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D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5'-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC 50  = 13 ± 1 μM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine β-synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures., (© 2023. Springer Nature Limited.)

Organoids are regarded as physiologically relevant cell models and useful for compound screening for drug development; however, their applications are currently limited because of the high cost of their culture. We previously succeeded in reducing the cost of human intestinal organoid culture using conditioned medium (CM) of L cells co-expressing Wnt3a, R-spondin1, and Noggin. Here, we further reduced the cost by replacing recombinant hepatocyte growth factor with CM. Moreover, we showed that embedding organoids in collagen gel, a more inexpensive matrix than Matrigel, maintains organoid proliferation and marker gene expression similarly when using Matrigel. The combination of these replacements also enabled the organoid-oriented monolayer cell culture. Furthermore, screening thousands of compounds using organoids expanded with the refined method identified several compounds with more selective cytotoxicity against organoid-derived cells than Caco-2 cells. The mechanism of action of one of these compounds, YC-1, was further elucidated. We showed that YC-1 induces apoptosis through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, the mechanism of which was distinct from cell death caused by other hit compounds. Our cost-cutting methodology enables large-scale intestinal organoid culture and subsequent compound screening, which could expand the application of intestinal organoids in various research fields., (© 2023. The Author(s).)

Mycobacterium abscessus species (MABS) is the most commonly isolated rapidly growing mycobacteria (RGM) and is one of the most antibiotic-resistant RGM with rapid progression, therefore, treatment of MABS is still challenging. We here presented a new combination treatment with sitafloxacin that targeted rough morphotypes of MABS, causing aggressive infections. Thirty-four clinical strains of MABS were isolated from various clinical samples at the Juntendo university hospital from 2011 to 2020. The susceptibility to a combination of sitafloxacin and antimicrobial agents was compared to that of the antimicrobial agents alone. Out of 34 MABS, 8 strains treated with sitafloxacin-amikacin combination, 9 of sitafloxacin-imipenem combination, 19 of sitafloxacin-arbekacin combination, and 9 of sitafloxacin-clarithromycin combination showed synergistic effects, respectively. Sitafloxacin-arbekacin combination also exhibited the synergistic effects against 10 of 22 Mycobacterium abscessus subspecies massiliense (Mma) strains and 8 of 11 Mycobacterium abscessus subspecies abscessus (Mab) strains, a highly resistant subspecies of MABS. The sitafloxacin-arbekacin combination revealed more synergistic effects in rough morphotypes of MABS (p = 0.008). We demonstrated the synergistic effect of the sitafloxacin-arbekacin combination against MABS. Further, this combination regimen might be more effective against Mab or rough morphotypes of MABS., (© 2023. The Author(s).)

The study of cooperation has been extensively studied in game theory. Especially, two-player two-strategy games have been categorized according to their equilibrium strategies and fully analysed. Recently, a grand unified game covering all types of two-player two-strategy games, i.e., the weightlifting game, was proposed. In the present study, we extend this two-player weightlifting game into an [Formula: see text]-player game. We investigate the conditions for pure strategy Nash equilibria and for Pareto optimal strategies, expressed in terms of the success probability and benefit-to-cost ratio of the weightlifting game. We also present a general characterization of [Formula: see text]-player games in terms of the proposed game. In terms of a concrete example, we present diagrams showing how the game category varies depending on the benefit-to-cost ratio. As a general rule, cooperation becomes difficult to achieve as group size increases because the success probability of weightlifting saturates towards unity. The present study provides insights into achieving behavioural cooperation in a large group by means of a cost-benefit analysis., (© 2022. The Author(s).)

