Effect of calcium on extraction of Z-band proteins from I-Z-I brushes of rabbit striated muscle.

1. When rabbit striated muscle I-Z-I brushes were subjected to eleven extractions with three different extracting solutions, relatively more amount of proteins was extracted in the presence of 1 nM CaCl2 than in the presence of 5 mM EDTA or 5 mM ethyleneglycol-bisp(beta-aminoethylether)-N,N,N1,N1-tetra-acetic acid (EGTA). Among proteins extracted in the presence of 1 mM CaCl2, the protein components with molecular weights of 85,000, 95,000 and 220,000 were included, whereas these were not extracted in the other two. 2. Co-electrophoreses of 220,000 dalton protein and myosin heavy chain showed that these two protein components were distinct from each other. 3. Roles of Ca2+ are discussed on disintegration processes of I-Z-I brushes in special reference to its co-operative action with calcium-activated factor enzyme.     

Cell surface protein decreases microvilli and ruffles on transformed mouse and chick cells.

Transformation of cultured fibroblasts usually results in a decrease in a high molecular weight cell surface glycoprotein (LETS protein) and often in increased numbers of surface microvilli and ruffles. We have isolated such a major cell surface glycoprotein from chick embryo fibroblasts; this protein, CSP, is decreased after transformation. Treatment of a mouse tumor cell line (SV1), L929 cells, and transformed chick fibroblasts with CSP results in a decrease in the number of microvilli and marginal ruffles, accompanied by restoration of a more normal morphology.     

Effects of 2,4-substituents of deuteroheme upon the stability of the oxy-form and compound I of horseradish peoxidases.

The stability of oxyperoxidases increased in the order meso- < proto < chlorocruoro- < diacetylperoxidases, which was an increasing order of electron-withdrawing capacities of 2,4-substituents of deuteroheme and the ratio of Δlogk₁toΔpK₃ was approximately 0.6 in the two series of isoenzyme preparations, horseradish peroxidases A and (B + C), where k₁andpK₃ represent a rate constant for conversion from an oxyperoxidase to the ferric enzyme and a measure of basicity of pyrrole nitrogen of the substituted deuterohemes, respectively. Deutero-oxyperoxidases A and (B + C) were definitely more stable than expected from the above linear relationship. The stability of peroxidase Compound I also varied with the 2,4-substituents, but it did not necessarily correlate with electron-withdrawing capacities of the substituents. Natural peroxidases formed relatively stable Compound I in both series of the isoenzymes. From these results it was concluded that the stability of oxyperoxidases was affected by the electron density at the iron atom of the enzyme while steric factors might be involved in stabilizing Compound I.     

On the active streaming experiment of actomyosin solutions in glass microcapillaries.

The active-streaming experiments of Oplatka et al. (Oplatka, A. and Tirosh, R. (1973) Biochim. Biophys. Acta 305, 684-688 and Oplatka, A. Gadasi, H., Tirosh, R. Lamed, Y., Muhlrad, A. and Liron, N. (1974) J. Mechanochem. Cell Motil. 2, 295-306) with actomyosin solution in a glass microcapillary is reexamined under various conditions with several kinds of reference material. It is found that vigorous streaming took place in the actomyosin solution as reported by Oplatka et al. However, streaming which is indistinguishable from that observed in the actomyosin solution in the presence of actomyosin ATPase activity also occurred, even when the ATPase activity was blocked. The streaming also cannot be confirmed as being active when using acto-heavy meromyosin solution. There is a possibility that the streaming experiment provides interesting information on the microscopic state of solutions which is not directly related to the chemo-mechanical conversion.     

Endocrine function in a case of beta-adrenergic hyperdynamic circulatory state.

