+]. At constant [Ca2+], Po increases with depolarization. BK channels participate in many physiological processes, including repolarization of the action potential (Adams et al. 1982), frequency tuning in the inner ear (Hudspeth and Lewis 1988), and regulation of neurotransmitter release (Robitaille et al. 1993). The mechanism of BK channel gating is addressed by a recent paper in this journal (Rothberg and Magleby 1999) and by two papers in this issue (Horrigan et al. 1999; Horrigan and Aldrich 1999). The Magleby and Aldrich labs took very different approaches, but fortunately arrive at compatible conclusions. Rothberg and Magleby 1999 examined in detail the gating of single BK channels under a limited range of conditions: +30 mV, primarily at saturating [Ca2+]. Horrigan et al. 1999 and Horrigan and Aldrich 1999 examined macroscopic ionic and gating currents (respectively), over a wide voltage range but in the effective absence of [Ca2+]. Both find features of BK channel gating that favor allosteric models, resembling in some ways the classical MWC model (Monod et al. 1965) for activation of allosteric enzymes.

20 yr ago, a tribute appeared in this journal on the 70th anniversary of an animal model of disseminated encephalomyelitis, abbreviated EAE for experimental autoimmune encephalomyelitis. “Observations on Attempts to Produce Disseminated Encephalomyelitis in Monkeys” appeared in the Journal of Experimental Medicine on February 21, 1933. Rivers and colleagues were trying to understand what caused neurological reactions to viral infections like smallpox, vaccinia, and measles, and what triggered rare instances of encephalomyelitis to smallpox vaccines. The animal model known as EAE continues to display its remarkable utility. Recent research, since the 70th-anniversary tribute, helps explain how Epstein–Barr virus triggers multiple sclerosis via molecular mimicry to a protein known as GlialCAM. Proteins with multiple domains similar to GlialCAM, tenascin, neuregulin, contactin, and protease kinase C inhibitors are present in the poxvirus family. These observations take us a full circle back to Rivers’ first paper on EAE, 90 yr ago.

In this issue, two articles present major advances in the quantitative analysis of the molecular mechanisms underlying rod phototransduction. One is by Nikonov et al. (1998), the other by Calvert et al. (1998). These two papers are complimentary, but with substantial areas of intersection. At the present time, the activation cascade in rod phototransduction that leads to the hydrolysis of the internal transmitter, cyclic GMP (cGMP) and to the closure of light-sensitive channels is fairly well understood. The inactivation steps responsible for the termination of the photoresponse and the feedback mechanisms, which modulate sensitivity and kinetics and also contribute to response termination, are not understood nearly as well. The field of phototransduction has always been fraught with controversy: for every point, there has been a counterpoint. However, one can argue, with little fear of inciting controversy, that a complete understanding of phototransduction must include an understanding of the steps by which the photoresponse is initiated and the steps by which it is terminated. For this reason, the Nikonov et al. paper, “The Kinetics of Recovery of the Dark-adapted Salamander Rod Photoresponse” is especially significant. This paper moves us closer to a definitive answer to an old, controversial question: What is the rate-limiting biochemical reaction that determines the time course of recovery of the photocurrent from a flash bright enough to temporarily shut off all light-sensitive current? Two main contenders have been the inactivation of rhodopsin and the inactivation of the activated phosphodiesterase–G-protein complex. The authors present a case for the latter. A highlight of the paper is a new approach to quantify the extent of guanylyl cyclase activation in a feedback pathway mediated by calcium. The role of cyclase in determining the time at which photocurrent recovery begins and its role in sculpting the waveform of recovery are quantified. This analysis supports the existence of at least one more significant target for calcium feedback. A notable feature of the paper is that the authors make stunning progress largely through powerful new theoretical analysis applied to data gathered with stateof-the-art techniques. A number of important observations and conclusions are stated in a formal manner in mathematical language, which includes theorems, lemmas, and proofs. The rigorous approach in this paper has the advantages of clarity and completeness. The aspects of phototransduction that can now be well understood are highlighted by a mathematical model, and the gaps in our knowledge are set off in stark contrast. Fortunately for the reader with less mathematical background, sufficient explanatory discussion surrounds the mathematical statements, such that a reader may even choose to skip the theorems entirely without major loss of information.