A delay in the diagnosis of urinary tract infection (UTI) is not uncommon. Atypical presentation is often cited as one of the causes of diagnostic delays. However, few studies have investigated the prevalence of atypical presentation and determined factors associated with atypical presentation at initial contact among patients with UTI. Therefore, a retrospective and prospective cohort study using chart review was conducted in two acute care hospitals. We included 285 consecutive patients hospitalized for bacteremic UTI. The primary outcome was atypical presentation, defined as the absence of any urinary tract symptom or sign at initial contact. Of all patients, the median age was 82 years, 186 (65.3%) were women, and 53 (18.6%) had dementia. Urinary tract symptoms and signs were absent at initial contact in 144 patients (50.5%; 95% CI 44.7-56.4%). The multivariable analysis revealed that older age, male sex, dementia, and early visit from symptom onset were significantly associated with an increased risk of atypical presentation. Patients with atypical presentation were less likely to receive a correct diagnosis at initial contact than patients with urinary tract symptoms and signs (OR 0.30; 95% CI 0.17-0.51). Atypical presentation in patients with bacteremic UTI is common and negatively affects the correct diagnosis of UTI., (© 2022. The Author(s).)

Novel coronavirus disease 2019 (COVID-19) has emerged as a global pandemic with far-reaching societal impact. Here we demonstrate that Pin1 is a key cellular molecule necessary for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) propagation. In this study, siRNA-mediated silencing of Pin1 expression markedly suppressed the proliferation of SARS-CoV-2 in VeroE6/TMPRSS2 cells. In addition, several recently generated Pin1 inhibitors showed strong inhibitory effects on SARS-CoV-2 proliferation, measured by both viral mRNA and protein synthesis, and alleviated the cytopathic effect (CPE) on VeroE6/TMPRSS2 cells. One compound, termed H-77, was found to block SARS-CoV-2 proliferation at an EC 50 below 5 μM regardless of whether it was added to the culture medium prior to or after SARS-CoV-2 infection. The inhibition of viral N protein mRNA synthesis by H-77 implies that the molecular mechanism underlying SARS-CoV-2 inhibition is likely to be associated with viral gene transcription or earlier steps. Another Pin1 inhibitor, all-trans retinoic acid (ATRA)-a commercially available drug used to treat acute promyelocytic leukemia (APL) and which both activates the retinoic acid receptor and inhibits the activity of Pin1-similarly reduced the proliferation of SARS-CoV-2. Taken together, the results indicate that Pin1 inhibitors could serve as potential therapeutic agents for COVID-19., (© 2021. The Author(s).)

Insecticide resistance has recently become a serious problem in the agricultural field. Development of insecticides with new mechanisms of action is essential to overcome this limitation. Juvenile hormone (JH) is an insect-specific hormone that plays key roles in maintaining the larval stage of insects. Hence, JH signaling pathway is considered a suitable target in the development of novel insecticides; however, only a few JH signaling inhibitors (JHSIs) have been reported, and no practical JHSIs have been developed. Here, we established a high-throughput screening (HTS) system for exploration of novel JHSIs using a Bombyx mori cell line (BmN_JF&AR cells) and carried out a large-scale screening in this cell line using a chemical library. The four-step HTS yielded 69 compounds as candidate JHSIs. Topical application of JHSI48 to B. mori larvae caused precocious metamorphosis. In ex vivo culture of the epidermis, JHSI48 suppressed the expression of the Krüppel homolog 1 gene, which is directly activated by JH-liganded receptor. Moreover, JHSI48 caused a parallel rightward shift in the JH response curve, suggesting that JHSI48 possesses a competitive antagonist-like activity. Thus, large-scale HTS using chemical libraries may have applications in development of future insecticides targeting the JH signaling pathway.

There are a few studies about the clinical impacts of plasma B-type natriuretic peptide (BNP) at discharge with the occurrence of worsening renal function (WRF) on mortality in patients with heart failure (HF). We divided total 301 patients with acute decompensated HF into four groups by the median value (278.7 pg/mL) of BNP level at discharge and by the occurrence of WRF. WRF developed in 100 patients (33.2%). Cardiovascular mortality was significantly different between the four groups (P = 0.0002). Patients with WRF and elevated BNP had a higher cardiovascular mortality than patients without WRF and elevated BNP in Cox proportional hazard models (hazard ratio [HR], 10.48; 95% confident interval [95% CI], 1.27-225.53; P = 0.03). Patients with either WRF or elevated BNP did not have an increased risk of cardiovascular mortality compared to patients without WRF and elevated BNP. Regarding HF readmission and cardiovascular mortality, patients with WRF and elevated BNP had the highest risk (HR, 5.17; 95% CI, 2.07-14.30, P = 0.0003) and patients with either WRF or elevated BNP had a higher risk than patients without WRF and elevated BNP. The occurrence of WRF combined with elevated BNP at discharge was associated with increased 1-year cardiovascular mortality and HF readmission.