Endocrine functions were investigated in a case of "beta-adrenergic hyperdynamic circulatory state". This state was diagnosed by (1) typical symptoms of cardiac awareness, (2) physical findings (increments of pulse rate and blood pressure by changing positions or walking), (3) increase in cardiac output (5.25 l/min leads to 14.03 l/min) and decrease in circulatory time (10.8 sec leads to 5.5 sec) by isoproterenol infusion (0.02 mug/min/kg body weight), (4) rapid loss of symptoms and above findings by propranolol treatment (30 mg per os daily) and reappearance by discontinuing medication. The mechanism of insulin response to glucose has been a controversy as to whether the secretion is transmitted by beta-receptor or independent glucose receptor. And in this physiologic beta-adrenergic state, it was found that insulin responses in IVGTT and OGTT were within normal limit. When beta-adrenergic condition was corrected by propranolol treatment, insulin responses were shown lowered, though in the normal range. This could be reproduced by discontinuing medication. Insulin, glucagon and growth hormone secretions caused by arginine were also found normal, but during the period the patient was on propranolol therapy, all responses were decreased, within the normal range. These results do not positively support the idea that glucose receptor is linked to beta-receptor. They do not either agree with the contention that secretions of insulin, glucagon and growth hormone induced by arginine are mediated through beta-receptors.     

The synergetic enzyme theory of muscular contraction: II Relation between Hill's equations and functions of two-headed myosin.

Based on the assumption of nonidentical two heads of myosin it is pointed out that a strong motive force is generated in actomyosin pair only when ATP-decomposition occurs co-operatively at the both heads and that the tension-independent part of shortening heat is liberated when an ATP molecule is decomposed only at the burst head. These two actions of actomyosin pair are related to the two states of force-generator in Huxley-Simmons' model. Elementary cycles at different positions in a sarcomere are interacted each other through feedback loop via sliding motion of muscular filaments. Due to this synergetic interaction the rate constant for the rate-determining step of elementary cycle has a dependence on velocity v of shortening such as k = k ° + κv. From these functions and properties of actomyosin system in vivo, the following properties of muscle are explained consistently in a quantitative manner: (1) Hill's equation on the relationship between tension and velocity of shortening, (2) damped oscillations in tension and in muscular length around steady state, (3) Hill's energy equation improved in 1964, (4) the chemical equivalence of shortening heat, (5) the influence of tension on the incorporation of radio-active phosphate into ATP and (6) the asymmetric activation by actomyosin system only for the forward reaction, the decomposition of ATP.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

SARS-CoV-2-specific T cells associate with inflammation and reduced lung function in pulmonary post-acute sequalae of SARS-CoV-2

As of January 2022, at least 60 million individuals are estimated to develop post-acute sequelae of SARS-CoV-2 (PASC) after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While elevated levels of SARS-CoV-2-specific T cells have been observed in non-specific PASC, little is known about their impact on pulmonary function which is compromised in the majority of these individuals. This study compares frequencies of SARS-CoV-2-specific T cells and inflammatory markers with lung function in participants with pulmonary PASC and resolved COVID-19 (RC). Compared to RC, participants with respiratory PASC had between 6- and 105-fold higher frequencies of IFN-γ- and TNF-α-producing SARS-CoV-2-specific CD4⁺ and CD8⁺ T cells in peripheral blood, and elevated levels of plasma CRP and IL-6. Importantly, in PASC participants the frequency of TNF-α-producing SARS-CoV-2-specific CD4⁺ and CD8⁺ T cells, which exhibited the highest levels of Ki67 indicating they were activity dividing, correlated positively with plasma IL-6 and negatively with measures of lung function, including forced expiratory volume in one second (FEV¹), while increased frequencies of IFN-γ-producing SARS-CoV-2-specific T cells associated with prolonged dyspnea. Statistical analyses stratified by age, number of comorbidities and hospitalization status demonstrated that none of these factors affect differences in the frequency of SARS-CoV-2 T cells and plasma IL-6 levels measured between PASC and RC cohorts. Taken together, these findings demonstrate elevated frequencies of SARS-CoV-2-specific T cells in individuals with pulmonary PASC are associated with increased systemic inflammation and decreased lung function, suggesting that SARS-CoV-2-specific T cells contribute to lingering pulmonary symptoms. These findings also provide mechanistic insight on the pathophysiology of PASC that can inform development of potential treatments to reduce symptom burden.