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Background: In the first-line treatment of elderly patients with advanced-stage non-small cell lung cancer (NSCLC) with high programmed death-ligand 1 (PD-L1) expression (tumor proportion score ≥ 50%), this study aimed to determine whether pembrolizumab monotherapy (MONO) or pembrolizumab plus platinum-based chemotherapy (COMB) should be selected., Methods: We performed a retrospective multicenter study (sub-analysis of the HOT/NJLCG2001 trial) of 299 patients with NSCLC with high PD-L1 expression who received MONO or COMB as the first-line treatment between December 2018 and January 2020. We selected patients aged 75 years and older and assessed the clinical efficacy and toxicity., Results: In total, 81 (median age: 79 years) and 19 (median age: 76 years) patients received MONO and COMB, respectively. Twenty patients with a performance status (PS) score of 2-3 were enrolled in the MONO group. The median progression-free survival (PFS) was 7.8 and 8.9 months in the MONO and COMB groups, respectively. The median overall survival (OS) was 14.6 and 20.3 months, and the 2-year survival rates were 38.8 and 49.9%, respectively. Furthermore, 29.6% and 26.3% of patients discontinued treatment due to adverse events, respectively. In MONO, patients with PS 0-1 had a longer PFS (10.5 months) and OS (21.7 months) than those with PS 2-3 (0.7 and 1.6 months, respectively)., Conclusion: Some elderly patients with NSCLC and high PD-L1 expression might benefit from COMB; however, MONO is considered the preferred treatment. MONO may not be an effective or feasible treatment for patients with PS 2-3, even with high PD-L1 expression., (© The Author(s) 2024. Published by Oxford University Press.)

The role of octamer-binding transcription factor 4 (OCT4) in human cancer is still debated. Although many studies have been published on human OCT4, determining which of the findings are accurate or which are false-positives is currently challenging. We thus developed the most reliable method to date for highly specific and comprehensive detection of genuine OCT4-transcript variants without false-positive results. Our results provided clear evidence that the transcripts of OCT4A, OCT4B, OCT4B1, and other novel splicing variants are indeed present in many cancer cell lines, but are rarely detected in normal tissue-derived differentiated cells. Using the tagged genomic transgene, we then verified endogenous OCT4A translation in cancer cell subpopulations. Moreover, analysis of possible other protein isoforms by enforced expression of OCT4B variants showed that the B164 isoform, designated human OCT4C, is preferentially produced in a cap-dependent manner. We confirmed that the OCT4C isoform, similar to OCT4A, can transform non-tumorigenic fibroblasts in vitro. Finally, ablation of OCT4-positive cells using promoter-driven diphtheria toxin A in high malignant cancer cells caused a significant decrease in migration and Matrigel invasion. These findings strongly suggest a significant contribution of OCT4 to the phenotype of human cancer cells. Stem Cells 2018., (© AlphaMed Press 2018.)

