The structural differences between bovine lens alphaA- and alphaB-crystallin.

Lens alphaA- and alphaB-crystallin have been reported to act differently in their protection against nonthermal destabilization of proteins. The nature of this difference, however, is not completely understood. Therefore we used a combination of thermally and solvent-induced structural changes to investigate the difference in the secondary, tertiary and quaternary structures of alphaA- and alphaB-crystallin. We demonstrate the relationship between the changes in the tertiary and quaternary structures for both polypeptides. Far-ultraviolet circular dichroism revealed that the secondary structure of alphaB-crystallin is more stable than that of alphaA-crystallin, and the temperature-induced secondary structure changes of both polypeptides are partially reversible. Tryptophan fluorescence revealed two distinct transitions for both alphaA- and alphaB-crystallin. Compared to alphaB-crystallin, both transitions of alphaA-crystallin occurred at higher temperature. The changes in the hydrophobicity are accompanied by changes in the quaternary structure and are biphasic, as shown by bis-1-anilino-8-naphthalenesulfonate fluorescence and sedimentation velocity. These phenomena explain the difference in the chaperone capacity of alphaA- and alphaB-crystallin carried out at different temperatures. The quaternary structure of alpha-crystallin is more stable than that of alphaA- and alphaB-crystallin. The latter has a strong tendency to dissociate under thermal or solvent destabilization. This phenomenon is related to the difference in subunit organization of alphaA- and alphaB-crystallin where both hydrophobic and ionic interactions are involved. We find that an important subunit rearrangement of alphaA-crystallin takes place once the molecule is destabilized. This subunit rearrangement is a requisite phenomenon for maintaining alpha-crystallin in its globular form and as a stable complex. On the base of our results, we suggest a four-state model describing the folding and dissociation of alphaA- and alphaB-crystallin better than a three-state model [Sun et al. (1999) J. Biol. Chem. 274, 34067-34071].     

Functional implications of structural differences between variants A and B of bovine beta-lactoglobulin

The structure of the trigonal crystal form of bovine beta-lactoglobulin variant B at pH 7.1 has been determined by X-ray diffraction methods at a resolution of 2.22 A and refined to values for R and Rfree of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 7.1, which was determined previously [Qin BY et al., 1998, Biochemistry 37:14014-14023], the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD (residues 61-67), there are small changes in main-chain conformation, whereas the substitution V118A on beta-strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH-dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.     

Sexing of murine and bovine embryos by developmental arrest induced by high-titer H-Y antisera

Murine and bovine embryos at the late morula stage were cultured in medium containing high-titer rat H-Y antisera. After 12h of incubation, embryos blocked at the late morulae stage were classified as males and those at the blastocyst stage were classified as females. Sexing of murine embryos by PCR and cytogenetics revealed that 83% of the embryos classified as males and 82% of those classified as females had their sex correctly predicted (P < 0.05). Bovine embryos were transferred to recipient females. Pregnancy rates were 71.4% (10/14) for embryos classified as males and 68.8% (11/16) for embryos classified as females. The sex was correctly predicted for 80% (8/10) of the embryos classified as males and for 81.8% (9/11) of those classified as females (overall accuracy, 80.9%, P < 0.05). Therefore, the induction of developmental arrest by high-titer male-specific antisera was an efficient strategy for non-invasive embryo sexing. The procedure was straightforward and has considerable commercial potential for sexing bovine embryos.     

Functional and structural differences between cultured outer and inner layer cells of bovine adrenal cortex

Summary Outer and inner layer cells of bovine adrenal cortex were cultured separately to compare cellular structural characteristics and functional differences. Outer layer cells were polygonal in shape with radially distributed lipid droplets in the cytoplasm, and produced mainly aldosterone and cortisol. The aldosterone production increased upon stimulation with angiotensin II or dibutyryl-cAMP. In contrast, inner layer cells were spindle-shaped and had fine diffused lipid droplets. They produced four times as much cortisol as outer layer cells but no aldosterone. Cortisol production increased with ACTH or dibutyryl-cAMP stimulation.When stimulated by ACTH or by dibutyryl-cAMP, both types of adrenocortical cells showed cellular retraction whereby the number of cytoplasmic lipid droplets decreased and microvilli on the cellular surface increased. At the same time, the transverse distribution of actin fibers disappeared and the microtubules changed their distribution pattern from circular to radial. Stimulation by angiotensin II, on the other hand, brought no marked structural changes.These results indicate that, in functional terms, the outer layer cells and the inner layer cells in this culture system reflect zona glomerulosa and zona fasciculata-reticularis, respectively.     

