Intact Triad Research Articles

Perceived Conflicting Desires to Delay the First Birth: A Household-Level Exploration in Nepal.

It is accepted as the norm that couples in South Asia begin childbearing immediately after marriage and that, even if they would like to delay, they are pressured to have children by household members. Little research, however, has explored the desire to delay childbearing among newly married couples and their household members in Nepal-a setting with changing marriage formation patterns, increasing women's education and falling fertility. To explore the dynamics of current childbearing desires, in-depth interviews of 20 intact triads of newly married women, their husbands and their mothers-in-law were conducted in one district of Nepal in February-March 2017. Using thematic analysis, interviews were read and coded separately by type (wives, husbands, mothers-in-law), and then the triads were read together and coded to determine household-level patterns and themes. Most newly married women and men want to delay their first birth, but have not communicated with each other about this. Even though couples are often in agreement about delaying, they feel pressured by in-laws and society to bear children early. Contrary to expectations, some mothers-in-law support delaying childbearing to allow their daughter-in-law to mature, continue her education or earn wages; however, they too perceive societal pressure. Male migration for work also contributes to early childbearing pressure. Helping couples to sort through conflicting fertility norms and desires may be important to delay childbearing when desired. Programs should engage all household members, and work to increase couples' and household communication to address misperceptions about fertility desires.

Semi-arranged marriages and dowry ambivalence: Tensions in the changing landscape of marriage formation in South Asia

Marriage is a point of change in young people’s lives, especially in parts of the world that place high value on it, such as in South Asian countries including Nepal. However, marriage practices are changing, with a move towards more love marriage; this is likely to have important implications on women’s status and agency, household and couple dynamics, and mental and physical health. The aim of this paper is to describe how changing marriage formation patterns and traditional practices such as co-residence and dowry are intersecting and impacting relationships post-marriage. In-depth qualitative interviews took place with 20 intact triads of newly married women, their husbands and their mothers-in-law, in one district of Nepal in 2017. Many marriages remain arranged; however, couples often talk or meet before marriage and feel that they are able to build a foundation of love before marrying. Access to technology facilitates this practice, although some couples are reluctant to admit their communication, suggesting stigma about this practice. Husbands have growing ambivalence about dowry, leading to confusion and negatively impacting on relationships post-marriage. A clash of traditional and modern ideas and practices is occurring in Nepal, influencing newly married women’s household status and relationship quality, and potentially impacting women’s health.

Overlapping patterns of neural activity for different forms of novelty in fMRI.

When stimuli are presented multiple times, the neural response to repeated stimuli is reduced relative to novel stimuli (repetition suppression). Responses to different types of novelty were examined. Stimulus novelty was examined by contrasting first vs. second presentation of triads of objects during memory encoding. Semantic novelty was contrasted by comparing unrelated (semantically novel) triads of objects to triads in which all three objects were related (e.g., all objects were tools). In recognition, associative novelty was examined by contrasting rearranged triads (previously seen objects in a new association) with intact triads. Activity was observed in posterior regions (occipital and fusiform), with the largest extent of activity for stimulus novelty and smallest for associational novelty. Frontal activity was also observed in stimulus and semantic novelty. Additional analysis indicated that the hemodynamic response in voxels identified in the stimulus and semantic novelty contrasts was modulated by reaction time on a trial-by-trial basis. That is, the duration of the hemodynamic response was driven by reaction time. This was not the case for associative novelty. The high level of overlap across different forms of novelty suggests a similar mechanism for reduced neural activity, which may be related to reduced visual processing time. This is consistent with a facilitation model of repetition suppression, which posits a reduced peak and duration of neuronal firing for repeated stimuli.

Disrupted Membrane Structure and Intracellular Ca2+ Signaling in Adult Skeletal Muscle with Acute Knockdown of Bin1

Efficient intracellular Ca2+ ([Ca2+]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca2+ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short –hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca2+ release. Voltage-induced Ca2+ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca2+ current and SR Ca2+ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca2+ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca2+ sparks' amplitude that was attributed to decreased total Ca2+ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca2+]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy.