Aims: We aimed to determine whether elevated serum high-sensitive cardiac troponin T (hs-TnT) levels predict atrial fibrillation (AF) recurrence after pulmonary vein isolation (PVI)., Methods and Results: We included 125 consecutive patients with AF (paroxysmal, n = 79; persistent, n = 46) who underwent first-time PVI. Serum hs-TnT, high-sensitive C-reactive protein (hs-CRP), atrial natriuretic peptide, and plasma B-type natriuretic peptide levels were measured in venous samples collected before PVI. Elevated hs-TnT was diagnosed in patients with levels ≥0.014 μg/L. All patients underwent multidetector computed tomographic examinations before PVI to measure left atrial volume (LAV) and left ventricular (LV) mass, which were indexed to body surface area. Arrhythmia recurrence was defined as AF/atrial tachycardia episodes lasting for ≥30 s after a 2-month blanking period from the PVI procedure. Elevated hs-TnT levels were observed in 22 (17.6%) patients. Age, diabetes mellitus, LV mass index, estimated glomerular filtration rate, and hs-CRP were independently associated with serum hs-TnT levels (all P < 0.05). During a mean follow-up of 12.9 ± 8.5 months after a single PVI procedure, the clinical recurrence rate was 33% (n = 41). Multivariate Cox proportional hazard analysis revealed that a greater LAV index (P = 0.01) and elevated serum hs-TnT level (P = 0.01) were significant predictors of AF recurrence after PVI., Conclusion: This study demonstrated that elevated serum hs-TnT levels are associated with AF recurrence independent of traditional risk factors and left atrial enlargement., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Most blood vessels contain elastin that provides the vessels with the resilience and flexibility necessary to control hemodynamics. Pathophysiological hemodynamic changes affect the remodeling of elastic components, but little is known about their structural properties. The present study was designed to elucidate, in detail, the three-dimensional (3D) architecture of delicate elastic fibers in small vessels, and to reveal their architectural pattern in a rat model. The fine vascular elastic components were observed by a newly developed scanning electron microscopy technique using a formic acid digestion with vascular casts. This method successfully visualized the 3D architecture of elastic fibers in small blood vessels, even arterioles and venules. The subendothelial elastic fibers in such small vessels assemble into a sheet of meshwork running longitudinally, while larger vessels have a higher density of mesh and thicker mesh fibers. The quantitative analysis revealed that arterioles had a wider range of mesh density than venules; the ratio of density to vessel size was higher than that in venules. The new method was useful for evaluating the subendothelial elastic fibers of small vessels and for demonstrating differences in the architecture of different types of vessels.

We identified two compounds that demonstrated 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity from cultures of Lactobacillus plantarum. Spectroscopic analyses proved these compounds to be L-3-(4-hydroxyphenyl) lactic acid (HPLA) and L-indole-3-lactic acid (ILA). The respective EC50 values for HPLA and ILA were 36.6 ± 4.3 mM and 13.4 ± 1.0 mM.

One kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP ≧ 4 than KP from Thermoanaerobacter brockii ATCC35047.

The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.

Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99, 95-103]. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled anti-S100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.

There have been some attempts to develop immunotoxins utilizing human RNase as a cytotoxic domain of antitumor agents. We have recently shown that only human RNase 3 (eosinophil cationic protein, ECP) among five human pancreatic-type RNases excels in binding to the cell surface and has a growth inhibition effect on several cancer cell lines, even though the RNase activity of RNase 3 is completely inhibited by the ubiquitously expressed cytosolic RNase inhibitor. This phenomenon may be explained by that RNase 3 is very stable against proteolytic degradation because RNase 3 internalized through endocytosis could have a longer life time in the cytosol, resulting in the accumulation of enough of it to exceed the concentration of RNase inhibitor, which allows the degradation of cytosolic RNA molecules. Thus, we compared the stabilities of human pancreatic-type RNases (RNases 1-5) and bovine RNase A by means of guanidium chloride-induced denaturation experiments based on the assumption of a two-state transition for unfolding. It was demonstrated that RNase 3 is extraordinarily stabler than either RNase A or the other human RNases (by more than 25 kJ/mol). Thus, our data suggest that in addition to its specific affinity for certain cancer cell lines, the stability of RNase 3 contributes to its unique cytotoxic effect and that it is important to stabilize a human RNase moiety through protein engineering for the design of human RNase-based immunotoxins.

Cationization of a protein is considered to be a powerful strategy for internalizing a functional protein into cells. Cationized proteins appear to adsorb to the cell surface by electrostatic interactions, then enter the cell in a receptor- and transporter-independent fashion. Thus, in principle, all cell types appear to take up cationized proteins. Since ribonucleases (RNases) have a latent cytotoxic potential, cationized RNases could be useful cancer chemotherapeutics. In this study, we investigated the effect of the degree of cationization on the cytotoxicity of RNase A by modifying carboxyl groups with ethylenediamine. We found that there is an optimum degree of modification for cytotoxicity, in which 5 to 7 out of 11 carboxyl groups in RNase A are modified, toward MCF-7 and 3T3-SV-40 cells. More interestingly, the cytotoxicity of cationized RNase As correlates well with the value of [RNase activity] x [estimated concentration of RNase free from RNase inhibitor], mimicking the practical enzymatic activity of cationized RNase As in cytosol. The results indicate that cationization of a protein to an optimum level is important for maintaining protein function in the cytosol. Sophisticated protein cationization techniques will help to advance protein transduction technology.

