Search

Objective: Elizabethkingia anophelis causes meningitis, bloodstream infections, and respiratory infections in immunocompromised individuals. We examined two E. anophelis strains isolated from the first life-threatening cases caused by this species in Japan to determine the phylogenetic origin and genomic features of them., Methods: We performed whole genome-based analysis to clarify the genetic relationship for the two strains (EK0004 and EK0079) and Elizabethkingia sp. strains isolated from worldwide and to characterize the genomic features such as the prevalence of virulence- and antimicrobial resistance (AMR)-related genes., Patients: A 29-year-old man with hepatosplenic T-cell lymphoma and a 52-year-old man with systemic lupus erythematosus developed fatal bacteremia and meningitis due to E. anophelis, respectively., Results: Two strains, EK0004 and EK0079, were genetically different but most closely related to the strains isolated from the largest outbreak in Wisconsin, USA from 2015 to 2016, and the strain isolated from cerebrospinal fluid of a patient in Florida, USA in 1982, respectively. The two strains contained AMR-related genes such as those encoding for an extended-spectrum β-lactamase and multiple metallo-β-lactamases and several virulence-related genes such as capsular polysaccharide synthesis gene clusters., Conclusions: Although further functional analyses are required to understand the virulence of these clones, these finding suggests that enough caution of E. anophelis infection in immunocompromised patients is required since the number of infections by this species is increasing outside Japan., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Age-related muscle atrophy is associated with decreased protein anabolic capacity. Dietary intervention is an important strategy for the treatment of age-related muscle atrophy. This study examined the effect of Lactococcus cremoris subsp. cremoris FC-fermented milk on muscle mass and protein anabolic signaling in middle-aged mice. Male C57BL/6J mice (18-month-old) were divided into the control and Lactococcus cremoris subsp. cremoris FC-fermented milk supplementation groups. Mice were administered unfermented or fermented milk (300 μL/day) by gavage every alternate day for 8 weeks; thereafter, muscle weight, protein metabolic signaling factors, and inflammatory factors were investigated. Soleus muscle weight was higher in the fermented milk group than in the control group. Expression of insulin growth factor-1, a typical anabolic factor, and phosphorylation levels of anabolic signaling factors (mTOR and p70S6K) were higher after fermented milk supplementation. Levels of tumor necrosis factor-α, an inhibitor of protein anabolism, were lower in the fermented milk group. These data suggest that the daily intake of Lactococcus cremoris subsp. cremoris FC-fermented milk increased skeletal muscle mass as well as protein synthesis in the middle-aged mice, which may be mediated by reduction in the levels of inflammatory factors. Therefore, accelerated protein synthesis, induced by the consumption of fermented milk, has a potential role in counteracting muscle atrophy., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: We had previously reported an increase in trimethylamine N-oxide (TMAO) levels in patients with both compensated and decompensated heart failure (HF) and alteration in gut microbiota composition using 16S rRNA gene amplicon analysis. Although a metagenome-wide analysis showed that choline-TMA lyase levels increased in HF patients, which TMA generation pathway from choline, carnitine, or betaine contributes to the increase in TMAO levels in HF needs to be elucidated., Methods: We conducted a metagenome-wide shotgun sequencing analysis of gut microbiota and measured the TMAO levels in plasma of 22 HF patients during the compensated phase and 11 age-, sex-, and comorbidity-matched control subjects, whose gut microbiota compositions were reported in a previous 16S rRNA-based analysis., Results: The abundance of cntA/B was positively correlated with TMAO, especially in HF patients, whereas that of cutC/D or betaine reductase was not correlated either in controls or HF patients. The abundance of cntA/B was mainly derived from the genera Escherichia and Klebsiella either in controls or HF patients., Conclusion: TMAO levels in plasma depend on the abundance of cntA/B in HF. Although it is difficult to exclude the involvement of confounding factors, microbial dysbiosis connecting the abundance of cntA/B in the gut and the increase of TMAO in plasma can be a therapeutic target for HF., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We have reported that cytoskeletal proteins such as desmin and vimentin are expressed on the surface of muscle, mesenchymal and cancer cells, and possess N-acetyl-β-D-glucosamine (β-GlcNAc) residue-binding properties. As cell-recognizable β-GlcNAc residue-bearing biopolymer, we prepared glycoconjugates (SF-GlcNAc) composed of silk fibroin (SF) and monosaccharide N-acetyl-D-glucosamine (GlcNAc) by chemical modification using cyanuric chloride. The covalent immobilization of GlcNAc into SF was assessed by 1 H-NMR measurements. The 1 H-NMR spectrum of SF-GlcNAc conjugates showed new peaks attributed to the methyl protons of the N-acetyl group in GlcNAc, and the integration of these peaks revealed that the GlcNAc content in the conjugates was 9 wt%. The existence of β-GlcNAc residues in SF-GlcNAc was examined by the criteria using lectins such as wheat germ agglutinin (WGA). Addition of WGA to SF-GlcNAc solution caused an increase in the turbidity of the solution due to lectin-mediated aggregation. Solid-phase lectin binding assay based on the biotin-avidin interaction showed that biotinylated succinylated WGA bound more strongly onto SF-GlcNAc conjugate-coated wells compared to SF-coated well. Following the establishment