Due to the globalization and computerization of financial and economic activities, numerous repetitive leveraged investments have become possible in stock markets and currency exchanges. In reality, repeated leveraged investments up to 100 times/day are possible via online access. With computer-aided programs, this repetition number may easily increase 1000 times/day. The possibility of bankruptcy in repeated leveraged investments has never been considered in actual practices because the probability of bankruptcy in a single investment trial is almost negligible. Here, we show that the extremely numerous repetitions have a considerable chance of bankruptcy overall, even if the probability of bankruptcy for a single investment is extremely close to zero. The exact relationship between the repetitions and the probability of bankruptcy is approximated well by n(0.63) = m, where 10 n is the number of repetitions, 10 -m is the bankruptcy probability of a single investment, and n(0.63) is the 63% chance of bankruptcy. Thus, extremely rare events can always lead to bankruptcy in continuously repeated investment, even if the possibility of such an event is almost null. We suggest that the avoidance measure of bankruptcy is necessary in numerous repeated investments even if a single trial is almost certain to win.

In grassland studies, an intermediate level of grazing often results in the highest species diversity. Although a few hypotheses have been proposed to explain this unimodal response of species diversity to grazing intensity, no convincing explanation has been provided. Here, we build a lattice model of a grassland community comprising multiple species with various levels of grazing. We analyze the relationship between grazing and plant diversity in grasslands under variable intensities of grazing pressure. The highest species diversity is observed at an intermediate grazing intensity. Grazers suppress domination by the most superior species in birth rate, resulting in the coexistence of inferior species. This unimodal grazing effect disappears with the introduction of a small amount of nongrazing natural mortality. Unimodal patterns of species diversity may be limited to the case where grazers are the principal source of natural mortality.

Melanoma is an aggressive skin cancer that originates from melanocytes and, especially in the case of early-stage melanoma, is distributed adjacent to the epidermis and superficial dermis. Although early-stage melanoma can be distinguished from benign nevus via a dermoscopy, it is difficult to distinguish invasive melanoma in its early stages from in situ melanoma. Because invasive melanoma must undergo a sentinel lymph node biopsy to be diagnosed, a non-invasive method to detect the micro-invasion of early-stage melanoma is needed for dermato-oncologists. This paper proposes a novel quantitative melanoma identification method based on accurate measurements of thermal conductivity using a pen-shaped device. This method requires skin temperature data for one minute to determine the effective thermal conductivity of the skin, allowing it to distinguish melanoma lesions from healthy skin. Results suggest that effective thermal conductivity was negative for in situ melanoma. However, in accordance with tumour progression, effective thermal conductivity was larger in invasive melanoma. The proposed thermal conductivity measurement is a novel tool that detects the micro-invasion of melanoma.

Predator-prey systems have been studied intensively for over a hundred years. These studies have demonstrated that the dynamics of Lotka-Volterra (LV) systems are not stable, that is, exhibiting either cyclic oscillation or divergent extinction of one species. Stochastic versions of the deterministic cyclic oscillations also exhibit divergent extinction. Thus, we have no solution for asymptotic stability in predator-prey systems, unlike most natural predator-prey interactions that sometimes exhibit stable and persistent coexistence. Here, we demonstrate that adding a small immigration into the prey or predator population can stabilize the LV system. Although LV systems have been studied intensively, there is no study on the non-linear modifications that we have tested. We also checked the effect of the inclusion of non-linear interaction term to the stability of the LV system. Our results show that small immigrations invoke stable convergence in the LV system with three types of functional responses. This means that natural predator-prey populations can be stabilized by a small number of sporadic immigrants.