Activin A is a multifunctional cytokine essential for cell differentiation and apoptosis including erythroid cell differentiation in the bone marrow. In addition, activin A is induced by inflammation and exerts anti-inflammatory effects. However, the mechanism of activin A induction is still unclear, especially by inflammatory processes. Here we show that activin A secretion from monocytes and bone marrow stromal fibroblasts, its major sources in the bone marrow, is markedly enhanced by cognate interaction with activated T cells. This process is mediated by CD40/CD40 ligand interaction as well as concomitantly secreted T cell-derived cytokines, granulocyte macrophage-colony stimulating factor, and interferon-gamma. Furthermore, stromal fibroblasts as well as monocytes provide a costimulatory signal to anti-CD3-treated T cells via CD80 and CD86 to maintain the enhanced activin A production. These findings suggest that activin A is potently induced in the bone marrow and may play a role in the suppression of inflammatory or immune processes.

The gene encoding alpha-L-arabinofuranosidase (STX-IV), located upstream of the previously reported stxI gene, was cloned and sequenced. The gene is divergently transcribed from the stxI gene, and the two genes are separated by 661 nucleotides. The stxIV gene consists of a 1,092-bp open reading frame encoding 363 amino acids. The deduced amino acid sequence of the gene showed that STX-IV was an enzyme consisting of only a catalytic domain, and that the enzyme had significant similarity with alpha-L-arabinofuranosidases belonging to family 62 of glycosyl hydrolases. The stxIV gene was expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. Arabinoxylan and oat spelt xylan were good substrates for STX-IV, however, the enzyme showed a low activity with p-nitrophenyl alpha-L-arabinofuranoside. The optimum pH and temperature were 5.0 and 60 degrees C, respectively.

Activin A is a multi-functional cytokine with a potent stimulation on erythroid cell differentiation in the bone marrow. The actions of activin A are determined by a balance of the levels of activin A and its inhibitor, follistatin (FS). However, the regulation of its actions in the bone marrow has been unclear. Here we show that bone marrow-derived stromal fibroblasts are the major source of activin A and FS in the bone marrow, and that the production of activin A is enhanced by interleukin-1beta (IL-1beta) and lipopolysaccharide (LPS), whereas interferon-gamma (IFN-gamma) inhibits the secretion of activin A by stromal fibroblasts. Concomitantly, IL-1beta as well as LPS inhibits and IFN-gamma stimulates FS secretion from stromal fibroblasts. Thus, these cytokines potently regulate activin A actions by reciprocal modulation of activin A and FS secretion from stromal fibroblasts. Because activin A exhibits anti-inflammatory effects in various tissues, up-regulation of activin A actions by IL-1beta and endotoxin in the bone marrow may play a protective role against inflammatory processes as well as anaemia. The present results also suggest that the inhibitory effect of IFN-gamma on erythropoiesis is mediated at least in part by a suppression of activin A actions in bone marrow.

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.

We evaluated an enzyme-linked immunosorbent assay (ELISA) for detecting anti-Jo-1 antibodies in patients with polymyositis (PM) or dermatomyositis (DM) by use of the recombinant fusion protein Jo-1. Sera from 64 patients with PM or DM, from 80 patients with other connective tissue diseases, and from 64 healthy subjects matched for age, sex and race, were studied by the ELISA and by the double immunodiffusion (DID) method. Eight patients with myositis (six PM, one DM and one DM with malignancy) with positive anti-Jo-1 by DID also showed positive results by the ELISA method, whereas five patients with positive anti-Jo-1 by this ELISA showed negative results on DID. One of the five had non-specific results. The incidence of positive results for anti-Jo-1 with the ELISA (18.8%) was greater than that for DID (12.5%), but the difference was not statistically significant. All patients with positive results for anti-Jo-1 by DID were also positive by the ELISA. The ELISA system with the recombinant Jo-1 antigen was useful in the detection of anti-Jo-1 antibodies in patients with PM/DM.

