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Two antigenically distinct types of myosin heavy chain, referred to as α and β, have been identified in autoptic and bioptic specimens of human heart using specific antimyosin antibodies. By immunofluorescence heavy chain α was present in all atrial myocytes and in a variable number of ventricular myocytes. Heavy chain β was present in all ventricular myocytes and in a number of atrial myocytes. Ventricular hypertrophy in patients with aortic stenosis, systemic hypertension or tetralogy of Fallot was characterized by an almost complete absence of fibres reactive with anti-α. A striking decrease in α chain reactivity and a parallel increase in β chain reactivity was apparent in the hypertrophied left atria of patients with mitral stenosis. To quantify these myosin changes a novel procedure was developed whereby myosin was extracted from single cryosections serial to those processed for immunofluorescence and the relative amount of α and β heavy chain was determined by enzyme immunoassay. Heavy chain a was less than 5% in most normal ventricular specimens and disappeared completely under the effect of pressure overload. On the other hand heavy chain β was generally undetectable in the left atrial myocardium but increased up to 90% in biopsies of hypertrophied atria. [ABSTRACT FROM PUBLISHER]

Multiple myeloma (MM) is a malignant plasma cell (PC) neoplasm, which also displays pathological bone involvement. Clonal expansion of MM cells in the bone marrow causes a perturbation of bone homeostasis that culminates in MM-associated bone disease (MMABD). We previously demonstrated that the S/T kinase CK1α sustains MM cell survival through the activation of AKT and β-catenin signaling. CK1α is a negative regulator of the Wnt/β-catenin cascade, the activation of which promotes osteogenesis by directly stimulating the expression of RUNX2 , the master gene regulator of osteoblastogenesis. In this study, we investigated the role of CK1α in the osteoblastogenic potential of mesenchymal stromal cells (MSCs) and its involvement in MM-MSC cross-talk. We found that CK1α silencing in in vitro co-cultures of MMs and MSCs modulated RUNX2 expression differently in PCs and in MSCs, mainly through the regulation of Wnt/β-catenin signaling. Our findings suggest that the CK1α/RUNX2 axis could be a potential therapeutic target for constraining malignant PC expansion and supporting the osteoblastic transcriptional program of MSCs, with potential for ameliorating MMABD. Moreover, considering that Lenalidomide treatment leads to MM cell death through Ikaros, Aiolos and CK1α proteasomal degradation, we examined its effects on the osteoblastogenic potential of MSC compartments.

Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin lymphoma, characterized by frequent relapses. In the last decade, the pro-survival pathways related to BCR signaling and Bcl-2 have been considered rational therapeutic targets in B cell derived lymphomas. The BTK inhibitor Ibrutinib and the Bcl-2 inhibitor Venetoclax are emerging as effective drugs for MCL. However, primary and acquired resistance also to these agents may occur. Protein Kinase CK2 is a S/T kinase overexpressed in many solid and blood-derived tumours. CK2 promotes cancer cell growth and clonal expansion, sustaining pivotal survival signaling cascades, such as the ones dependent on AKT, NF-κB, STAT3 and others, counteracting apoptosis through a "non-oncogene" addiction mechanism. We previously showed that CK2 is overexpressed in MCL and regulates the levels of activating phosphorylation on S529 of the NF-κB family member p65/RelA. In the present study, we investigated the effects of CK2 inactivation on MCL cell proliferation, survival and apoptosis and this kinase's involvement in the BCR and Bcl-2 related signaling. By employing CK2 loss of function MCL cell models, we demonstrated that CK2 sustains BCR signaling (such as BTK, NF-κB and AKT) and the Bcl-2-related Mcl-1 expression. CK2 inactivation enhanced Ibrutinib and Venetoclax-induced cytotoxicity. The demonstration of a CK2-dependent upregulation of pathways that may antagonize the effect of these drugs may offer a novel strategy to overcome primary and secondary resistance., Competing Interests: FP is in the Advisory Board of Roche and Janssen and reports personal fees from Roche and Janssen. LT reports grants, personal fees from Janssen and Abbvie, grants from Gilead. GS has reported consultancy or advisory board for Janssen and Celgene and has received research support from Roche and Novartis, all outside of the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor AV declared a shared parent affiliation with the authors at the time of review., (Copyright © 2022 Manni, Pesavento, Spinello, Saggin, Arjomand, Fregnani, Quotti Tubi, Scapinello, Gurrieri, Semenzato, Trentin and Piazza.)