Bin1, A Bar Domain Protein, Is Necessary For The Maintenance Of T-tubule Structure And Intracellular Ca2+ Homeostasis In Skeletal Muscle

Efficient intracellular Ca2+ homeostasis in skeletal muscle requires establishment and maintenance of an intact triad junctional complex. Bin1, an amphiphysin family protein contains a conserved BAR domain that facilitates membrane curvature. Bin1 also contains a unique phosphatidylinositol-4,5-bisphosphate (PIP2) binding domain that is required for transverse-tubule membrane biogenesis during C2C12 myogenic cell differentiation. The lethality associated with Bin1 knockout limits the opportunities to study the function of Bin1 in adult skeletal muscle. Using in vivo electroporation to deliver shRNA against Bin1, we knockdown Bin1 expression in the FDB muscle of adult mice. We find that transient loss of Bin1 alters the properties of Ca2+ sparks induced by osmotic stress. Specifically, we observe that the mean frequency of Ca2+ sparks per minute is reduced in fibers that exhibit a high level of Bin1 knockdown (33.1 ± 5.7), compared to control fibers (138.1 ± 21.7). Kinetic analysis of individual Ca2+ sparks shows that spark amplitude (ΔF/F0) is reduced in the Bin1 knockdown fibers (0.67 ± 0.02) when compared to control (0.85 ± 0.01). Bin1 knockdown also alters the full duration at half maximal (FDHM) of Ca2+ sparks. A two-exponential decay function fit of the FDHM histograms indicates that, in the control fiber, the corresponding time constants for Ca2+ sparks (t01) are 38.0 ± 2.8 ms and 281.3 ± 40.0 ms for Ca2+ bursts (t02). The time constant for Ca2+ bursts (t02) are significantly reduced in the high Bin1 knockdown muscle fiber (140.9 ± 41.2 ms). Electron microscopy reveals Bin1 knockdown fibers exhibit vacuolation and swelling of t-tubule structures. Thus, alteration of triad junction structure can potentially affect the resting cytosolic Ca2+ levels and internal Ca2+ stores in Bin1 knockdown fibers.

Peptide and protein modulation of local Ca2+ release events in permeabilized skeletal muscle fibers

Local discrete elevations in myoplasmic Ca2+ (Ca2+ sparks) arise from the opening of a small group of RyRs. Summation of a large number of Ca2+ sparks gives rise to the whole cell Ca2+ transient necessary for muscle contraction, Unlike sarcoplasmic reticulum vesicle preparations and isolated single channels in artificial membranes, the study of Ca2+ sparks provides a means to understand the regulation of a small group of RyRs in the environment of a functionally intact triad and in the presence of endogenous regulatory proteins. To gain insight into the mechanisms that regulate the gating of RyRs we have utilized laser scanning confocal microscopy to measure Ca2+ sparks in permeabilized frog skeletal muscle fibers. This review summarizes our recent studies using both exogenous (ImperatoxinA and domain peptides) and endogenous (calmodulin) modulators of RyR to gain insight into the number of RyR Ca2+ release channels underlying a Ca2+ spark, how domain-domain interactions within RyR regulate the functional state of the channel as well as gating mechanisms of RyR in living muscle fibers.

Disulfide bonds, N-glycosylation and transmembrane topology of skeletal muscle triadin.

Native triadin is a disulfide linked homopolymer of variable subunit number. Two monoclonal antibodies (mAbs), AE8.91 and GE4.90, recognize cytoplasmic regions of triadin between amino acids 110 and 163 and at the C-terminal 34 amino acids, respectively. Triadin in intact triads is largely unaffected by trypsin, while triads whose membrane has been disrupted by hypotonicity or by treatment with the detergent Triton X-100 yield both soluble and membrane bound fragments. Soluble fragments monitored by mAb GE4.90 appear to be formed sequentially during the course of proteolysis at 28, 16, 10 and 7 kDa in the presence of mercaptoethanol. Higher molecular weight bands are observed under nonreducing conditions. A two-dimensional electrophoresis immunoblot (first nonreducing; second reducing) of the soluble fragments developed with mAb GE4.90 shows the presence of several bands which can be interpreted as containing a dimer formed by a combination of any two of the fragments of 16, 10, or 7 kDa present in the digest. MAb AE8.91 does not detect these fragments. This observation indicates that one of the intermolecular disulfide bonds is formed between the identical domains of two triadin molecules at cysteine 671. Immunoblots performed with and without mercaptoethanol of the insoluble fragments using mAb AE8.91 indicate the presence of a dimer formed between identical domains of two triadin intermolecular disulfide linkage at cysteine 270. The glycosidase endo F/N-glycosidase F changed the mobility of intact triadin in TC/triads and its proteolytic fragments detected by mAb GE4.90.(ABSTRACT TRUNCATED AT 250 WORDS)