Aspergillus oryzae is an industrially important filamentous fungus used for Japanese traditional food fermentation and heterologous protein production. Although cell fusion is important for heterokaryon formation and sexual/parasexual reproduction required for cross breeding, knowledge on cell fusion and heterokaryon incompatibility in A. oryzae is limited because of low cell fusion frequency. Therefore, we aimed to develop a BiFC system to specifically visualise fused cells and facilitate the analysis of cell fusion in A. oryzae. The cell fusion ability and morphology of 15 A. oryzae strains were investigated using heterodimerising proteins LZA and LZB fused with split green fluorescence protein. Morphological investigation of fused cells revealed that cell fusion occurred mainly as conidial anastomosis during the early growth stage. Self-fusion abilities were detected in most industrial A. oryzae strains, but only a few strain pairs showed non-self fusion. Protoplast fusion assay demonstrated that almost all the pairs capable of non-self fusion were capable of heterokaryon formation and vice versa, thus providing the first evidence of heterokaryon incompatibility in A. oryzae. The BiFC system developed in this study provides an effective method in studying morphology of fused cells and heterokaryon incompatibility in the filamentous fungal species with low cell fusion efficiency.

Classical Lotka-Volterra (LV) competition equation has shown that coexistence of competitive species is only possible when intraspecific competition is stronger than interspecific competition, i.e., the species inhibit their own growth more than the growth of the other species. Note that density effect is assumed to be linear in a classical LV equation. In contrast, in wild populations we can observed that mortality rate often increases when population density is very high, known as crowding effects. Under this perspective, the aggregation models of competitive species have been developed, adding the additional reduction in growth rates at high population densities. This study shows that the coexistence of a few species is promoted. However, an unsolved question is the coexistence of many competitive species often observed in natural communities. Here, we build an LV competition equation with a nonlinear crowding effect. Our results show that under a weak crowding effect, stable coexistence of many species becomes plausible, unlike the previous aggregation model. An analysis indicates that increased mortality rate under high density works as elevated intraspecific competition leading to the coexistence. This may be another mechanism for the coexistence of many competitive species leading high species diversity in nature.

Quantum entanglement in magnetic materials is expected to yield a quantum spin liquid (QSL), in which strong quantum fluctuations prevent magnetic ordering even at zero temperature. This topic has been one of the primary focuses of condensed-matter science since Anderson first proposed the resonating valence bond state in a certain spin-1/2 frustrated magnet in 1973. Since then, several candidate materials featuring frustration, such as triangular and kagome lattices, have been reported to exhibit liquid-like behavior. However, the mechanisms that stabilize the liquid-like states have remained elusive. Here, we present a QSL state in a spin-1/2 honeycomb lattice with randomness in the exchange interaction. That is, we successfully introduce randomness into the organic radial-based complex and realize a random-singlet (RS) state (or valence bond glass). All magnetic and thermodynamic experimental results indicate the liquid-like behaviors, which are consistent with those expected in the RS state. Our results suggest that the randomness or inhomogeneity in the actual systems stabilize the RS state and yield liquid-like behavior.

[This corrects the article DOI: 10.1038/s41522-017-0026-1.].

Formation of bacterial biofilms on medical devices can cause severe or fatal infectious diseases. In particular, biofilm-associated infections caused by methicillin-resistant Staphylococcus aureus are difficult to eradicate because the biofilm is strongly resistant to antibiotics and the host immune response. There is no effective treatment for biofilm-associated infectionss, except for surgical removal of contaminated medical devices followed by antibiotic therapy. Here we show that norgestimate, an acetylated progestin, effectively inhibits biofilm formation by staphylococcal strains, including methicillin-resistant S. aureus , without inhibiting their growth, decreasing the selective pressure for emergence of resistance. 17-Deacetyl norgestimate, a metabolite of norgestimate, shows much weaker inhibitory activity against staphylococcal biofilm formation, indicating that the acetyl group of norgestimate is important for its activity. Norgestimate inhibits staphylococcal biofilm formation by inhibiting production of polysaccharide intercellular adhesin and proteins in the extracellular matrix. Proteome analysis of S . aureus indicated that norgestimate represses the expression of the cell wall-anchored protein SasG, which promotes intercellular adhesion, and of the glycolytic enzyme enolase, which plays a secondary role in biofilm formation. Notably, norgestimate induces remarkable changes in cell wall morphology, characterized by increased thickness and abnormal rippled septa. Furthermore, norgestimate increases the expression level of penicillin binding protein 2 and resensitizes methicillin-resistant S. aureus to β-lactam antibiotics. These results suggest that norgestimate is a promising lead compound for the development of drugs to treat biofilm-associated infections, as well as for its ability to resensitize methicillin-resistant S. aureus to β-lactam antibiotics.