Free light chains (FLC) are a natural product of B lymphocytes and, as such, represent a quantifiable biomarker of cellular proliferation. Accurate measurement of the concentrations of these components in serum and urine provides a unique means of ascertaining B cell immunoglobulin synthesis during physiologic and, especially, pathologic states, where such information has important diagnostic and therapeutic implications. Previously, use of such quantitative assays has been limited due to the lack of potent serologic reagents specific for these components. We have immunized mice with kappa- and lambda-type monoclonal human light chains (Bence Jones proteins (BJP)) and have obtained monoclonal antibodies (MoAbs) that differentiate between unbound and bound light chains. These highly specific MoAbs were used to measure by ELISA the concentrations of FLC in the serum of 22 normal individuals and in urine from 16 of these subjects. The mean serum kappa and lambda FLC concentrations were found to be 16.6+/-6.1 microg/ml and 33.8+/-14.8 microg/ml, respectively. In contrast, the values for urinary kappa and lambda FLC were 2.96+/-1.84 microg/ml and 1.07+/-0.69 microg/ml, respectively. In each case studied, the serum kappa:lambda ratio was consistently less than that of urine (mean values, serum approximately 1:2; urine approximately 3:1). That the rate of synthesis of lambda-type FLC exceeded that of kappa was evidenced in assays of culture fluid supernatants of unstimulated normal peripheral blood mononuclear cells (PBMC), where the mean kappa:lambda ratio was determined to be 1:1.4. Metabolic studies in which mice were injected with pools of kappa- and lambda-type BJP prepared in ratios of 1:1, 1:2 and 1:4 demonstrated that, regardless of the proportion, kappa FLC were preferentially excreted. Our studies provide the first evidence that lambda FLC are secreted by normal PBMC at a greater rate than are kappa FLC, as evidenced in biosynthetic studies and by measurement of their serum concentrations. Further, we posit that quaternary structural differences between the two light-chain isotypes may account for the predominance of kappa versus lambda components in urine.

The inhibiting activity of serum on haematopoiesis has been described in patients with SLE. To explore further the features of serum inhibitor, we first examined the suppression of granulocytic and erythroid colony formation in vitro by serum from patients with SLE using methylcellulose culture. The potent inhibiting activity was demonstrated in six of 20 patients. All of these six patients were associated with leukocytopenia and/or anaemia. Five of 10 sera from patients with active SLE suppressed the colony formation of both burst-forming units of erythrocyte (BFU-E) and colony-forming units of granulocyte/macrophage (CFU-GM), and one serum suppressed BFU-E only. IgG fraction isolated from sera with inhibiting activity suppressed colony formation without complement involvement. The elimination of monocytes and lymphocytes from target mononuclear cells did not affect the suppression by the IgG fractions. The suppressive effect was completely eliminated after incubation of the IgG fractions with progenitor-enriched mononuclear cells. Flow cytometric analysis showed these IgG bound to CD34+ haematopoietic progenitor cells, but not to CD33+ cells. These data suggest that (i) the inhibitor of colony formation in serum was observed in IgG fraction; (ii) its suppressive effect on colony formation was mediated by neither monocytes and lymphocytes nor complements; and (iii) IgG fraction could bind to primitive haematopoietic progenitor cells and suppress the growth of these cells. Thus, IgG autoantibodies to primitive haematopoietic progenitor cells are demonstrated to be present in the sera of a significant proportion of active SLE patients with anaemia and leukocytopenia and to suppress the progenitor cell growth.