of the existence of β-GlcNAc residues in SF-GlcNAc, the interaction of SF-GlcNAc with desmin was examined by enzyme-linked immunosorbent assay using anti-desmin antibody. The stronger binding of desmin was observed for SF-GlcNAc conjugate-coated wells compared to SF-coated wells. The use of SF-GlcNAc conjugates as a substrate for culturing desmin-expressing human cardiac myocytes demonstrated an increase in the numbers of attached cells and proliferating cells on the conjugate-coated wells compared to SF-coated wells. These results suggest that the immobilization of monosaccharide GlcNAc is a useful method for the versatile functionalization of SF as an application in tissue engineering., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The glucagon-like peptide-1 receptor (GLP-1R) is an important physiologic regulator of insulin secretion and a major therapeutic target for diabetes mellitus. GLP-1 (7-36) amide (active form of GLP-1) is truncated to GLP-1 (9-36) amide, which has been described as a weak agonist of GLP-1R and the major form of GLP-1 in the circulation. New classes of positive allosteric modulators (PAMs) for GLP-1R may offer improved therapeutic profiles. To identify these new classes, we developed novel and robust primary and secondary high-throughput screening (HTS) systems in which PAMs were identified to enhance the GLP-1R signaling induced by GLP-1 (9-36) amide. Screening enabled identification of two compounds, HIT-465 and HIT-736, which possessed new patterns of modulation of GLP-1R. We investigated the ability of these compounds to modify GLP-1R signaling enhanced GLP-1 (9-36) amide- and/or GLP-1 (7-36) amide-mediated cyclic adenosine monophosphate (cAMP) accumulation. These compounds also had unique profiles with regard to allosteric modulation of multiple downstream signaling (PathHunter β-arrestin signaling, PathHunter internalization signaling, microscopy-based internalization assay). We found allosteric modulation patterns to be obviously different among HIT-465, HIT-736, and Novo Nordisk compound 2. This work may enable the design of new classes of drug candidates by targeting modulation of GLP-1 (7-36) amide and GLP-1 (9-36) amide., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Na(+)/Ca(2+) exchanger (NCX) is a bidirectional transporter that is controlled by membrane potential and transmembrane gradients of Na(+) and Ca(2+). Although two isoforms of NCX1 and NCX2 are coexpressed on the basolateral membrane of the distal nephron, the functional significance of these isoforms is not entirely clear. Therefore, we used NCX1- and NCX2-heterozygote knockout mice (KO) and their double KO, as well as isoform-selective NCX inhibitors, to determine the roles of NCX isoforms in urine formation and electrolyte excretion in mice. NCX inhibitors, particularly NCX2-sensitive inhibitors, caused a dose-dependent natriuresis and in a higher dose, moreover, hypercalciuria. Consistently, NCX1-KO possessed normal renal function similar to wild-type mice (WT), whereas NCX2-KO and double KO exhibited moderate natriuresis and hypercalciuria. Notably, renal responses to YM-244769 were equivalently observed in NCX1-KO and WT, but disappeared in NCX2-KO and double KO. Thus, functional inhibition of NCX2 initially causes natriuresis, and further inhibition of NCX2 produces hypercalciuria, suggesting that the functional significance of NCX2 lies in Na(+) and Ca(2+) reabsorption of the kidney., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aralin from Aralia elata is a newly identified type II ribosome- inactivating protein, which preferentially induces apoptosis in cancer cells. In this study, we identified that the aralin receptor is a 110-kDa high-density lipoprotein-binding protein (HDLBP), which functions as a HDL receptor. The sensitivities of tumor cell lines to aralin were dependent on the expression levels of the 110-kDa HDLBP and its forced expression in aralin-resistant Huh7 cells conferred aralin sensitivity. HDLBP-knockdown HeLa cells showed a significant aralin resistance in vitro and in vivo. Conversely, ectopic expression of the 150-kDa HDLBP resulted in increased aralin sensitivity in vivo, accompanying enhanced expression of the 110-kDa HDLBP. Thus, these results showed that the 110-kDa HDLBP in lipid rafts acted as an aralin receptor and that its expression levels determined aralin sensitivity, suggesting that aralin could be a promising anticancer drug for HDLBP-overexpressing tumors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The aim of this study was to estimate genetic correlations between milk yield, somatic cell score (SCS), mastitis, and claw and leg disorders (CLDs) during first lactation in Holstein cows by using a threshold-linear random regression test-day model. We used daily records of milk, fat and protein yields; somatic cell count (SCC); and mastitis and CLD incidences from 46 771 first-lactation Holstein cows in Hokkaido, Japan, that calved between 2000 and 2009. A threshold animal model for binary records (mastitis and CLDs) and linear animal model for yield traits were applied in our multiple trait analysis. For both liabilities and yield traits, additive genetic effects were used as random regression on cubic Legendre polynomials of days on milk. The highest positive genetic correlations between yields and disease incidences (0.36 for milk and mastitis, 0.56 for fat and mastitis, 0.24 for protein and mastitis, 0.32 for milk and CLD, 0.44 for fat and CLD and 0.31 for protein and CLD) were estimated at about the time of peak milk yield (36 to 65 days in milk). Selection focused on early lactation yield may therefore increase the risk of mastitis and CLDs. The positive genetic correlations of SCS with mastitis or CLD incidence imply that selection to reduce SCS in the early stages of lactation would decrease the incidence of both mastitis and CLD.