Resurrected Ceriodaphnia quadrangula highlight differences between pheno‐ and genotypic expressions

The hatching of cladoceran ephippia from a 15‐cm long sediment core was investigated, and Ceriodaphnia quadrangula clones were isolated from different sediment layers. Bosmina microfossil data were also analyzed, and compared with the corresponding data from a Pb210 dated core, which allowed us to infer the age of the sediment layers. Using changes in Bosmina microfossil morphologies, we were, furthermore, able to infer the presence of different regimes of fish predation. C. quadrangula was found to hatch in layers with an inferred age of approximately a century. Newly hatched individuals had smaller eye‐size in sediment layers corresponding to high predation by young‐of‐the‐year perch. Newly hatched individuals also generally had a marked neck‐spine. In contrast, morphological characters of C. quadrangula clones reared in the laboratory over several generations showed no variation in relation to predation regime, indicating the absence of fixed genotype level changes. Furthermore, the laboratory grown clones only rarely produced a neck‐spine. The results suggest phenotypic variation in response to the regime under which ephippia were produced.     

Tertiary and quaternary structural differences between two genetic variants of bovine casein by small-angle X-ray scattering

The casein complexes of bovine milk consist of four major protein fractions, alpha s1, alpha s2, beta, and kappa. Colloidal particles of casein (termed micelles) contain inorganic calcium and phosphate; they are very roughly spherical with an average radius of 650 A. Removal of Ca2+ leads to the formation of smaller protein aggregates (submicelles) with an average radius of 94 A. Two genetic variants, A and B, of the predominant fraction, alpha s1-casein, result in milks with markedly different physical properties, such as solubility and heat stability. To investigate the molecular basis for these differences, small-angle X-ray scattering was performed on the respective colloidal micelles and submicelles. Scattering curves for submicelles of both variants showed multiple Gaussian character; data for the B variant were previously interpreted in terms of two concentric regions of different electron density, i.e., a "compact" core and a relatively "loose" shell. For the submicelle of A, there was a third Gaussian, reflecting a negative contribution due to interparticle interference. Molecular parameters for submicelles of both A and B are in agreement with hydrodynamic data in the literature. Data for the micelles, for which scattering yields cross-sectional information, were fitted by a sum of three Gaussians for both variants; for these, the corresponding two lower radii of gyration represent the two concentric regions of the submicelles, while the third reflects the average packing of submicelles within the micellar cross section. Most of the molecular parameters obtained showed small but consistent differences between A and B, but for submicelles within the micelle several differences were particularly notable: A has a greater molecular weight for the "compact" region of the constituent submicelle (82,000 vs 60,000) and a much greater submicellar packing number (6:1 vs 3:1). Reasons for these and other differences are to be sought in sequence differences and in differences in calcium-binding sites and charge distribution.     

Active site comparisons highlight structural similarities between myosin and other P-loop proteins.

The phosphate binding loop (P-loop) is a common feature of a large number of enzymes that bind nucleotide whose consensus sequence is often used as a fingerprint for identifying new members of this group. We review here the binding sites of nine purine nucleotide binding proteins, with a focus on their relationship to the active site of myosin. This demonstrates that there is considerable conversation in the distribution and nature of the ligands that coordinate the triphosphate moiety. This comparison further suggests that at least myosin and the G-proteins utilize a similar mechanism for nucleotide hydrolysis.     