Two recent papers published in the Journal of General Physiology (Guo and Lu, 2003; Guo et al., 2003) address the mechanism of inward rectification by polyamines in Kir2.1 channels. In these two papers, Guo et al. (2003) extensively analyze channel block by monoamines, diamines (DAs), and polyamines of varying length and charge, using thermodynamic mutant cycles to calculate interaction energies of these compounds with acidic residues in the channel. Based on their findings, a physical model for the blocking action of polyamines is proposed that incorporates the most recent structural information determined for Kir channels. We write to suggest a different and, in our view, more plausible physical interpretation, which we believe better reconciles their results with previous electrophysiological as well as structural findings (Pearson and Nichols, 1998; Kubo and Murata, 2001; Xie et al., 2002, 2003; Chang et al., 2003; Kuo et al., 2003). DAs are alkyl chains with positively charged amine groups at each end, whereas polyamines such as spermidine and spermine have amine groups at both ends and interspersed within. In their model, Guo et al. (2003) propose that when intracellular DAs or polyamines enter the pore, the leading amine group first interacts with a ring of negative charges formed by E224 and E299 in the cytoplasmic pore, occluding the ion-conducting pathway with a shallow voltage dependence. The leading amine then moves deeper into the transmembrane pore toward the negative charge at D172, with the trailing amine group remaining stabilized by an electrostatic interaction with E224 and E299. They postulate that as the alkyl chain length of the DA increases, the leading amine approaches more closely to D172, displacing more K ions from the pore and thereby increasing the voltage dependence of block. For DAs, this effect plateaus at a chain length of nine alkyl groups (DA9), which is also the DA length at which the interaction energy (relative to DA2) peaks with respect to the D172N mutant. Although this schema explains their findings from the biophysical standpoint, a problem arises when the recent structural information is considered. From the crystal structure of closed KirBac1.1 (Kuo et al., 2003), the distance between D172 and E224/E299 is ∼35 A, whereas DA9 has a total extended length of only ∼12 A. Assuming similar dimensions for Kir2.1, then if the leading amine of DA9 binds close at D172 (as required for it to sweep K ions from the transmembrane pore), then its trailing amine would be some 23 A away from E224 and E299. To rationalize this distance problem, Guo et al. (2003) hypothesize a long-range electrostatic interaction between the trailing amine group of D9 and the negative ring of charges at E224 and E299, so that the actual position of the trailing amine is only 12 A or so from D172. Thus, longer or shorter DA cannot be as effectively stabilized with their leading amine close to D172 to sweep K ions from the pore. We suggest that an alternative scenario for polyamine binding is equivalently consistent with the biophysical evidence, but, in our view, more compatible with available structural information (Fig. 1) and previous observations by other investigators (Pearson and Nichols, 1998; Kubo and Murata, 2001; Xie et al., 2002, 2003; Chang et al., 2003). This model was previously suggested for Kir6.2 (Phillips and Nichols, 2003) and Kir3 (Dibb et al., 2003) channels as well as Kir2.1 channels (Chang et al., 2003), and is based on the leading amine group of the DA or polyamine first occluding the pore when it interacts electrostatically with D172 in the transmembrane pore, rather than at E224/E299. The leading amine then moves deeper toward the selectivity filter (SF), thereby displacing K ions from the pore, whereas the trailing amine is stabilized by electrostatic interaction with D172. The distance between I138 (D172 in Kir2.1) and V111 (V143 in Kir2.1), which is just at the intracellular side of the SF signature sequence (GYG) in KirBac1.1, is estimated at ∼12 A, which matches almost exactly the length of DA9 (∼12 A). This explains why the interaction energy peaks for DA9, and falls off for longer or shorter DAs. For example, if the extracellular movement of the leading amine group of DA12 (length ∼16 A) is physically obstructed by the SF, then its alkyl chain will be too long for the trailing amine to interact as effectively with D172. Like DA9, spermidine (∼11 A) has a nearly ideal length for its leading and trailing amine groups to bind between D172 and the SF. Spermine (∼16 A) is too long for the leading and trailing amine groups to fit between D172 and the SF, but the distance between its leading and third amine groups, which is the same as spermidine, is optimal. The middle amine groups in spermine appear to further increase its