We investigated the relationships between conception rates (CRs) at first service in Japanese Holstein heifers (i.e. animals that had not yet had their first calf) and cows and their test-day (TD) milk yields. Data included records of artificial insemination (AI) for heifers and cows that had calved for the first time between 2000 and 2008 and their TD milk yields at 6 through 305 days in milk (DIM) from first through third lactations. CR was defined as a binary trait for which first AI was a failure or success. A threshold-linear animal model was applied to estimate genetic correlations between CRs of heifers or cows and TD milk yield at various lactation stages. Two-trait genetic analyses were performed for every combination of CR and TD milk yield by using the Bayesian method with Gibbs sampling. The posterior means of the heritabilities of CR were 0.031 for heifers, 0.034 for first-lactation cows and 0.028 for second-lactation cows. Heritabilities for TD milk yield increased from 0.324 to 0.433 with increasing DIM but decreased slightly after 210 DIM during first lactation. These heritabilities from the second and third lactations were higher during late stages of lactation than during early stages. Posterior means of the genetic correlations between heifer CR and all TD yields were positive (range, 0.082 to 0.287), but those between CR of cows and milk yields during first or second lactation were negative (range, -0.121 to -0.250). Therefore, during every stage of lactation, selection in the direction of increasing milk yield may reduce CR in cows. The genetic relationships between CR and lactation curve shape were quite weak, because the genetic correlations between CR and TD milk yield were constant during the lactation period.