The immunological and structural comparisons of deoxyribonucleases I. Glycosylation differences between bovine pancreatic and parotid deoxyribonucleases

A rabbit antiserum against bovine pancreatic DNase A is used to study the immunological reaction of DNases I. As shown by double immunodiffusion, bovine pancreatic DNases A, B, C, and D are immunologically identical, so are DNases from bovine pancreas and parotid and from ovine pancreas. These DNases also behave similarly in immunotitration of DNase activity and all are tightly bound to the immunoaffinity medium, requiring an acidic buffer with 10% ammonium sulfate to dissociate. On the other hand, porcine pancreatic and malted barley DNases that do not form precipitin lines remain active in solution with the antibody; however, in spite of the lack of inhibition these DNases are retarded (but not tightly bound) in immunoaffinity chromatography, suggesting interaction with the antibody. In thin layer isoelectric focusing, the parotid DNase, purified with the immunoaffinity technique, shows only two major active components whose isoelectric points correspond to those of DNases A and C of bovine pancreas. As estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of parotid DNase is 34,000, approximately 3,000 more than that of the pancreatic enzyme. However, both parotid and pancreatic DNases have the same NH2-terminal leucine, an identical COOH-terminal amino acid sequence, nearly identical amino acid compositions, and almost the same peptide maps. The molecular weight difference is due to differences in the carbohydrate side chains. Results of peptide analyses indicate that parotid DNase contains two glycopeptides; pancreatic DNase has only one. In addition, both parotid glycopeptides contain glucosamine and galactosamine while the pancreatic glycopeptide has only glucosamine.     

Simple structural differences between coding and noncoding DNA

Background

The study of large-scale genome structure has revealed patterns suggesting the influence of evolutionary constraints on genome evolution. However, the results of these studies can be difficult to interpret due to the conceptual complexity of the analyses. This makes it difficult to understand how observed statistical patterns relate to the physical distribution of genomic elements. We use a simpler and more intuitive approach to evaluate patterns of genome structure.

Methodology/Principal Findings

We used randomization tests based on Morisita''s Index of aggregation to examine average differences in the distribution of purines and pyrimidines among coding and noncoding regions of 261 chromosomes from 223 microbial genomes representing 21 phylum level groups. Purines and pyrimidines were aggregated in the noncoding DNA of 86% of genomes, but were only aggregated in the coding regions of 52% of genomes. Coding and noncoding DNA differed in aggregation in 94% of genomes. Noncoding regions were more aggregated than coding regions in 91% of these genomes. Genome length appears to limit aggregation, but chromosome length does not. Chromosomes from the same species are similarly aggregated despite substantial differences in length. Aggregation differed among taxonomic groups, revealing support for a previously reported pattern relating genome structure to environmental conditions.

Conclusions/Significance

Our approach revealed several patterns of genome structure among different types of DNA, different chromosomes of the same genome, and among different taxonomic groups. Similarity in aggregation among chromosomes of varying length from the same genome suggests that individual chromosome structure has not evolved independently of the general constraints on genome structure as a whole. These patterns were detected using simple and readily interpretable methods commonly used in other areas of biology.     

Chemical differences between bovine pineal antigonadotropin and arginine vasotocin

Studies were conducted in order to characterize chemically a partially purified antigonadotropic factor extracted from bovine pineal glands (PAG). Because several reports have appeared recently suggesting a role for arginine vasotocin (AVT) as a pineal antigonadotropin, our experiments were designed to determine the presence or absence of this nonapeptide in our material. A comparison of biologically active PAG and synthetic AVT revealed dissimilar UV absorption and fluorescence maxima, different mobilities on thin layer chromatography and paper electrophoresis, as well as different elution patterns on DEAE Sephadex ion-exchange chromatography. Amino acid analysis using 2 different methods revealed dissimilar amino acid compositions. On the basis of these and other chemical data, it is concluded that our preparations of PAG do not contain AVT.     

Sequence and structural differences between enzyme and nonenzyme homologs

To improve our understanding of the evolution of novel functions, we performed a sequence, structural, and functional analysis of homologous enzymes and nonenzymes of known three-dimensional structure. In most examples identified, the nonenzyme is derived from an ancestral catalytic precursor (as opposed to the reverse evolutionary scenario, nonenzyme to enzyme), and the active site pocket has been disrupted in some way, owing to the substitution of critical catalytic residues and/or steric interactions that impede substrate binding and catalysis. Pairwise sequence identity is typically insignificant, and almost one-half of the enzyme and nonenzyme pairs do not share any similarity in function. Heterooligomeric enzymes comprising homologous subunits in which one chain is catalytically inactive and enzyme polypeptides that contain internal catalytic and noncatalytic duplications of an ancient enzyme domain are also discussed.     