The main components of the quorum-sensing system are expected to be favorable targets for drug development to combat various chronic infectious diseases. ComA of Streptococcus is an ATP-binding cassette transporter containing a peptidase domain (PEP), which is essential for the quorum-sensing signal production. Using high-throughput screening, we found a potent small molecule that suppressed the S. mutans quorum-sensing pathway through inhibition of PEP activity. The compound effectively attenuated the biofilm formation and competence development of S. mutans without inhibiting cell growth. The kinetic and structural studies with this molecule and a related compound unexpectedly revealed an allosteric site of PEP. This relatively hydrophobic site is thought to undergo large structural changes during the catalytic process. These compounds inhibit PEP activity by binding to and suppressing the structural changes of this site. These results showed that PEP is a good target for inhibitors of the Streptococcus quorum-sensing system.

Bivalve, ammonite and snail shells are described by a small number of geometrical parameters. Raup noted that the vast majority of theoretically possible shell forms do not occur in nature. The constraint factors that regulate the biased distribution of natural form have long since been an open problem in evolution. The problem of whether natural shell form is a result of optimization remains unsolved despite previous attempts. Here we solve this problem by considering the scaling exponent of shell thickness as a morphological parameter. The scaling exponent has a drastic effect on the optimal design of shell shapes. The observed characteristic shapes of natural shells are explained in a unified manner as a result of optimal utilization of shell material resources, while isometric growth in thickness leads to impossibly tight coiling., Competing Interests: The authors declare no competing financial interests.

Very recent studies indicate that sulfur atoms with oxidation state 0 or -1, called sulfane sulfurs, are the actual mediators of some physiological processes previously considered to be regulated by hydrogen sulfide (H 2 S). 3-Mercaptopyruvate sulfurtransferase (3MST), one of three H 2 S-producing enzymes, was also recently shown to produce sulfane sulfur (H 2 S n ). Here, we report the discovery of several potent 3MST inhibitors by means of high-throughput screening (HTS) of a large chemical library (174,118 compounds) with our H 2 S-selective fluorescent probe, HSip-1. Most of the identified inhibitors had similar aromatic ring-carbonyl-S-pyrimidone structures. Among them, compound 3 showed very high selectivity for 3MST over other H 2 S/sulfane sulfur-producing enzymes and rhodanese. The X-ray crystal structures of 3MST complexes with two of the inhibitors revealed that their target is a persulfurated cysteine residue located in the active site of 3MST. Precise theoretical calculations indicated the presence of a strong long-range electrostatic interaction between the persulfur anion of the persulfurated cysteine residue and the positively charged carbonyl carbon of the pyrimidone moiety of the inhibitor. Our results also provide the experimental support for the idea that the 3MST-catalyzed reaction with 3-mercaptopyruvate proceeds via a ping-pong mechanism.

At first glance, the trailing tentacles of a jellyfish appear to be randomly arranged. However, close examination of medusae has revealed that the arrangement and developmental order of the tentacles obey a mathematical rule. Here, we show that medusa jellyfish adopt the best strategy to achieve the most uniform distribution of a variable number of tentacles. The observed order of tentacles is a real-world example of an optimal hashing algorithm known as Fibonacci hashing in computer science.

Plant leaves are arranged around a stem axis in a regular pattern characterized by common fractions, a phenomenon known as phyllotaxis or phyllotaxy. As plants grow, these fractions often transition according to simple rules related to Fibonacci sequences. This mathematical regularity originates from leaf primordia at the shoot tip (shoot apical meristem), which successively arise at fixed intervals of a divergence angle, typically the golden angle of 137.5°. Algebraic and numerical interpretations have been proposed to explain the golden angle observed in phyllotaxis. However, it remains unknown whether phyllotaxis has adaptive value, even though two centuries have passed since the phenomenon was discovered. Here, I propose a new adaptive mechanism explaining the presence of the golden angle. This angle is the optimal solution to minimize the energy cost of phyllotaxis transition. This model accounts for not only the high precision of the golden angle but also the occurrences of other angles observed in nature. The model also effectively explains the observed diversity of rational and irrational numbers in phyllotaxis.