The Na(+)/Ca(2+) exchanger (NCX), an ion-transporter located in the plasma membrane of neuronal cells, contributes to intracellular Ca(2+) homeostasis. Within the brain, three isoforms (NCX1, NCX2, and NCX3) are widely distributed. However, it is not clear to what extent these isoforms are involved in ischemic brain damage in mammals. We therefore used genetically altered mice and isoform-selective NCX inhibitors in a model of transient focal ischemia to investigate the role of each NCX isoform in ischemic brain damage. NCX isoform-mutant mice (NCX1(+/-), NCX2(+/-), and NCX3(+/-)) and wild-type mice were subjected to 90min of middle cerebral artery occlusion (MCAO) followed by 24h of reperfusion. One of three NCX inhibitors [SN-6, KB-R7943, or SEA0400 (3 or 10mgkg(-1), i.p.)] was administered to ddY mice at 30min before more prolonged (4-h) MCAO followed by 24h of reperfusion. After transient MCAO reperfusion, the cerebral infarcts in NCX1(+/-) mice, but not those in NCX2(+/-) or NCX3(+/-) mice, were significantly smaller than those in wild-type mice. SN-6 and SEA0400, which are more selective for the NCX1 isoform, significantly reduced the infarct volume at 10mg/kg. In contrast, KB-R7943, which is more selective for NCX3, did not. These results suggest that the NCX1 isoform may act preferentially (vs. the NCX2 and NCX3 isoforms) to exacerbate the cerebral damage caused by ischemic insult in mice, and that NCX1-selective inhibitors warrant investigation as a potential therapeutic agents for stroke., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The nuclear transcription factor NF-kappaB is crucial to the expression of numerous cytokines, enzymes, and cell adhesion molecules, all of which can drive inflammatory and autoimmune disorders such as rheumatoid arthritis. The IKK complex plays the most important role in the signal cascade leading to NF-kappaB activation. Recently, inhibition of the interaction between NEMO (NF-kappaB essential modulator) and the catalytic subunits of IKK, especially IKKbeta, has received particular attention as a possible new therapeutic approach to treatment of inflammatory disorders, and several reports have shown the efficacy of cell permeable NEMO binding domain (NBD)-containing peptides in blocking the IKK/NF-kappaB pathway. In this article, we describe in detail the development and validation of two novel binding assays, a homogeneous time-resolved fluorescence (HTRF)-based assay and an enzyme-linked immunosorbent assay (ELISA)-based assay, suitable for the discovery of small molecules that inhibit IKKbeta-NEMO interaction. Using the HTRF-based assay, we screened approximately 15,000 compounds from our chemical library and eliminated false positive hits by the ELISA-based assay and IKK complex kinase assay. As a result, seven positive hit compounds that inhibit IKK complex activity through inhibition of IKKbeta-NEMO interaction were identified. These hit compounds may have a good potential in the treatment of inflammatory and autoimmune disorders such as rheumatoid arthritis., (Copyright 2010 Elsevier Inc. All rights reserved.)

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates intestinal immunological homeostasis. However, precise expression patterns of CEACAM1 isoforms remain poorly understood in the intestinal epithelia. Focusing on the small intestinal epithelium of BALB/c mice, we identified three novel splice variants encoding CEACAM1(a)-2, -2C1, and -4C1 by RT-PCR. CEACAM1(a)-2, -2C1, and -4C1 demonstrated secretory properties by transfection experiments in vitro. Among them, CEACAM1(a)-4C1 was the major secreted isoform in vivo due to the soluble/secreted CEACAM1(a) with a frameshift sequence in the C-terminus, specific for CEACAM1(a)-2C1 and -4C1. CEACAM1(a)-4C1 was capable of binding murine hepatitis virus (MHV) and was detected at approximately 120kDa in the small intestinal secretions. Neutralizing effects of the soluble CEACAM1(a) on MHV infectivity in vitro were demonstrated by using recombinant CEACAM1(a)-4C1. Our data suggest an intrinsic mechanism operated by free CEACAM1 for surveillance of pathogens and maintenance of homeostasis in the intestine.

To study the spinning condition of natural biopolymer silk, the silk fibers were directly acquired from Bombyx mori silkworm, N140 x C140 by a simple artificial forcibly silking method at the speed of 60, 120, 180 and 240 cm min(-1), respectively and its microstructure and physical properties were evaluated. The fine silk fibers (about 8 microm) were obtained at faster spinning speed, 240 cm min(-1). The tensile properties of silk fibers were remarkably increased with raising the forcibly spinning speeds. The beta-sheet structure contents of silk fibers obtained at higher speed were considerably increased. The fibers obtained by different spinning speeds exhibited a fairly similar X-ray crystallinity, while the degree of molecular orientation increased with decreasing the fiber diameter. The fine silk fibers obtained at higher speed (240 cm min(-1)) exhibited a slightly higher thermal stability, as shown by the upward shift of differential scanning calorimetry (DSC) decomposition temperature.