Ten hybridomas producing monoclonal antibodies (Mabs) against rabbit platelet factor 4 (PF4) were obtained from the fusion of splenocytes from mice immunized with purified rabbit PF4 and NSO mouse myeloma cells. When the reactivities of these monoclonal antibodies were determined by enzyme-linked immunosorbent assay and immunoblotting with human and rabbit PF4, they showed a high degree of specificity. Only one Mab recognized an epitope common to the human and rabbit molecules, the other nine reacted only with the rabbit protein. All the antibodies recognized, in crude platelet lysates, a band that comigrates with the purified PF4 protein. None of these antibodies cross-reacted with major rabbit or human platelet-poor plasma proteins. The significance of the Mabs in immunological and physiological studies is discussed.

We have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4's ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.

Fetal rat skeletal muscles express a troponin T (TnT) isoform similar to the TnT isoform expressed in the embryonic heart with respect to electrophoretic mobility and immunoreactivity with cardiac TnT-specific monoclonal antibodies. Immunoblotting analyses reveal that both the embryonic and the adult isoforms of cardiac TnT are transiently expressed during the neonatal stages. In addition, other TnT species, different from both cardiac TnTs and from the TnT isoforms expressed in adult muscles, are present in skeletal muscles during the first two postnatal weeks. By immunocytochemistry, cardiac TnT is detectable at the somitic stage and throughout embryonic and fetal development, and disappears during the first weeks after birth, persisting exclusively in the bag fibers of the muscle spindles. Cardiac TnT is re-expressed in regenerating muscle fibers following a cold injury and in mature muscle fibers after denervation. Developmental regulation of this TnT variant is not coordinated with that of the embryonic myosin heavy chain with respect to timing of disappearance and cellular distribution. No obligatory correlation between the two proteins is likewise found in regenerating and denervated muscles.

P19 embryonal carcinoma cells are multipotential stem cells that differentiate into striated muscle as well as some other cell types when aggregated and exposed to dimethyl sulfoxide (DMSO). Immunofluorescence experiments using monospecific antibodies indicated that the majority of muscle cells were mononucleate and contained four myosin isoforms normally found in cardiac muscle; atrial and ventricular myosin heavy chains, ventricular myosin light chain 1, and atrial myosin light chain 2. Northern blot analysis of RNA isolated from differentiating cultures indicated that cardiac actin and skeletal actin mRNAs were expressed at similar levels and with identical kinetics during the differentiation of P19-derived myocytes. These results demonstrate that most of the P19-derived myocytes are of the cardiac type and suggest that they closely resemble the cells of the early embryonic myocardium.

Prothrombin was assayed using chromogenic substrate of S-2238 for patients who were being treated with coumarin, for patients who had liver disease, and for patients who had congenital hypoprothrombinemias and dysprothrombinemias. In coumarin therapy and in patients with liver disease the levels found correlated well with the one-stage clotting methods. The same was true for heterozygous and homozygous "true" prothrombin deficiency. In the case of congenital dysprothrombinemias the levels observed with the chromogenic substrate were higher than the clotting counterparts, particularly so in the case of prothrombin Padua. In the latter case the levels observed were always about 100% of normal, as compared with the levels of about 50% of normal found with clotting methods. These data indicate that chromogenic substrates are not always equivalent to "clotting" substrates, namely, that amidolytic activity is not always equivalent to clotting activity. Therefore the two methods cannot be used interchangeably, lest some defects escape detection.

Factor X was assayed in 30 patients on long-term anticoagulation by means of a chromogenic substrate (S-2222) for a total of 120 determinations. The average level found was 25.38 +/- 9.38% of normal. A satisfactory correlation was found between the factor X chromogenic level and the prothrombin time or Thrombotest percental values. However, factor X levels as determined by the chromogenic assay were usually higher than the global tests percental figures. The factor X chromogenic level was also slightly higher than the RVV-cephalin factor X assay. Since the chromogenic assay does not allow any new information to be made on the coumarin induced defect and since the costs of the assay are about 20-30 times higher than those of a simple prothrombin time, the test seems to play no part in the follow-up of coumarin therapy.

Prothrombin antigen concentration was evaluated by means of laser nephelometry in 10 patients on coumarin therapy, in 17 patients with cirrhosis of the liver, and in four patients with congenital hypo- or dysprothrombinemias. The average values obtained were 46.4, 37.7, and 35.6%, respectively, for anticoagulated, cirrhotic, and congenitally abnormal patients. These values correlated well with those obtained by means of electroimmunoassay (Laurell) and immunodiffusion (Mancini) methods. Similarly, satisfactory results were obtained in eight normal subjects. Multiple evaluations at different incubation times, also allowed the authors to construct kinetic curves of the interaction between antigen and antibody. However, an abnormal kinetic curve was demonstrated only for coumarin-treated patients.

