大黄素对豚鼠结肠带平滑肌细胞电和收缩性能的影响

联合应用平滑肌肌力张力测量技术和细胞内微电极记录技术,同步地现测豚鼠结肠带平滑肌自发的肌源性电活动和力学活动,研究了大黄素的药物作用。大黄素能缩短膜电位的波动周期,从而缩短峰电位集簇发放的周期;相应地,可使平滑肌的分节律收缩加快,幅值指数升高。大黄素又能促使细胞膜电位自发的周期性波动的出现,导致峰电位的集簇发放;相应地,可使强直收缩转化为分节律收缩,即促进收缩形式向有利于肠道推进功能的方向转化。以上结果表明,大黄素能有效地提高豚鼠结肠带平滑肌细胞的电兴奋性和收缩功能,并且对其电学和力学活动的影响之间有明确的对应关系。     

Parallel measurements of bound calcium and force in glycerinated rabbit psoas muscle fibers

A simple double-isotope procedure has been developed for making simultaneous measurements of bound Ca²⁺ and relative force in glycerinated rabbit psoas bundles containing two fibers. With this preparation it is possible to study Ca²⁺-troponin interactions coincident with MgATP-induced force development. Over the free [Ca²⁺] range 6 · 10^?8–1.2 · 10^?5 M the bound Ca²⁺ varied from 0.25 to 1.65 μmol/g protein. The free [Ca²⁺] at half-maximal Ca²⁺ saturation was 2 · 10^?7 M while that a half-maximal force was 5 · 10^?7 M. Half-maximal Ca²⁺ saturation was associated with 20% maximal force. The force-[Ca²⁺] saturation curve showed a steep rise in slope at greater than half saturation. The observed relationship was consistent with a model in which multiple occupancy of troponin Ca²⁺-binding sites is essential for initiation of cross-bridge cycling.     

Motory effects of perforating peripheral cell layers ofAmoeba proteus

Summary Perforation of peripheral cell layers ofA. proteus in any place provokes immediate endoplasm efflux, what supports the view that the hydrostatic pressure is higher in the cell interior than outside. The local effusion of endoplasm results in the reversal of flow in formerly advancing pseudopodia, in agreement with the pressure gradient theories of protoplasmic streaming. Amoebae with destroyed frontal zones squeeze all their endoplasm out through the breach, what disproves the frontal contraction hypothesis of amoeboid movement, but supports the concept of a general contraction of cell cortex.Study supported by the Research Project II.1 of the Polish Academy of Science.     

Cytoplasmic actomyosin fibrils in tissue culture cells

Summary A special cell line derived from a rat mammary adenocarcinoma (RMCD cells) displays a distinct pattern of actomyosin fibrils (AM fibrils) visible with phase contrast, Nomarski interference and polarized light optics. It was shown that the cytoplasmic AM fibrils are arranged as bundles of highly parallel F-actin filaments. The chemical nature of the filaments was identified by incubation with heavy meromyosin from rabbit skeletal muscle.These cytoplasmic actomyosin fibrils actively contract under isotonic conditions. This was shown by contraction experiments under polarized light optics, by cinematographic analysis and by direct proof of the contractility of AM fibrils isolated by laser micro-dissection. Thus, cytoplasmic AM fibrils can be assumed to represent structures essential for motive force generation in contraction processes in non-muscle cells.We thank Dr. W. Meier-Ruge and Mr. W. Bielser (Basic Medical Research Departments, Sandoz AG, Basle, Switzerland) for making the laser equipment available to us and for their kind cooperation during this investigation. Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Structural changes in smooth muscle cells during isotonic contraction

Summary Smooth muscle cells of the guinea-pig taenia coli were studied in light and electron microscopy, in condition of mild stretch or of isotonic contraction. During contraction the cells increase in transverse sectional area and their packing density passes from 94,000 · mm^-2 to 18,000 · mm^-2. The percentage increase in transverse sectional area of the taenia is approximately the same as the percentage decrease in length. Measurements of cell transverse sectional area suggest that the individual cells shorten and fatten more than the taenia as a whole. Whereas stretched muscle cells run parallel to each other and show a fairly smooth surface, isotonically contracted cells are twisted and entwine around each other. Their surfaces are covered with myriad processes and folds. Longitudinal, transverse or oblique stripes are seen in light microscopy in the contracted muscle cells and it is suggested that they are related to the characteristics of the cell surface. In electron microscopy a complex pattern of interdigitating finger-like and laminar processes is observed. Caveolae are mainly found on the evaginated parts of the cell surface, dense patches are mainly (but not always) found on the invaginated parts. Desmosome-like attachments between contracted cells are frequent. The collagen fibrils run approximately parallel to the stretched muscle cells; on the other hand, they run obliquely and transversely around the isotonically contracted cells.This work is supported by the Medical Research Council. I thank Miss E.M. Franke and Mr S.J. Sarsfield for excellent technical assistance     

Acute Hypoxemia Does Not Increase the Oxidative Stress in Resting and Contracting Muscle in Humans

In healthy humans sustaining static handgrip at 60% of maximal voluntary contraction (MVC) until exhaustion, we measured the venous blood concentration of reduced ascorbic acid (RAA) and thiobarbituric acid reactive substances (TBARS), respectively, used as markers of the post-exercise oxidative stress and lipid peroxidation. Measurements were conducted in normoxemia, then during a 30-min period of hypoxemia (PaO 2 =56 mmHg) produced by inhalation of an hypoxic gas mixture. Compared to normoxemia, hypoxemia did not significantly modify the resting concentrations of TBARS and RAA, and did not affect the consumption of ascorbic acid after 60% MVC but suppressed the post-exercise TBARS increase. We conclude that acute hypoxemia does not modify the production of oxygen free radicals after strenuous static efforts and even seems to attenuate the lipid peroxidation.     