The concentration of polyacrylate (PA) used as a scale inhibitor was potentiometrically determined with a solid state copper ion-selective electrode after addition of Cu2+ as a probe. While the conventional methods monitor only the total concentration of PA, the proposed method measures the free, "effective" concentration of PA in equilibrium with species like Ca2+ and CaCO3. The slope of a potential response to PA was -40 mV decade(-1) and the limit of detection was 10(-6.3) M (= mol dm(-3)) at a probe concentration of 10(-6) M. The system could be used to control the PA concentration just enough to prevent the scale formation in various circulating water systems.

Cortical neural precursor cells (NPCs) sequentially undergo expansion, neurogenic and gliogenic phases during development, although the underlying mechanisms are poorly understood. Recent studies have identified a number of extrinsic factors that regulate the fate of NPCs. For example, we have shown that Wnt signaling induces neuronal differentiation of NPCs in an instructive manner. Importantly, Wnt signaling does so in late stage (neurogenic phase) of NPCs but not in early stage (expansion phase) of NPCs. Instead, Wnt signaling promotes proliferation of early NPCs. Likewise, STAT3-activating ligands induce astrocytic differentiation in late (gliogenic phase) but not in early (expansion and neurogenic phases) NPCs. These stage-dependent responses of NPCs might play a central role in determining the timing of differentiation and the size of final population of each differentiated cell type.

During development of the mammalian brain, many neural precursor cells (NPCs) undergo apoptosis. The regulation of such cell death, however, is poorly understood. We now show that the survival of mouse embryonic NPCs in vitro was increased by culture at a high cell density and that this effect was attributable to activation of Notch signaling. Expression of an active form of Notch1 thus markedly promoted NPC survival. Hes proteins, key effectors of Notch signaling in inhibition of neurogenesis, were not sufficient for the survival-promoting effect of Notch1. This effect of Notch1 required a region of the protein containing the RAM domain and was accompanied by up-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1. Moreover, knockdown of these proteins by RNA interference resulted in blockade of the Notch1-induced survival. These results reveal a new function of Notch, the promotion of NPC survival.

PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor alpha (TNFalpha) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-kappaB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580-584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-kappaB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-kappaB signaling pathway.

The aim of this study was to examine the changes in the tissue Cu/Zn- and the Mn-SOD contents and gene expression following mild and severe burns in a rodent burn model. Thirty-eight male Wistar rats, weighing 208-278g, were divided into a sham burn group and two burn groups, with one receiving burns to 35% of the body surface and the other to 60%. Twenty animals of the burn groups were monitored daily for 7 days after injuries to examine survival. Six animals in the sham, 35 or 60% burn group were sacrificed at 3h postburn, and the blood, lungs and kidneys were collected for a biological analysis. The Cu/Zn- and Mn-SOD contents of the tissue and plasma specimens were measured using ELISA. The mRNA expressions of Cu/Zn- and Mn-SOD were determined by a Northern blot analysis. The survival rate of the 60% burn group for 7 days was 30%, whereas the survival rate of the 35% burn group was 100%. The mRNA expressions of Mn-SODs in the lung and the kidney were significantly higher in the 60% burn group than in 35% burn or sham burn group, as was the mRNA expression of lung Cu/Zn-SOD. Nevertheless, the tissue SOD contents in the 60% burn group (mortality 70%) did not exceed those in the 35% group. Based on these findings, tissue SOD synthesis is thus suggested to be inhibited in lethal burns in spite of a strong mRNA expression of SOD.

For field emitters with a given surface condition, but different apex radii, there exists an empirical linear relationship between the slopes and intercepts of their Fowler-Nordheim plots. This behavior is quite different from that predicted by the theory with the assumption of a smooth surface. We have already characterized the possible reasons for this discrepancy; one possibility is that the emission area rapidly increases with increase in the apex radius. The previous report (Jpn. J. Appl. Phys. Part II 32 (1993) L342), however, showed a curved relation between the slope and the intercept of Fowler-Nordheim plots. In the present article, we try to extract the relationship between the emission area and the apex radius needed in order to give a linear relationship between slope and intercept of the F-N plots. As an additional result of the present analysis, the change in work function of the field emitting surface is demonstrated.