When We Don’t See Eye to Eye: Discrepancies between Supervisors and Subordinates in Absence Disciplinary Decisions

The present study provided a within-subjects assessment of the factors associated with absence disciplinary decisions for both supervisors and subordinates. In addition, this study examined discrepancies in disciplinary decisions between supervisors and their subordinates based on differences in psychological and demographic attributes. A sample of non-academic employees from 19 intact triads (one supervisor; two subordinates) at a large Midwest university responded to hypothetical scenarios describing factors that might contribute to absence disciplinary decisions. The results demonstrated that both supervisors and subordinates generally place similar weights on factors that are relevant to disciplinary decisions. Perhaps more importantly, a number of psychological and demographic differences between supervisors and subordinates related positively to discrepancies in disciplinary decisions.

When we don't see eye to eye: Discrepancies between supervisors and subordinates in absence disciplinary decisions

The present study provided a within-subjects assessment of the factors associated with absence disciplinary decisions for both supervisors and subordinates. In addition, this study examined discrepancies in disciplinary decisions between supervisors and their subordinates based on differences in psychological and demographic attributes. A sample of non-academic employees from 19 intact triads (one supervisor; two subordinates) at a large Midwest university responded to hypothetical scenarios describing factors that might contribute to absence disciplinary decisions. The results demonstrated that both supervisors and subordinates generally place similar weights on factors that are relevant to disciplinary decisions. Perhaps more importantly, a number of psychological and demographic differences between supervisors and subordinates related positively to discrepancies in disciplinary decisions.

Primary structure and topological analysis of a skeletal muscle-specific junctional sarcoplasmic reticulum glycoprotein (triadin)

The primary amino acid sequence for a highly abundant junctional sarcoplasmic reticulum glycoprotein (triadin) has been deduced from the cDNA sequence. Based on both biochemical analysis and the predicted amino acid sequence we suggest that this protein is an intrinsic membrane glycoprotein containing a single transmembrane domain that separates the protein into cytoplasmic and luminal domains. The cytoplasmic domain is proposed to contain the amino-terminal 47 amino acids. The remainder of the protein including the carboxyl terminus is proposed to be found within the lumen of the sarcoplasmic reticulum and contains an extremely high concentration of basic residues. Protease analysis of intact triads was consistent with the topological predictions. Western and Northern blots suggest that the protein is specifically expressed in skeletal muscle and not cardiac muscle or brain. The abundance and localization of this protein suggest that it plays an important regulatory or structural role in excitation-contraction coupling in skeletal muscle.

Strength and co-operativity of contributions of surface salt bridges to protein stability

Many of the interactions that stabilize proteins are co-operative and cannot be reduced to a sum of pairwise interactions. Such interactions may be analysed by protein engineering methods using multiple thermodynamic cycles comprising wild-type protein and all combinations of mutants in the interacting residues. There is a triad of charged residues on the surface of barnase, comprising residues Asp8, Asp12 and Arg110, that interact by forming two exposed salt bridges. The three residues have been mutated to alanine to give all the single, double and triple mutants. The free energies of unfolding of wild-type and the seven mutant proteins have been determined and the results analysed to give the contributions of the residues in the two salt bridges to protein stability. It is possible to isolate the energies of forming the salt bridges relative to the solvation of the separated ions by water. In the intact triad, the apparent contribution to the stabilization energy of the protein of the salt bridge between Asp12 and Arg110 is -1.25 kcal mol-1, whereas that of the salt bridge between Asp8 with Arg110 is -0.98 kcal mol-1. The strengths of the two salt bridges are coupled: the energy of each is reduced by 0.77 kcal mol-1 when the other is absent. The salt-linked triad, relative to alanine residues at the same positions, does not contribute to the stability of the protein since the favourable interactions of the salt bridges are more than offset by other electrostatic and non-electrostatic energy terms. Salt-linked triads occur in other proteins, for example, haemoglobin, where the energy of only the salt-bridge term is important and so the coupling of salt bridges could be of general importance to the stability and function of proteins.