We have recently shown the presence of activin A in human thyroid cells. To determine whether activin A affects the growth and function of the thyroid, we investigated its effects on DNA synthesis, iodine metabolism, and cAMP accumulation in cultured porcine thyroid cells. Activin A increased DNA synthesis. This effect was abolished by addition of follistatin but additively enhanced by epidermal growth factor. In contrast, activin A significantly reduced uptake and release of iodide by the thyroid and also TSH-induced cAMP accumulation but did not inhibit the cAMP accumulation induced by forskolin. These data indicate that activin A stimulates thyroid growth irrespective of cAMP accumulation and inhibits thyroid function.

A novel S-alkylating reagent, N-(3-bromopropyl)-N,N,N',N',N'-pentamethyl-1,3-propanedi(ammonium bromide) (TAP2-Br) which carries two positive charges in the molecule, was prepared to increase the solubility or to decrease the hydrophobicity of cysteine-containing denatured proteins (or peptides). S-Alkylation with TAP2-Br introduces two positive charges per cysteine residue, which will effectively shift the net charge of a protein in the positive direction. Disulfide-containing proteins, such as hen egg-white lysozyme, RNase A, BSA, and soybean trypsin inhibitor (Kunitz type), were reduced and S-alkylated with TAP2-Br to evaluate the potential of this reagent compared with other S-alkylating reagents such as monoiodoacetic acid, bromosuccinic acid and (3-bromopropyl)trimethylammonium bromide. The solubilities of these denatured proteins in the pH range of 2-10 indicated that S-alkylation with TAP2-Br effectively solubilized not only basic proteins (lysozyme and RNase) but also an acidic protein containing a fairly large number of cysteine residues (BSA). Moreover, the retentions of cysteine-containing tryptic peptides derived from lysozyme on reversed-phase HPLC were greatly reduced by S-alkylation with TAP2-Br. These results indicate that TAP2-Br is very useful to increase the solubility of some cysteine-containing denatured proteins and to decrease the hydrophobicity of peptides containing cysteine residue(s).

The authors report the clinical features and the results of immunochemical studies of patients with angioimmunoblastic lymphadenopathy, primary amyloidosis, and multiple myeloma, each of whom had in the serum and urine multiple forms of Bence Jones protein (BJP). The BJPs were isolated and purified and were shown by electrophoretic, gel filtration, and ultracentrifugal analyses to exist as tetramers and dimers. The components in two cases were kappa type and in one lambda type. The kappa tetramers consisted of two covalent dimers and the lambda tetramer two noncovalently bound dimers present in both serum and urine.

Since we had experience of eight patients with adult T-cell leukemia lymphoma from 130 cases of non-Hodgkin's lymphoma between 1978 and 1986, a sero-epidemiological survey of anti human lymphotropic virus type-I (HTLV-I) antibody was performed using a gelatin particle agglutination test, an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay for 2,190 adults (768 men and 1,422 women) aged between 21 and 86 years, in the southern district of Tokushima Prefecture. The inconclusive data obtained using the above mentioned methods have been re-examined by western blotting analysis using enzyme-labelled anti IgM and anti IgG. There was an overall prevalence rate of 6.0% (132/2, 190) but higher rate were found in village C (10.9%) and village D (14.2%). These two villages together with town K (6.1% seropositive) form one community unit with a small population in a mountain area, which could be prone to producing a cluster of HTLV-I carriers. Town G with 8.0% sero-positivity is on the Pacific Ocean coast neighboring one of the endemic areas in the eastern section of Kohchi Prefecture. Interestingly, only IgM antibody was detected in 17 of 137 carriers (13%), all females who had never had a blood transfusion, suggesting them to h ave been in a sort of immunodeficient state, as reported in cases of HTLV-I infection. In 13 of 29 HTLV-I carriers (44.8%) from villages C and D, the viral antigen was detected in 1-9% (0.41 +/- 0.19%) of the peripheral blood mononuclear cells after being cultured with phytohemagglutinin for three days. The data indicate that those carriers had evidently been infected with the HTLV-I virus.