The reason for the increase of R21 which is defined by the intensity ratio of the doubly charged ions to the singly charged ions, was studied. Based on the conventional field evaporation theory, an increase in the electric field and/or the source temperature is considered to be attributable. We took the fact that R21 turned to decrease at the higher current regime into consideration and examined whether the change of R21 due to change in the field or temperature would finally meet the criterion for the decrease of R21. It was concluded that an increase of the source temperature may be a possible reason for the increase of R21.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) system was developed for human soluble interleukin-2 receptor (sIL-2R) with an ELISA-amplification system (ELAST((R))). The sensitivity of the new method was 20-fold higher than that without the amplification. Thus very low concentrations of sIL-2R in urine can be detected. With this method, serum and urine concentrations of sIL-2R were analyzed for healthy Japanese subjects with age 1-67 years. Mean sIL-2R concentrations in both serum and urine of children were significantly higher than those of adults. However, the concentrations of children showed a progressive decline to those of adults by the age of 15 years. There was no difference in the values between males and females. The results provide a control value of sIL-2R against those in disease states such as nephrotic syndrome. Since the trends in serum and urine were found to be similar, urinary sIL-2R measurement may provide sufficient information, without measuring the blood concentration.

This paper describes the chemical transformation of the basic 16-membered macrolides, tylosin derivatives, into neutral macrolides having a 3'-methoxyl group. 2',4'-Di-O-acetyl-3,23-bis(O-tert-butyldimethylsilyl)mycaminosyltylon olide 9,20-bis(ethylene acetal) N-oxide (1b) was treated with Ac2O-pyridine in CH2Cl2 to afford the 3'-ketone 1c and the 3'-N-acetyl-3'-N-demethyl derivative 1d in 67 and 5% yield; respectively. Reduction of 1c with Zn(BH4)2 gave the 3'-alcohol 1e in 84% yield stereoselectively. O-Methylation of 1e with MeOTf and 2,6-di-tert-butylpyridine gave the 3'-methyl ether 1f in 49% yield in spite of the presence of the adjacent acetoxyl groups. Deprotection of 1f provided the desired neutral macrolide 1g. Similar synthetic routes were also used for transformation of the suitably protected 4'-deoxymycaminosyltylonolide 2b and desmycosin 3c into neutral macrolides having a 3'-methoxyl group. It was found that the mycinose moiety of a neutral macrolide plays an important role in its antimicrobial activity.

The arginyl residue of solubilized silk fibroin was chemically modified with 1,2-cyclohexanedione in aqueous alkaline medium to form a stable imidazolidinone ring, and its positive charge was masked. CD spectra of the modified silk fibroin in aqueous solution showed an increase in the fraction of random coil conformation. The increase may be caused by the exposure to alkaline medium in the modification reaction. FT-IR and CD spectra of the silk fibroin films before and after the modification indicated that the conformational change in the modified silk fibroin in the solid state did not occur by the modification of its arginyl residue with 1,2-cyclohexanedione. The chemical stability of the modified silk fibroin film was investigated in vitro with phosphate-buffered saline solution. The modified arginyl residue in the film was stable in the phosphate-buffered solution.

N-Acetyl-chito-oligosaccharides (NACOS)-silk fibroin (SF) conjugates (NACOS-CY-SF) were prepared by the reaction of solubilized SF and cyanuric chloride (CY)-activated NACOS modifier (NACOS-CY). N-Acetyl-D-glucosamine (NAG), as a model compound, was reacted with CY to clarify the chemical structure of the modifier. The 1H- and 13C-NMR spectra of the reactant suggest that the anomeric hydroxyl group of NACOS reacted with the chlorine atom of CY. The content of NACOS in the NACOS-CY-SF conjugates was calculated by comparing the integral values of the signals in the 1H-NMR spectra of the conjugates and the mixture of NACOS and SF. As the 1H-NMR spectrum of the conjugates showed a downfield shift of the aromatic protons of the tyrosine residue, the tyrosine residue in SF reacted with another chlorine atom of the triazine ring of the modifier. The result of the amino acid analysis of the conjugates suggests that lysine residues also reacted with the modifier.