The covalent differences between bovine alpha- and beta-thrombin. A structural explanation for the changes in catalytic activity.

The partial covalent structure of bovine beta-thrombin has been determined by the use of automated Edman degradation and carboxypeptidase digestion of the component polypeptide chains separated by gel filtration following either reduction and carboxymethylation or performic acid oxidation. beta-Thrombin has been found to contain three peptide chains derived by proteolysis of the parent alpha-thrombin molecule. The A chain of alpha-thrombin has been cleaved at two points yielding a peptide (A1 chain) which contains 17 amino acids, beginning with threonine 14 and ending with lysine 30. The B chain of alpha-thrombin has been cleaved at two positions to yield a B1 chain which begins with the NH2-terminal isoleucine and terminates with lysine 65 and a B2 chain which begins with lysine 74 and continues through COOH-terminal serine 259. The A1 chain and B2 chain are linked by a disulfide bridge. Although there is no evidence for a covalent bond between the B1 chain and the B2-A1 chains, the B1 chain is tightly bound to the remainder of the molecule, for separation is achieved only under denaturing conditions.     

Functional and structural differences between skinned and intact muscle preparations

Myofilaments and their associated proteins, which together constitute the sarcomeres, provide the molecular-level basis for contractile function in all muscle types. In intact muscle, sarcomere-level contraction is strongly coupled to other cellular subsystems, in particular the sarcolemmal membrane. Skinned muscle preparations (where the sarcolemma has been removed or permeabilized) are an experimental system designed to probe contractile mechanisms independently of the sarcolemma. Over the last few decades, experiments performed using permeabilized preparations have been invaluable for clarifying the understanding of contractile mechanisms in both skeletal and cardiac muscle. Today, the technique is increasingly harnessed for preclinical and/or pharmacological studies that seek to understand how interventions will impact intact muscle contraction. In this context, intrinsic functional and structural differences between skinned and intact muscle pose a major interpretational challenge. This review first surveys measurements that highlight these differences in terms of the sarcomere structure, passive and active tension generation, and calcium dependence. We then highlight the main practical challenges and caveats faced by experimentalists seeking to emulate the physiological conditions of intact muscle. Gaining an awareness of these complexities is essential for putting experiments in due perspective.