A critical issue in polymer-based solar cells (PSCs) is to improve the power conversion efficiency (PCE) as well as the stability. Here, we describe the development of new semiconducting polymers consisting of thiophene, thiazolothiazole and naphthobisthiadiazole in the polymer backbone. The polymers had good solubility and thus solution-processability, appropriate electronic structure with narrow band gaps of ~1.57 eV and low-lying HOMO energy levels of ~-5.40 eV, and highly ordered structure with the favorable face-on backbone orientation. Solar cells based on the polymers and PC71BM exhibited quite high PCEs of up to 9%. More interestingly, the cells also demonstrated excellent stability as they showed negligible degradation of PCE when stored at 85˚C for 500 hours in the dark under nitrogen atmosphere. These results indicate that the newly developed polymers are promising materials for PSCs in the practical use.

During screening for inhibitors of ras-mediated differentiation of PC12 cells, trichostatin A (TSA) was isolated from the metabolites of Streptomyces as a potent inhibitor. TSA blocked both oncogenic ras- and NGF-induced neurite outgrowth from PC12 cells. However, addition of TSA 1 h after NGF-stimulation did not inhibit neuronal differentiation, suggesting that TSA affects an early step in the NGF-signaling pathway mediated by ras. Northern blotting analysis showed that TSA prolonged the maximum expression period of c=fos mRNA triggered by NGF and delayed its return to the basal level. TSA reduced c-jun mRNA induction by NGF but greatly enhanced c-myc mRNA induced by NGF. Yoshida et al. (J. Biol. Chem, 265, 17174-17179, 1990) showed that TSA inhibits histone deacetylation, which might influence the gene expression involved in cellular differentiation. In this study, we also found that TSA prevents histone deacetylation in PC12 cells as well as other cell lines, suggesting that inhibition of histone deacetylation by TSA might affect the expression of early-response genes. We also demonstrated that TSA induced reversion of oncogenic ras-transformed NIH3T3 cells to a normal morphology, suggesting that inhibitors of ras-mediated differentiation of PC12 cells may be effective as anticancer agents.

Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [3H]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression. tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.

BE-23372M, a novel protein tyrosine kinase inhibitor, was isolated from the culture broth of a fungus. The producing strain, F23372, was identified as Rhizoctonia solani, based on the cultural and morphological characteristics. The active principle was extracted from the mycelium with acetone and purified by solvent extraction, silica gel column chromatography and Sephadex LH-20 column chromatography. BE-23372M showed strong inhibitory activity against EGF receptor kinase with IC50 values of 0.02 and 0.03 microM on two different substrates, whereas IC50 values against protein kinase C and cAMP-dependent protein kinase were 4.5 and > 20 microM, respectively. The compound inhibited the growth of A431 human epidermoid carcinoma and MKN-7 human stomach cancer cell lines with IC50 values of 8 and 24 microM, respectively.

BE-23372M, a new protein tyrosine kinase inhibitor, has been obtained as a reddish orange solid. The compound, C17H12O6, HRFAB-MS: m/z 312.0625 (M)+, is an acidic substance, showing UV (MeOH) lambda max (epsilon) 266 (8,800), 426 nm (20,400), and IR (KBr)vmax 1752(C = O) and 3298(OH) cm-1. The structure of BE-23372M, (E)-3-(3,4-dihydroxybenzylidene)-5-(3,4-dihydroxyphenyl)-2(3H)-fur anone, has been elucidated by 1H and 13C NMR studies.