The attachment and growth of fibroblast cells (L-929) on matrices of silk fibroin from Bombyx mori domestic silkworm (DSF) and Antheraea pernyi wild silkworm (WSF) were studied by a cell culture method. The performance of the two kinds of silk fibroin was compared to that of collagen. DSF exhibited as high a cell attachment and growth as collagen did. The cells attached to DSF were extensively spread out and their filopodia were visible in the SEM pictures. WSF, which contains the tripeptide sequence Arg-Gly-Asp (believed to be a specific interaction site for cell-attachment), displayed much higher cell attachment and growth compared to DSF. The cells attached on WSF became virtually flat and their filopodia could be seen, indicating that the cells were very strongly held on the surface.

Chicken bone powder, sequentially extracted with 4M guanidine HCl (NG), 0.3M HCl (H), 0.5 NaCl at neutral pH (NS), and 4M guanidine HCl pH 7.4 (G), preferentially solubilized osteocalcin (OC), osteopontin (OPN), alpha 2 HS-glycoprotein, bone sialoprotein (BSP), and osteonectin (ON). These studies provide the first amino acid sequences for a non-mammalian form of ON and both N-terminal and internal amino acid sequences obtained for ON demonstrated that the avian form of this protein is more than 80% conserved for all the peptides sequenced in this study. A novel approximately 60 kDa protein was identified with a unique N-terminal amino acid sequence and several internal amino acid sequences. Amino acid composition of this protein indicates that it is similar to other acidic glycosylated phosphoprotein of bone, and it is phosphorylated principally on serine residues, although a small amount of phosphorylated threonine residues were also detected. Solubility characteristics of this protein differed from those of ON, OPN and Decorin, and, it was not recognized by polyclonal antibodies to ON, BSP, OPN or by a monoclonal antibody to Decorin. This protein was not present in chicken serum and no comparable sequences were found in the GenBank or EMBL protein sequence database. These studies provide the first sequence for a non-mammalian form of ON and identify a novel acidic bone protein.

Chemical modifications of silk fibroin were attempted in order to add new properties and functions to silk fibroin. The arginyl residue in solubilized silk fibroin was chemically modified with the reaction of 1,2-cyclohexanedione in borate buffer. FT-i.r. and c.d. spectra of the silk fibroin before and after the modification indicated that the fraction of random coil conformation increased with the modification. The chemical stability of the modified silk fibroin membrane was investigated in vitro with phosphate buffer. The modified arginyl residue in the membrane was considerably regenerated with the treatment in phosphate buffer.

Two phosphorylated proteins of approximately 66 kDa and approximately 60 kDa mass with different DEAE-Sephacel elution patterns were isolated from chicken bone and were shown to be genetically distinct by both biochemical and immunological analysis. A tryptic peptide from the 60 kDa protein was identified that was similar to a sequence of the rat bone sialoprotein II. Both proteins showed RGD inhibited cell-attachment with the MG-63 osteosarcoma cell, and the approximately 66 kDa phosphoprotein appeared to promote cell adhesion better than human vitronectin. The two phosphoproteins appear to share functional and biochemical characteristics and to be homologous to the mammalian bone phosphoproteins, osteopontin and bone sialoprotein II.

To evaluate the importance of the surrounding nucleotide sequence in the selection of a splice site for mRNA, we have carried out computer studies of eukaryotic protein genes whose entire nucleotide sequences were available. A splice site-like sequence that has a significant homology to the consensus splice junction sequences is frequently found within an intron and exon. It is found that the higher the homology of a candidate donor site sequence to the nine-nucleotide consensus sequence, the higher is its probability of being a donor site. For most of the donors, the stability of presumed base-pairing with U1-RNA is higher than that of donor-like sequences, if any, in the adjacent exon and intron. However, homology of a candidate acceptor sequence to the 15-nucleotide consensus is a poor criterion of an acceptor site. The presence of a sequence that could serve as a branch-point 18 to 37 nucleotides before an acceptor does not seem to be critical in distinguishing it from an acceptor-like sequence. For genes of human, rat, mouse and chicken, respectively, nucleotide frequencies around splice junctions of many genes have been calculated. They seem to be different at some positions around a donor site from species to species. The acceptors for these vertebrates have longer pyrimidine-rich regions than the previous consensus sequence. The newly derived nucleotide frequencies were used as the standard to calculate the weighted homology score of a candidate splice site sequence in a gene of the four species. This weighted homology score of the 40 to 60-nucleotide intron-exon sequence is a much better criterion of an acceptor. These results suggest that the most important signal in the selection of a splice resides in the surrounding nucleotide sequence. It is also suggested that the surrounding nucleotide sequence alone is not generally sufficient for the selection.