IntroductionIn striated muscle, force is generated by sarcomeres located within myocytes (Bers, 2001, 2002). The sarcomere is located within the selectively permeable cell membrane, which supports intracellular ionic homeostasis. Within this highly regulated space, sarcomere force generation is activated by dynamic changes in cytosolic Ca²⁺. The sarcomeric protein troponin C (TnC) binds to Ca²⁺, which prompts the formation of myosin cross-bridges between the sarcomere thick (myosin) and thin (actin) filaments. These myofilaments are arranged in a regular lattice oriented along the muscle fiber direction and form the main structural basis of myocyte contraction. The contraction process is regulated by many other intracellular molecules and ions, in particular Mg²⁺ and H⁺, as well as by cellular and sarcomeric morphologies.To identify the ionic and molecular mechanisms that regulate the sarcomere, it is necessary to control the chemical environment it is exposed to. The biochemistry of the sarcomere proteins can be studied using in vitro biochemistry assays. However, these fail to account for the regular structure of the sarcomere, which is important for both biochemistry and function. Alternatively, the sarcomeres can be accessed by skinning the muscle, i.e., removing the sarcolemma membrane (or making it permeable to compounds and ions), while preserving sarcomere functionality (Curtin et al., 2015). Exposing the sarcomeres to tailored ionic conditions provides a means to observe and control molecular behavior in a setting that more closely resembles native structures. After skinning, the sarcomere system is effectively isolated from the other cellular subsystems (except in some skeletal muscle experiments that remove the sarcolemma while preserving intracellular organelles and structures; Donaldson, 1985; Fill and Best, 1988; Posterino et al., 2000). This facilitates the study of contraction and its regulation separately from the sarcolemma. The central assumption of skinned muscle experiments is that the response of the sarcomeres to changes in the natural cytosol can be reproduced artificially and controllably through analogous changes in the bathing solution.In skinning protocols (typically used with skeletal muscle) where the SR is preserved, applying caffeine liberates the intracellular Ca²⁺ reserves to stimulate contraction (Donaldson, 1985). In cases where the T tubules are preserved in the skinning process, ionic substitution in the bathing solution may induce T-tubule membrane depolarization and hence Ca²⁺ release from the SR (Fill and Best, 1988). An alternative approach to releasing SR calcium is by electric-field stimulation, with the electric field applied transversely relative to the fiber direction (Posterino et al., 2000).The principal readouts of skinned-muscle experiments are contraction kinetics, adenosine triphosphatase (ATPase) activity, and generated force. Their value therefore rests on the premise that the structural integrity of the sarcomeres is preserved. Under this condition, skinned muscle may be viewed as an intermediary experimental system, straddling intact muscle and in vitro molecular experiments.Skinned preparations allow the probing of muscle behavior beyond the current reach of experiments on intact systems. In experiments where contraction is elicited by controlling the bath [Ca²⁺], the influence of “cytosolic” conditions on Ca²⁺ sensitivity, in the steady-state, is typically presented in terms of Hill-type force-[Ca²⁺] relationships, or “F-pCa,” where pCa ≡ − log₁₀[Ca²⁺]/(mol/liter). Other intracellular molecular structures that fulfill structural and mechanical roles (e.g., titin [Cazorla et al., 2001; Fukuda and Granzier, 2005; Fukuda et al., 2005; Li et al., 2016; Tonino et al., 2017] or the cytoskeleton [Roos and Brady, 1989]) can also be investigated. The controlled progression of the system from one equilibrium state to another has helped to reveal, for example, hysteresis in F-pCa, which may potentially fulfill a physiological role but would be difficult to identify in the dynamic natural system (Bers, 2001; Harrison et al., 1988). Dynamic mechanical experiments also yield insight into myofilament kinetics (Breithaupt et al., 2019; Palmer et al., 2020; Stelzer et al., 2006; Terui et al., 2010). In some (mechanical) skinning methods that preserve the T tubules, further details of the excitation–contraction coupling become experimentally accessible (Fill and Best, 1988; Posterino et al., 2000). The ability to perform protein-exchange manipulations (e.g., cardiac versus skeletal TnC; Babu et al., 1988; Gulati and Babu, 1989), to include fluorescent proteins (e.g., troponin; Brenner et al., 1999), and to perform time-resolved dynamics measurements through the flash photolysis of caged compounds (ATP [Goldman et al., 1982, 1984], inorganic phosphate [Araujo and Walker, 1996; Dantzig et al., 1992; Millar and Homsher, 1990; Tesi et al., 2000], and Ca²⁺ chelators [Luo et al., 2002; Wahr et al., 1998]) provide additional handles for probing molecular mechanisms. Overall, much of our understanding of striated muscle generally and cytosolic conditions (temperature, pH, etc.) is derived from skinned-muscle experiments (Bers, 2001).Historically, skinning has been performed in a wide array of animal species and striated muscle systems, ranging from single cells to multicellular fibers of cardiac, skeletal, and smooth muscle. Various skinning techniques have been proposed. In “mechanical” skinning, the sarcolemma is effectively peeled off (entirely or partially; Cassens et al., 1986; Endo, 1977; Trube, 1978) by microdissection (Azimi et al., 2020; Donaldson, 1985; Fabiato, 1985b; Fabiato and Fabiato, 1975, 1977, 1978a, 1978b; Fill and Best, 1988; Godt, 1974; Godt and Maughan, 1977; Jewell, 1977; Lamb and Stephenson, 2018; Matsubara and Elliott, 1972; Moisescu, 1976; Rebbeck et al., 2020), while preserving the structural integrity and function of the T tubules and the SR (Lamb and Stephenson, 1990; Posterino et al., 2000; Stephenson, 1981). However, the technique is difficult and no longer used routinely. In contrast, “chemical” skinning involves dissolving or permeabilizing