Evidence for the association of dystrophin with the transverse tubular system in skeletal muscle.

Polyclonal antibodies to dystrophin (the protein product of the human Duchenne muscular dystrophy gene) were used to identify and characterize dystrophin in isolated triads from rabbit skeletal muscle. Anti-dystrophin antibodies recognize an approximately 400,000-Da protein in isolated triads or heavy microsomes from skeletal muscle. Treatment of heavy microsomes with buffers containing high salt or EDTA to remove peripheral or extrinsic membrane proteins does not remove dystrophin; however, treatment of intact triads with trypsin shows that dystrophin is extremely sensitive to mild proteolytic digestion. Isolation of junctional complexes from skeletal muscle triads indicates that dystrophin is tightly associated with the triadic junction. Fractionation of the triadic junction into junctional transverse tubular membranes and junctional sarcoplasmic reticulum membranes has shown that dystrophin is enriched in junctional transverse tubular membranes. Thus, our results suggest that dystrophin is a component of the triad junction which is exposed to the cytoplasm and embedded in or attached to the transverse tubular membrane.

6] Isolation of triads from skeletal muscle

This chapter describes methodology for the isolation of morphologically intact triad structures. Excitation-contraction coupling in skeletal muscle is initiated at the neuromuscular junction. The action potential then proceeds longitudinally along the length of the fiber and transversely to within the fiber by way of invaginations from the plasmalemma, referred to as transverse tubules. The latter are junctionally associated with the terminal cisternae of sarcoplasmic reticulum via "feet" structures. By an unknown mechanism, Ca 2+ is then released from the terminal cisternae, elevating the myoplasmic calcium concentration, and—thereby, triggering muscle contraction. The structure formed by the junctional association of the transverse tubule with the terminal cisternae is referred to as the "triad," and the junction is the triad junction. The methodology is presented in two sections: (1) Procedures for triad isolation, and (2) characterization of the isolated triads. A method of sample preparation for electron microscopy was developed to estimate the purity and morphological integrity of our subcellular fractions. Dextran is added as a nonosmophilic spacer to the prefixed sample and filtration is used in the preparation for thin sectioning.

Biochemical properties of purified transverse tubules isolated from skeletal muscle triads

Transverse tubules (t-tubules) were prepared from muscle by dissociation of intact triads during centrifugation in ion-free sucrose gradients. They were further purified by the removal of contaminating sarcoplasmic reticulum after loading with calcium phosphate. Purification was accompanied by enrichment in markers specific for t-tubules, e.g., nitrendipine binding sites. According to gel electrophoresis the purified t-tubules contained three major protein bands of 104, 70, and 30 kDa. When solubilized with detergents there was a two- to threefold increase in Mg 2+-ATPase activity, and a corresponding increase in the 30-kDa protein band. The 104-kDa protein was shown to be a (Na + + K +)-ATPase because of its phosphorylation by [γ- 32P]ATP in the presence of sodium ions. The orientation of the t-tubule membrane was predominantly inside-out.

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes.

Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.

Biochemical characterization, integrity, and sidedness of purified skeletal muscle triads.

The release of Ca2+ from the terminal cisternae of sarcoplasmic reticulum in muscle fiber triggers muscle contraction. The signal for Ca2+ release is mediated via the triad junction, i.e. the junctional association of terminal cisternae and transverse tubule. Recently, highly purified morphologically intact triads were isolated from rabbit skeletal muscle (Mitchell, R. D., Palade, P., and Fleischer, S. (1983) J. Cell Biol. 96, 1008-1016). In this study, biochemical characterization of two variants of purified triad preparations (Pyrophosphate and Standard) is provided. Terminal cisternae of triads sequester Ca2+ at rates comparable to those of purified heavy sarcoplasmic reticulum which is referable to terminal cisternae (Meissner, G. (1975) Biochim. Biophys. Acta 389, 51-68). The permeability for calcium ions, as reflected by a 2-3-fold stimulation of (Ca2+, Mg2+)-ATPase activity in the presence of the Ca2+ ionophore A23187, and by the Ca2+ leak rate, is comparable in triads and heavy sarcoplasmic reticulum. Several transverse tubule characteristics are present in triads. Four of them, i.e. cholesterol content, ouabain binding, dihydroalprenolol binding (beta-adrenergic receptor), and ouabain-sensitive (Na+, K+)-ATPase activity, are comparably enriched in the Pyrophosphate triads and therefore appear to be quantitative indices of the amount of transverse tubule. Adenylate cyclase and basal ATPase are unreliable in this regard. Methodology for analyzing membrane integrity and sidedness was applied (adenylate cyclase activity) and modified (ouabain-sensitive (Na+, K+)-ATPase activity) to characterize the transverse tubule of the triad. In addition, a new method was developed making use of ouabain binding to study sidedness. These studies show that the transverse tubule is largely sealed and inside out in orientation, i.e. with the cytoplasmic face exposed. This report indicates that the t-tubule and sarcoplasmic reticulum components of the triads possess transport capability and retain permeability barriers for ions. Therefore, the isolated triads appear to be suitable for studying the physiological Ca2+ release process in vitro.