Intracellular signalling mechanisms regulating glucose transport in insulin-sensitive tissues

The rate of glucose transport into cells is of fundamental importance in whole body homeostasis and adaptation to metabolic stresses, and this review examines the signalling mechanisms controlling this process. The events that mediate the action of insulin on glucose transport, which is by far the best characterized paradigm for glucose transport regulation, are discussed. There are several excellent reviews on various aspects of this subject, which are referred to while highlighting very recent developments in the field, including the recently described CAP pathway, and emerging mechanisms for feedback regulation of insulin signalling. The manner in which hormonal signalling is modulated by stimuli such as oxidative and osmotic stress is then discussed. The second major physiological event where glucose transport regulation is critical is the contraction of skeletal muscle, due to the large metabolic demands of this activity. The mechanism of this regulation is distinct from that initiated by insulin, and recent developments will be examined that have begun to clarify how contraction stimulates glucose transport in skeletal muscle, including the roles performed by AMP-activated protein kinase and nitric oxide synthase.     

Biochemical characterization of distinct regions of SPEC molecules and their role in phagocytosis

Cdc42 signaling pathways play important roles in immune cell polarization and cytoskeletal changes. Although the small Cdc42-binding proteins SPEC1 and SPEC2 play a role in F-actin accumulation in activated T lymphocytes, little is known about their precise activities in other cell types. Here, we mapped the Cdc42-binding activity of SPEC1 to the CRIB sequence and a downstream alpha helical region. Biochemical studies revealed that SPEC1 did not interact with a Rac1 switch-of-function mutant capable of inducing Cdc42-like filopodia, potentially eliminating a role for SPECs in this process. A phosphoinositide-binding region was identified within a basic region N-terminal to the CRIB sequence of SPEC1. Using an anti-SPEC2 antibody, we found that endogenous SPEC2 colocalized with Cdc42 at the phagocytic cup of macrophages internalizing zymosan A particles prior to significant F-actin accumulation. Overexpression studies of the related SPEC1 protein induced marked macrophage contraction and prevented particle binding and phagocytosis. Although a Cdc42-binding mutant of SPEC1 still caused macrophage contraction, mutations within the N-terminal cysteines and phosphoinositide-binding region reversed macrophage contraction but still resulted in impaired phagocytosis. These results identify three distinct structural and functional regions within SPECs and demonstrate their likely role in early contractile events in phagocytosis.     

How to correct relative voxel scale factors for calculations of vector-difference Fourier maps in cryo-EM

The atomic coordinates derived from cryo-electron microscopy (cryo-EM) maps can be inaccurate when the voxel scaling factors are not properly calibrated. Here, we describe a method for correcting relative voxel scaling factors between pairs of cryo-EM maps for the same or similar structures that are expanded or contracted relative to each other. We find that the correction of scaling factors reduces the amplitude differences of Fourier-inverted structure factors from voxel-rescaled maps by up to 20–30%, as shown by two cryo-EM maps of the SARS-CoV-2 spike protein measured at pH 4.0 and pH 8.0. This allows for the calculation of the difference map after properly scaling, revealing differences between the two structures for individual amino acid residues. Unexpectedly, the analysis uncovers two previously overlooked differences of amino acid residues in structures and their local structural changes. Furthermore, we demonstrate the method as applied to two cryo-EM maps of monomeric apo-photosystem II from the cyanobacteria Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus. The resulting difference maps reveal many changes in the peripheral transmembrane PsbX subunit between the two species.     

Mechanical analysis of intestinal contractility in a neonatal maternal deprivation irritable bowel syndrome rat model

The aims of the present study are to investigate biomechanical properties and provide mechanical analysis of contractility in ileum and colon in a neonatal maternal deprivation (NMD) irritable bowel syndrome (IBS) rat model. Mechanical testing was done on segments from ileum and colon in 25 IBS rats and 13 Control rats. Morphometric data were obtained from digitized images of the segments at no-load and zero-stress states. Pressure and diameter changes were measured during flow and ramp distensions under active and passive experimental conditions. Circumferential stresses (force per area) and strains (deformation) were computed with referenced to the zero-stress state. The contraction frequency was analyzed. Contraction thresholds and maximum contraction amplitude were calculated in terms of mechanical stress and strain. Compared with controls, the IBS rats had lower body weight (P < 0.01), smaller colonic opening angle (P < 0.05), higher colonic contraction frequency (P < 0.05 and P < 0.01) and lower contraction thresholds of pressure, stress and strain in both ileum and colon (P < 0.05 and P < 0.01). The maximum contraction pressure, stress and strain did not differ between IBS and Control groups (P > 0.05). In conclusion, the pressure, stress, and strain to evoke contractility in ileum and colon were lower whereas the frequency of induced colon contractions was higher in NMD IBS rats compared to normal rats. Furthermore, zero-stress state remodeling occur in colon in NMD IBS rats. Further studies on the association between intestinal biomechanical properties, hypersensitivity and afferent signaling in the IBS animal models are warranted.