In a preceding paper, the physico-chemical properties and structural elucidation of BE-23372M, a potent novel protein tyrosine kinase inhibitor, were described. In this paper, we report the synthesis of BE-23372M from 3-(3,4-dimethoxybenzoyl)propionic acid and veratraldehyde or 3,4-diacetoxy-benzaldehyde. The structure of BE-23372M was confirmed to be (E)-3-(3,4-dihydroxybenzylidene)-5-(3,4-dihydroxyphenyl)-2(3H)-fur anone.

We screened cdc2 kinase inhibitors from cultured mediums of micro organisms using purified mouse cyclin B-cdc2 kinase and a specific substrate peptide for cdc2 kinase. A selective inhibitor of cdc2 kinase was isolated from the cultured medium of Aspergillus species F-25799, and identified as butyrolactone I. Butyrolactone I inhibited cdc2 and cdk2 kinases but it had little effect on mitogen-activated protein kinase, protein kinase C, cyclic-AMP dependent kinase, casein kinase II, casein kinase I or epidermal growth factor-receptor tyrosine kinase. Its inhibitory effect was found to be due to competition with ATP. Butyrolactone I selectively inhibited the H1 histone phosphorylation in nuclear extracts. It also inhibited the phosphorylation of the product of retinoblastoma susceptibility gene in nuclear extracts and intact cells. Thus butyrolactone I should be very useful for elucidating the function of cdc2 and cdk2 kinases in cell cycle regulation.

The growth-suppressive activity of the retinoblastoma (RB) protein is suggested to be regulated by phosphorylation. In studies on the kinase that phosphorylates the RB proteins, we have previously found that RB proteins can be phosphorylated by purified cdc2 kinase. In this study, we noted that RB proteins immunoprecipitated from human cell lysates are weakly phosphorylated in the absence of purified cdc2 kinase. Immunoblot analysis showed the presence of p34cdc2 in the immunoprecipitates with anti-RB monoclonal antibody. In addition, the coprecipitated kinase was found to have the same substrate specificity as cdc2 kinase. The associated kinase activity was particularly high in cells arrested in G1/S and S phase by aphidicolin. Furthermore, RB proteins were shown to be phosphorylated in nuclear extracts by some endogenous cdc2-like kinase(s). These results suggest that cdc2-like kinase is the main kinase for phosphorylation of RB proteins in vivo.

The relationship between the activity and the chemical structure of cadeguomycin (CDM, 7-carboxy-7-deazaguanosine) was studied with six analogs of CDM. Both activities of CDM, enhancing the incorporation of [3H]thymidine in K562 cells and potentiating the cytotoxicity of cytosine arabinoside for K562 cells, were significantly augmented by the replacement of the 7-carboxyl group with cyano (CDM-CN) or formyl (CDM-CHO), but they were not changed by the replacement with methyl. The activities were almost completely diminished by the replacement of ribose with arabinose, but the simultaneous replacement of carboxyl and ribose with formyl and arabinose showed higher activities than those of CDM. The replacement of 7-carboxy-7-deazaguanine with 7-carboxy-7-deazainosine markedly weakened the activity. CDM-CN and CDM-CHO at 0.2 micrograms/ml significantly potentiated the activity of cytosine arabinoside against MOLT-3 cells but CDM at 1 micrograms/ml did not. These results indicate that the ribose and guanine moieties in the CDM molecule are very important for its activity. Also replacing the carboxyl group at the C-7 position with cyano or formyl group is a useful way to strengthen the CDM activity. These compounds would effectively potentiate cytosine arabinoside against various kinds of tumor cells which CDM could not do.

Resorthiomycin was revealed to be a new antibiotic with a molecular weight of 284 and a chemical formula of C14H20O4S as determined by MS and elemental analysis. The structure of resorthiomycin was determined to be 6-acetyl-4-(3-hydroxybutyl)-2-methyl-5-methylthioresorcinol by IR spectrum and 1H and 13C NMR.

Resorthiomycin, a novel antitumor antibiotic, was isolated from the fermentation broth of a strain of Streptomyces collinus by ethyl acetate extraction, silica gel chromatography and HPLC. Resorthiomycin exhibited an in vitro cytotoxic activity against mouse leukemia L5178Y cells (IC50, 15.5 micrograms/ml) and also inhibited the clonogenic activity of a multidrug-resistant mutant of human hepatoma PLC/PRF/5 cells to a greater extent than that of the parental cells. On the other hand, this antibiotic does not possess any antibacterial or antifungal activity.