the membrane by applying a chemical agent. The most common agent is Triton X-100 (Solaro et al., 1971), but alternatives include Brij (Hibberd and Jewell, 1982), lubrol (Scheld et al., 1989), glycerol, and saponin (Edes et al., 1995; Endo and Iino, 1980; Gwathmey and Hajjar, 1990; Launikonis and Stephenson, 1997; Patel et al., 2001). Chemical skinning is particularly appropriate for multicellular tissue preparations. Controlling the precise protocol and chemical agent reportedly allows the selective dissolution of the sarcolemma membrane while leaving intracellular organelles (mitochondria and SR) intact. Nonetheless, treatment with (typically 1%) Triton X-100 frees the myofibrils of contamination by mitochondrial, sarcolemmal, and SR membranes while preserving ATPase activity and sensitivity to Ca²⁺ (Solaro et al., 1971). This straightforwardness makes Triton X-100 demembranation the predominantly used technique today. Other reported skinning approaches use propionate (Reuben et al., 1971) or the Ca²⁺ chelators EGTA or EDTA (Thomas, 1960; Winegard, 1971; Miller, 1979), but the uncertainty in the underlying mechanisms has undermined the reliability of these methods (Miller, 1979). For completeness, we also mention a less used “freeze drying” approach that arguably preserves the protein content of the fibers better than chemical skinning (De Beer et al., 1992; Schiereck et al., 1993; Stienen et al., 1983).Although, for many years, skinned muscle experiments have served as an invaluable method for investigating fundamental physiology, they are increasingly inspiring more ambitious practical applications. At a practical level, live human cells are inevitably a highly scarce resource, with facilities for collecting, storing, and measuring samples often being displaced both geographically and temporally. These issues are more realistically resolved with skinned cells, which can be preserved frozen for several months (Mosqueira et al., 2019). The development of new sarcomere drugs, including omecamtiv mecarbil and mavacamten, demonstrate that the sarcomere is a viable drug target (Tsukamoto, 2019). Similarly, Ca²⁺-sensitizing drugs (which act by increasing either the sensitivity to [Ca²⁺] or the magnitude of the generated force) such as levosimendan (Edes et al., 1995), pimobendan (Fitton and Brogden, 1994; Scheld et al., 1989), sulmazole (Solaro and Rüegg, 1982), isomazole (Lues et al., 1988), and EMD-57033 (Gross et al., 1993; Lee and Allen, 1997) have all been assessed using measurements on skinned fibers. Identifying further novel sarcomere modulator compounds requires large high-throughput screening, which is unrealistic using intact muscle.There is also a growing appetite for exploiting the quantitative value of skinned muscle experiments for more direct clinical applications, such as guiding patient-specific therapies. Much of this ambition relies on the integrative power of computational models to simulate human heart mechanics based on individual patients’ data, linking sub-cellular mechanisms with systemic behavior (Niederer et al., 2019a, 2019b). Building upon basic understanding of muscle behavior, recent developments in biomedical engineering extrapolate physiological processes at the cellular and tissue levels to predict global whole-heart function. As this field continues to grow in maturity, and as model predictions allow more meaningful comparisons with clinical data, efforts are increasingly focusing on quantitatively elucidating the interdependence between cellular behavior, tissue properties, and the anatomy. The quantitative accuracy of the subsystems at all these levels therefore becomes paramount.In both of these evolving applications, the relevance and value of skinned-muscle experiments hinges on their ability to reliably emulate the intact system (Land et al., 2017; Margara et al., 2021; Mijailovich et al., 2021). Skinned-muscle experiments conducted over the past decades confirm the fidelity, in many respects, of these preparations as valid experimental models. However, they also highlight caveats and significant interpretational challenges. Gaining an awareness of these issues is becoming all the more essential to avoid misinterpretations that may have practical consequences. This review therefore aims to highlight these challenges, to help users of skinned-based measurements put them in an appropriate perspective.The present review is structured as follows. We first compare measurements of the principal physiological properties of skinned and intact muscle, highlighting similarities and discrepancies. We focus primarily on chemical skinning, and in particular Triton X-100 (the predominantly used chemical agent). We then describe practical challenges involved in conducting experiments, insofar as they impact on measurement outcomes. We conclude with a summary of recommendations and main caveats.Comparing skinned and intact muscleSkinned muscle experiments aim to reveal and controllably reproduce features of the physiological function of sarcomeres. However, notable discrepancies arise between skinned- and intact-muscle measurements of basic muscle properties that govern overall muscle function. To establish these differences rigorously at the single-cell level encounters significant methodological challenges. Although it might seem obvious that this would require doing measurements systematically on both preparation types in tandem, many early experiments were done predominantly on skinned rather than on intact cells (King et al., 2011). This stems largely from the specific challenges of noninjurious cell attachment and performing small-force measurement on intact single cells (Brady, 1991). More recently, technical developments (e.g., involving the use of flexible carbon fibers to hold the cells at opposite ends; Iribe et al., 2007; Le Guennec et al., 1990; Yasuda et al., 2001) have made these measurements more practicable. Despite these advances, however, only a fraction of studies in the literature have systematically made direct comparisons between skinned and intact systems taken from the same species under optimally similar conditions (see the selection listed in