Effects of dietary cholesterol, beta-sitosterol and cholestyramine on hepatic HMG-CoA reductase activity and sterogenesis were examined in male rats refed different types and amounts of fats for 3 days after fasting 2 days. Safflower oil (10%) decreased reductase and sterogenic activities more than saturated fat or low fat. Reductase activity and sterogenesis decreased as dietary cholesterol increased; this was not influenced by the type of dietary fat. Although cholesterol as low as 0.01% depressed these activities, more cholesterol was required to deposit cholesterol in the livcreased cholesterol excretion as neutral steroids but failed to enhance reductase and sterogenic activities. In contrast, cholestyramine increased these activities to approximately the same level irrespective of the type and amount of dietary fat. Hepatic cholesterol decreased only in rats refed saturated fat; neutral or acidic steroid excretion was greatest in this group. Hepatic cholesterol and enterohepatically circulating cholesterol may not be critical factors in regulating HMG-CoA reductase in fasted-refed rats. Rather, the quantity of bile acids fluxed to the liver appears to influence the reductase activity in this situation. However, analyses of fecal acidic steroids provided no evidence for a relationship between HMG-CoA reductase activity, bile acid metabolism and dietary fat.

Chromosomes were studied in cultured T lymphocytes carrying adult T-cell leukemia-associated antigens (ATLA) that were obtained from five Japanese anti-ATLA seropositive healthy adults. Chromosomally abnormal cells were observed in three of the five healthy adults, and these cells were clonal in two subjects. All cells examined in one subject had rearrangements of chromosome nos. 7 and 14. Clonal cells from the second had a minute chromosome of unknown origin. A few cells in the third had nonclonal rearrangements of chromosomes. Thus, ATLA-positive T lymphocytes in some anti-ATLA seropositive healthy people have chromosome aberrations.

An improved and simpler plaque assay method for antibody-dependent cell-mediated cytotoxicity using Cunningham's chamber has been developed. Effector lymphocytes, target sheep red blood cells (SRBC) and anti-SRBC serum were incorporated together into a Cunningham's chamber and, immediately after making monolayers, incubated at 37 degrees C for 20 h. A hemolytic plaque of SRBC was formed around a lymphocyte in the presence of anti-SRBC serum, which was inhibited specifically by addition of aggregated IgG or Fc-fragment of human IgG in the culture. A hemolytic plaque formed around a monocyte-like cell was clearly differentiated from those around a lymphocyte (K-cell). The percentage of plaques in the purified lymphocytes from healthy individuals correlated significantly with ADCC activity measured by 51Cr-release. The method is a significant improvement on the earlier plaque assay methods with regard to: (1) making the monolayer, (2) observation and counting of plaques, and (3) shortening the time required for assay. Thus, the method should be valuable for clinical studies of cytotoxic activity in ADCC systems in healthy and diseased persons.

The clonally derived mouse osteoblast-like cell line MC3T3-E1 was shown to produce latent collagenase (approximately 0.2 units/ml) under stimulation with either heparin or parathyroid hormone in confluent cultures. However, it was found that MC3T3 E1 cultures which were first induced to undergo mineralization by the addition of beta-glycerophosphate and were subsequently stimulated with heparin showed an approximately ten-fold increase in collagenase synthesis. MC3T3-E1 cell collagenase from a small sample of serum-free culture medium was purified 49-fold to a specific activity of 200 units/mg protein with a yield of 14% by heparin-sepharose affinity chromatography and ion-exchange high performance liquid chromatography. This new mineralization-primed cell culture system may be a valuable model for the study of osteoblast collagenase.