Myosin heavy chain (MHC) composition of chemically-induced rhabdomyosarcoma (RMS) was analyzed by gel electrophoresis and Western blotting using a panel of monoclonal antimyosin antibodies specific for embryonic-, neonatal-, slow- and adult fast-type MHC isoforms. Myosin extracted from tumours and electrophoresed on 6%-sodium dodecyl sulfate (SDS)glycerol gels was found to migrate as three distinct MHC components. These polypeptides were present in different relative amounts in the five RMS studied. Western blotting experiments revealed that variable proportions of embryonic-, slow- and adult fast-, but not neonatal-type, MHC isoforms are consistently expressed in RMS. Indirect and double immunofluorescence procedures applied to cryosections of tumoral tissue showed that: (a) RMS cells were unreactive with antineonatal-type-MHC antibody, (b) the majority of neoplastic, desmin-positive, cells contained embryonic- as well as adult fast-type MHCs and (c) a minority of cells were labelled by anti-slow MHC antibody. The results of this study indicate that there is no obligatory sequence of MHC isoform expression in the molecular transition (emb----neo----adult) which occurs during rat skeletal myogenesis.

Isomyosin analyses by biochemical, immunochemical, and histochemical investigations have been carried out in five sheep following unilateral recurrent laryngeal nerve paralysis and direct functional electrostimulation of the denervated cricoarytenoid posterior muscle. Myosin light chains were identified by two-dimensional gel electrophoresis. Myosin heavy chains were analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis. Slow myosin heavy chain was identified by orthogonal peptide mapping and immunochemistry. The stimulation effect at cellular level was determined using adenosine triphosphatase (ATPase) histochemistry. A dramatic increase of the type 1 fiber area (slow, fatigue-resistant fibers) could be seen after many weeks of an increasing regime of low-frequency direct electrical stimulation. Biochemically, the amount of slow myosin was always higher than in normal muscles. Some muscles were transformed almost completely to the slow type. At the time they were studied and with the methods employed, the expression of embryonic isomyosin was not observed. In conclusion, after numerous weeks of maintained functional activity, elicited by direct electrostimulation, the denervated muscle regionally showed areas of hypertrophy or at least lack of atrophy of slow myofibers without major signs of muscle damage.

Factor X was assayed using chromogenic substrate S-2222 for four patients with severe factor X deficiency and for nine patients with homozygous or heterozygous factor X Friuli disorder. Factor X Friuli disorder is characterized by the presence of an abnormal factor X that is normally activated by Russell's viper venom, but is not activated by tissue thromboplastins. The levels of factor X found in factor X deficiency varied between 2 and 10% of normal and therefore were higher than those found in the same plasmas using "clotting" methods (1% or less than 1% of normal). The levels of factor X found in homozygous factor X Friuli patients varied between 4 and 11% of normal, and therefore were practically identical to those found by means of clotting methods that employed tissue thromboplastins (7-9% of normal). These values were definitely lower than those obtained using a Russell's viper venom and cephalin mixture as thromboplastin (82-92%). A similar pattern was observed for patients heterozygous for the abnormality. These findings indicate that "amidolytic" methods are not necessarily identical to clotting methods. Furthermore, they indicate that substrate S-2222 is not specific for factor X.

The immunologic concentration of two abnormal antithrombins III (AT III), namely antithrombin III Padua (AT III Padua) and antithrombin III Padua2 (AT III Padua2) and the kinetics of the reaction of these two ATs III with an anti AT III antiserum was investigated by means of a laser nephelometer. The immunologic concentration of these two AT III both in presence (0.2 IU/mL) or absence of heparin was normal. On the contrary, the analysis of kinetics behavior demonstrated that AT III Padua is radically different from pooled normal plasma both in presence or in absence of heparin. This was not the case for AT III Padua2, which showed no difference from pooled normal plasma regardless of the presence or absence of heparin. Both abnormal antithrombins III reached the plateau of the reaction at about the corresponding value of pooled normal plasma, indicating a normal antigen level. These experimental data were analyzed by means of a computer (Apple II 48 RAM) programmed to solve a four unknowns equation (Rodbard's equation). This analysis showed that the time needed to reach half of the maximum voltage, i.e., the parameter C, which is expressed in minutes, is clearly longer in the case of AT III Padua samples (heparinized or not) as compared with pooled normal plasma. Moreover, the time at which the maximum rate was reached was also longer. On the contrary, in the case of AT III Padua2 there is no difference from pooled normal plasma. These data confirm the view that a different kind of defect is present in these two AT III abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Two antigenically distinct types of myosin heavy chain, referred to as alpha and beta, have been identified in autoptic and bioptic specimens of human heart using specific antimyosin antibodies. By immunofluorescence heavy chain alpha was present in all atrial myocytes and in a variable number of ventricular myocytes. Heavy chain beta was present in all ventricular myocytes and in a number of atrial myocytes. Ventricular hypertrophy in patients with aortic stenosis, systemic hypertension or tetralogy of Fallot was characterized by an almost complete absence of fibres reactive with anti-alpha. A striking decrease in alpha chain reactivity and a parallel increase in beta chain reactivity was apparent in the hypertrophied left atria of patients with mitral stenosis. To quantify these myosin changes a novel procedure was developed whereby myosin was extracted from single cryosections serial to those processed for immunofluorescence and the relative amount of alpha and beta heavy chain was determined by enzyme immunoassay. Heavy chain alpha was less than 5% in most normal ventricular specimens and disappeared completely under the effect of pressure overload. On the other hand heavy chain beta was generally undetectable in the left atrial myocardium but increased up to 90% in biopsies of hypertrophied atria.