Evidence for structural homology between murine and human Ia antigens

The serological cross-reactivity and the structural homology of murine and human Ia alloantigens were analyzed. Both normal human peripheral blood B lymphocytes and chronic lymphocytic leukemia (CLL) cells were shown to be lysed in the presence of complement by both murine anti-Ia and human anti-HLA-DR alloantisera. A mouse A.TH anti-A.TL (anti-I ^k ) alloantiserum reacted with determinants expressed on all of the 20 normal human B cell populations tested. Only 3 of these 20 B cell populations were lysed with an A.TL anti-A.TH anti-I ^s alloantiserum. The frequency of cytotoxic cross-reactivity concordant with anti-I ^k appears to be greater for anti-I-EC ^k than for anti-I-A ^k alloreactivity. An immunochemical analysis demonstrated that Iaα-chain andβ-chain polypeptides may be immunoprecipitated from CLL cell lysates by either a mouse anti-I ^k alloantiserum or various human anti-HLA-DR alloantisera. The Ia molecules detected with the mouse and human antisera are coprecipitable as revealed by one-dimensional gel electrophoresis. Two-dimensional gel electrophoresis studies indicated that the human CLL cell Ia antigens analyzed possess considerable molecular heterogeneity. They are structurally more similar, with respect to molecular size and charge, to mouse Ia antigens determined by the murineH-2-linkedI-EC subregion rather than theI-A subregion. The structural, genetic and functional implications of these findings are discussed.     

Molecular structural differences between type-2-diabetic and healthy glycogen

Glycogen is a highly branched glucose polymer functioning as a glucose buffer in animals. Multiple-detector size exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis were used to examine the structure of undegraded native liver glycogen (both whole and enzymatically debranched) as a function of molecular size, isolated from the livers of healthy and db/db mice (the latter a type 2 diabetic model). Both the fully branched and debranched levels of glycogen structure showed fundamental differences between glycogen from healthy and db/db mice. Healthy glycogen had a greater population of large particles, with more α particles (tightly linked assemblages of smaller β particles) than glycogen from db/db mice. These structural differences suggest a new understanding of type 2 diabetes.     

Free-energy calculations highlight differences in accuracy between X-ray and NMR structures and add value to protein structure prediction

BACKGROUND: While X-ray crystallography structures of proteins are considerably more reliable than those from NMR spectroscopy, it has been difficult to assess the inherent accuracy of NMR structures, particularly the side chains. RESULTS: For 15 small single-domain proteins, we used a molecular mechanics-/dynamics-based free-energy approach to investigate native, decoy, and fully extended alpha conformations. Decoys were all less energetically favorable than native conformations in nine of the ten X-ray structures and in none of the five NMR structures, but short 150 ps molecular dynamics simulations on the experimental structures caused them to have the lowest predicted free energy in all 15 proteins. In addition, a strong correlation exists (r(2) = 0.86) between the predicted free energy of unfolding, from native to fully extended conformations, and the number of residues. CONCLUSIONS: This work suggests that the approximate treatment of solvent used in solving NMR structures can lead NMR model conformations to be less reliable than crystal structures. This conclusion was reached because of the considerably higher calculated free energies and the extent of structural deviation during aqueous dynamics simulations of NMR models compared to those determined by X-ray crystallography. Also, the strong correlation found between protein length and predicted free energy of unfolding in this work suggests, for the first time, that a free-energy function can allow for identification of the native state based on calculations on an extended state and in the absence of an experimental structure.