Purification of morphologically intact triad structures from skeletal muscle.

A procedure has been devised for isolation of triads (t-tubule/sarcoplasmic reticulum (SR) junctional complexes) from rabbit skeletal muscle. The procedure consists of preparation of a heavy microsomal fraction followed by two sequential 90-min sucrose gradient centrifugations to enrich the triads. A pyrophosphate/phosphate/magnesium buffer system was introduced to decrease aggregation in order to achieve effective separation. The preparation time is 12 h. Some differences between purified triads isolated by two variants of this method are noted. The purity of the triad fractions has been estimated by particle counting to be in the vicinity of 50%. There is good retention of morphology and Ca++-loading activity and enrichment in Na+,K+-ATPase and adenylate cyclase. The triads are practically devoid of contractile elements, mitochondria, and free plasmalemma, and low in content of light SR. The method for obtaining enriched triads is reproducible, and sufficient yields are obtained for structural, biochemical, and functional characterization.

Identification of a constituent of the junctional feet linking terminal cisternae to transverse tubules in skeletal muscle.

This study describes the biochemical composition of junctional feet in skeletal muscle utilizing a fraction of isolated triad junctions. [3H]Ouabain entrapment was employed as a specific marker for T-tubules. The integrity of the triad junction was assayed by the isopycnic density of [3H]ouabain activity (24-30% sucrose for free T-tubules, 38-42% sucrose for intact triads). Trypsin, chymotrypsin, and pronase all caused separation of T-tubules from terminal cisternae, indicating that the junction is composed as least in part of protein. Trypsin and chymotrypsin hydrolyzed four proteins: the Ca2+ pump, a doublet 325,000, 300,000, and an 80,000 Mr protein. T-tubules which had been labeled covalently with 125I were joined to unlabeled terminal cisternae by treatment with K cacodylate. The reformed triads were separated from free T-tubules and then severed by passage through a French press. When terminal cisternae were separated from T-tubules, some 125I label was transferred from the labeled T-tubules to the unlabeled terminal cisternae. Gel electrophoresis showed that, although T-tubules were originally labeled in a large number of different proteins, only a single protein doublet was significantly labeled in the originally unlabeled terminal cisternae. This protein pair had molecular weights of 325,000 and 300,000 daltons. Transfer of label did not occur to a substantial degree without K cacodylate treatment. We propose that the transfer of 125I label from T-tubules to terminal cisternae during reformation and breakage of the triad junction is a property of the protein which spans the gap between T-tubules and terminal cisternae.

Interconsequence generality of learned helplessness

The present study tested the interconsequence generality of the learned helplessness phenomenon. Thirty-six rats received escapable, inescapable, or no shock in a shuttlebox. Intact triads were then randomly assigned to two groups. The first group was required to learn to escape shock via barpressing; the second was required to learn a six-unit maze for food reward. The shock-escape triads demonstrated the standard learned helplessness effect, with inescapable subjects inferior to escapable and unshocked animals. However, the triads tested in the appetitive situation did not exhibit learned helplessness, but rather, they exhibited a trauma-like effect, with both escapable and inescapable subjects inferior to no-shock control subjects on early trials. These findings demonstrated a limit to the generality of learned helplessness. It was suggested that learned helplessness follows the rules of stimulus and response generalization.