stability in the pore, because its interaction energy was higher than any of the DAs. If, as originally postulated by Pearson and Nichols (1998) and later supported by Guo et al. (2003) as well as by our findings (Xie et al., 2002, 2003), the voltage dependence of polyamine block arises chiefly from the polyamines displacing K ions from the pore, then the similar effective valences for DA9, spermidine, and spermine can also be readily explained in this model, based on the following assumptions: (a) that the transmembrane voltage field is centered near the SF and K binding sites (such that the initial blocked state has a shallow voltage dependence); (b) it is primarily the leading amine group of DA9, spermidine, or spermine that electrostatically repels K ions and sweeps them from their binding sites between D172 and external side of the SF. The trailing amine groups of DA9, spermidine, or spermine would have little effect on valence, since they are for the most part located outside of the transmembrane voltage field, and do not contribute greatly to K ion displacement. Figure 1. Model of inward rectification. Structural model of the closed KirBac1.1 channel, outlining the transmembrane and cytoplasmic pore regions. For comparison with channel pore dimensions, DA4, DA9, DA12, and spermine (SPM) are shown from left to right as ... In this model, the lower valence of monoamines (including M9) occurs for the following reason: if the monoamine enters the pore with its uncharged nonpolar end leading, so that its trailing amine group interacts electrostatically with D172, then the nonpolar end will be less effective at repelling K ions near the SF. Conversely, if its amine group leads and it penetrates past D172 then the trailing nonpolar end cannot electrostatically stabilize the monoamine at D172, and the leading amine group will not penetrate as deeply toward the SF to repel K ions. In contrast, in their model, Guo et al. (2003) must postulate that monoamines can only enter the pore in the energetically less favorable orientation with the nonpolar end leading The orientation of DAs in the pore between the D172 and the SF that we propose here is compatible, unlike the model proposed by Guo et al. (2003), with the considerable evidence that polyamines can bind to the negative ring of charges at E224 and E299 without occluding the pore (Kubo and Murata, 2001; Xie et al., 2002, 2003; Chang et al., 2003). We (Xie et al., 2003) have presented evidence that DAs and polyamines of length ≥8 alkyl groups bind efficiently to the negative charges at E224 and E299 in the cytoplasmic pore without occluding the ion permeation pathway. This conclusion was based on the observation that DAs and polyamines with ≥8 alkyl groups were effective at reducing single-channel conductance over a wide range of voltage, which we attributed to reduction of net negative surface charge in the cytoplasmic pore. The distance between E224 and E299 on adjacent subunits, estimated from the Kir3.1 cytoplasmic structure, is 9.2 A, which corresponds closely to the length of DA8 (9.5 A). Thus, we postulated that initial binding of DAs and polyamines longer than 9 A, at E224 and E299 prepositions them in the wide (7–15 A) cytoplasmic pore, facilitating their access to the pore-blocking site at D172. In our model, this accounts for the kinetically rapid component of pore block, while the slow component is due to diffusion of untethered polyamines into the pore. This role of the negative charge ring in the cytoplasmic pore is consistent with the findings of Guo et al. (2003) that the alkyl chain length had no effect on the low interaction energy between DAs and E224 or E299. (We agree with their interpretation that the higher interaction energy of spermine with E224 or E299 compared with diamines is likely due to the additional amines in spermine acting as pseudodivalent cations.) In summary, we believe that the model of polyamine block proposed by Guo et al. (2003) is difficult to reconcile with all available structural and electrophysiological data. A caveat, of course, is that the structure of open Kir2.1 channels is not yet available to settle definitively which interpretation is correct. In addition, there are also controversies about the biophysical analyses (Ishihara and Ehara, 2004). Nevertheless, based on the available information, we agree fully with the comment by Stanfield and Sutcliffe (2003) in their accompanying editorial noting “how exquisitely well the channel and spermine match each other”, in the sense that our current best estimates of the distances between D172 and the SF, and between E224 and E299 on adjacent subunits in Kir2.1 appear to be exquisitely matched to the dimensions of the key biomolecules that cooperate synergistically to facilitate inward rectification so efficiently.