Using immunofluorescence procedures with specific polyclonal and monoclonal antimyosin antibodies we have found that embryonic and neonatal myosin heavy chains (MHCs), which in rat skeletal muscle disappear during the first weeks after birth, are reexpressed in adult muscle after denervation. Reactivity for embryonic and neonatal MHCs was detected in some fibers as early as 3 days after denervation, became more evident by 7 days, and occurred exclusively in the type 2A fiber population. Paralysis of innervated muscles by tetrodotoxin block of the sciatic nerve also resulted in the reappearance of embryonic and neonatal MHCs in type 2A fibers. Significant variation in the degree of immunoreactivity was observed in different segments of the same muscle fiber, suggesting that coordination of muscle fiber nuclei in the control of myosin heavy chain gene expression is partially lost following denervation.

The myosin heavy-chain composition of human smooth muscle has been investigated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, enzyme immunoassay, and enzyme-immunoblotting procedures. A polyclonal and a monoclonal antibody specific for smooth muscle myosin heavy chains were used in this study. The two antibodies were unreactive with sarcomeric myosin heavy chains and with platelet myosin heavy chain on enzyme immunoassay and immunoblots, and stained smooth muscle cells but not non-muscle cells in cryosections and cultures processed for indirect immunofluorescence. Two myosin heavy-chain isoforms, designated MHC-1 and MHC-2 (205 kDa and 200 kDa, respectively) were reactive with both antibodies on immunoblots of pyrophosphate extracts from different smooth muscles (arteries, veins, intestinal wall, myometrium) electrophoresed in 4% polyacrylamide gels. In the pulmonary artery, a third myosin heavy-chain isoform (MHC-3, 190 kDa) electrophoretically and antigenically distinguishable from human platelet myosin heavy chain, was specifically recognized by the monoclonal antibody. Analysis of muscle samples, directly solubilized in a sodium dodecyl sulfate solution, and degradation experiments performed on pyrophosphate extracts ruled out the possibility that MHC-3 is a proteolytic artefact. Polypeptides of identical electrophoretic mobility were also present in the other smooth muscle preparations, but were unreactive with this antibody. The presence of three myosin heavy-chain isoforms in the pulmonary artery may be related to the unique physiological properties displayed by the smooth muscle of this artery.

Mammalian skeletal muscles consist of three main fibre types, type 1,2A and 2B fibres, with different myosin heavy chain (MHC) composition. We have now identified another fibre type, called type 2X fibre, characterized by a specific MHC isoform. Type 2X fibres, which are widely distributed in rat skeletal muscles, can be distinguished from 2A and 2B fibres by histochemical ATPase activity and by their unique staining pattern with seven anti-MHC monoclonal antibodies. The existence of the 2X-MHC isoform was confirmed by immunoblotting analysis using muscles containing 2X fibres as a major component, such as the normal and hyperthyroid diaphragm, and the soleus muscle after high frequency chronic stimulation. 2X-MHC contains one determinant common to 2B-MHC and another common to all type 2-MHCs, but lacks epitopes specific for 2A- and 2B-MHCs, as well as an epitope present on all other MHCs. By SDS-polyacrylamide gel electrophoresis 2X-MHC shows a lower mobility compared to 2B-MHC and appears to comigrate with 2A-MHC. Muscles containing predominantly 2X-MHC display a velocity of shortening intermediate between that of slow muscles and that of fast muscles composed predominantly of 2B fibres.

Laser nephelometry was used to characterize an abnormal antithrombin III (AT III Padua) in comparison with normal antithrombin. Heparinized (0.2 IU/ml) or non-heparinized AT III Padua plasma reacts with Laser nephelometry antithrombin III antiserum in a different way compared with pooled normal plasma. There is in fact a slower antigen-antibody reaction during the first 40-45 min both in AT III Padua heparinized and non-heparinized plasmas, compared with pooled normal plasma; then the kinetics overlap. On the contrary the concentrations of antithrombin III Padua in percent correlate well with those obtained by Mancini's and Laurell's methods. These data indicate that Laser nephelometry is suitable for AT III antigen determinations and may also supply useful information for the characterization of abnormal clotting factors.