It's a truism that structures are helpful for mechanistic understanding of protein function, and nowhere is this dictum better illustrated than with ClC-type Cl− channels, a large molecular family found in virtually every type of cell in every biological niche. These channels, first observed over 20 yr ago (White and Miller, 1979; Miller, 1982), are now known from a barrage of knockout studies (Jentsch et al., 2002) to maintain the smooth operation of many varied physiological systems: skeletal muscle excitability, inhibitory interneuron responses, renal control of blood pressure, endosome acidification, and, well, the list goes on and on. Although the first ClC channel was cloned nearly 15 yr ago (Jentsch et al., 1990), our physical and mechanistic picture of these proteins at the molecular level languished at a frustrating level of murkiness until the first high-resolution structure of a ClC homologue burst onto the scene two years ago (Dutzler et al., 2002). Before this, we knew nearly nothing worthwhile about the molecular determinants of channel gating or selectivity; even the number of Cl−-permeation pores in the homodimeric channel was in contention (Maduke et al., 2000). This situation changed abruptly after the identification, overexpression, and functional reconstitution of a bacterial ClC channel (Maduke et al., 1999), followed by its crystallization and high-resolution structure determination (Dutzler et al., 2002, 2003). Suddenly, the pore became visible, with two Cl− ions sitting in it close to each other, mostly dehydrated, and engulfed by specific coordinating groups. Also evident was the fact that these ions are completely buried within the protein, i.e., that the structure is of a closed conformation. This structural work led to the proposal that the gate is merely a single glutamate sidechain located on the extracellular side of the two buried ions; in the closed channel, the glutamate occludes the pore, and when the channel opens, this sidechain rotates out of the way, according to this idea. In this issue, T.-Y. Chen and coworkers (Chen and Chen, 2003; Lin and Chen, 2003) attack the first question naturally arising from the structural work: how well does the bacterial channel structure help us understand the function of eukaryotic ClC channels? Can we read the electrophysiological behavior of eukaryotic ClCs in terms of the structure? This is a pertinent question since prokaryotic ClCs are stripped-down versions of their eukaryotic relatives; they are about half the mass and lack the large COOH-terminal intracellular domain common to all eukaryotic members of the family. We know that the bacterial channel is activated by low pH (Iyer et al., 2002), but we do not know if it shares with the eukaryotic channels features such as voltage dependence and Cl−-activation. An unfortunate problem here is that up until now, no electrophysiological recordings of the bacterial channel have been accomplished, and only a single eukaryotic ClC channel, ClC-0, has a unitary conductance large enough to be amenable to full electrophysiological analysis. So Chen and coworkers plunged into a detailed study of ClC-0 to see if its conduction and gating properties respond to mutations as anticipated from the bacterial channel structure. These two papers are separately aimed at Cl− permeation and channel gating. The bottom line is that on both of these fronts, ClC-0 seems to behave in harmony with the bacterial channel structure, at least in broad outline. The first paper (Chen and Chen, 2003) describes a series of electrostatic mutations in the region of the selectivity filter, i.e., at positions equivalent to those in the bacterial channel close to the two bound Cl− ions. We can crudely visualize this region, located toward the intracellular side of the protein, as a rather narrow kitchen-funnel held upside down, in which the completely dehydrated “central” ion is bound in the neck, and the partially hydrated “internal” ion resides right at the point where the funnel begins to open toward the cytoplasm. The wall of the widening funnel is decorated with several charged and polar groups known to influence Cl− permeation, and the influence of these groups on single-channel currents are examined in great detail here. In particular, Chen and Chen (2003) take an enzymological approach by heroically measuring single-channel conductance over a very wide range of Cl− concentrations, from 20 mM to >1.5 M, a range over which conductance saturates in a simple Michaelis-Menten fashion. This allows Km and Vmax effects of mutations to be distinguished. To my mind it is remarkable that these experiments could be done at all; five residues within this intricate selectivity region could be mutated singly and in pairs while preserving familiar ClC gating and selectivity and producing only small (