An actinomycete strain IM7912T was found to produce cadeguomycin, a new nucleoside analog antibiotic, together with tubercidin. Morphological, cultural and physiological studies showed that the organism belongs to the species Streptomyces hygroscopicus. Comparisons with S. hygroscopicus ISP 5578 revealed that both strains possess similar characteristics except for production of yellow or brownish yellow soluble pigment in some media. Cadeguomycin was isolated from the culture filtrate, separated from tubercidin, and purified.

Lactoquinomycin, a novel basic antibiotic, was isolated from the culture broth of a soil streptomyces by repeated solvent extraction and adsorption column chromatography. Morphological, cultural and physiological studies revealed that the organism belongs to the species Streptomyces tanashiensis. The antibiotic was active against bacteria, particularly Gram-positive organisms, and neoplastic cells in vitro. Antibiotic-resistant cell sublines of L5178Y lymphoblastoma were more significantly inhibited by lactoquinomycin than the parental cell line. Lactoquinomycin was effective against Ehrlich carcinoma in mice.

Cadeguomycin retarded growth of sc solid IMC carcinoma in CDF1 mice, and pulmonary metastasis of Lewis lung carcinoma in C57BL/6 mice. The antibiotic enhanced phagocytic activity of murine peritoneal macrophages and IL-1 production by P388D1 cells. Delayed type hypersensitivity was stimulated and interferon was induced by the drug. The results suggest that cadeguomycin inhibits tumor growth and metastasis in association with modification of the immune system. The cytotoxicity of arabinosylcytosine to K562 and YAC-1 cells was markedly enhanced by cadeguomycin in culture. The combined administration of arabinosylcytosine and cadeguomycin displayed potentiation in the inhibition of growth of ip-implanted P388 leukemia and metastasis of sc-implanted P388 leukemia to the regional lymph nodes. Cadeguomycin showed low toxicity for mice.

Lactoquinomycin, a new antibiotic, C24H27NO8, mp 151 approximately 159 degrees C (dec), FAB-MS: m/z 458 (MH+), is a basic substance, showing UV lambda MeOHmax (epsilon) 215 (37,600), 254 (10,700) and 432 nm (4,760), and IR nu CHCl3max 1790 (gamma-lactone), 1665 and 1650 (quinone) cm-1. The structure of lactoquinomycin has been elucidated by 1H NMR and ORD in comparison with those of kalafungin.

An antibiotic, identical with naphthomycin, was isolated from a soil Streptomyces. The antibiotic displayed significant therapeutic activity by ip administration against murine tumors: Ehrlich carcinoma and IMC carcinoma implanted ip. The maximum increase of life-span was more than 169% in Ehrlich carcinoma, and 128% in IMC carcinoma. The antibiotic exhibited a potent cytotoxicity against murine leukemic cells: P388, L1210, and L5178Y. IC50 was 0.4-1.3 microgram/ml in culture. The activity of naphthomycin was reversed by SH compounds: 2-mercaptoethanol, dithiothreitol, and glutathione. DNA and RNA syntheses were more markedly inhibited by naphthomycin than protein synthesis in L5178Y cells. Approximately 50% inhibition of nucleic acid syntheses was observed at an antibiotic concentration of 2 micrograms/ml. Naphthomycin blocked alkaline phosphodiesterase obtained from L5178Y cells: IC50 was ca. 7.6 micrograms/ml. The antibiotic neither caused metaphase arrest nor prevented tubulin polymerization. The results suggest that the mechanism of cytotoxicity of naphthomycin is the inhibition of various SH enzymes, particularly those involved in nucleic acid biosynthesis. The mode of action is unique in the ansamycin group of antibiotics.

Cadeguomycin markedly stimulated the uptake of thymidine, deoxycytidine and uridine into the acid-insoluble fraction of K562 human leukemic cells, but did not significantly affect adenosine incorporation. The enhancement of pyrimidine nucleoside uptake was 6 approximately 17 fold over the control. Aspartate incorporation into nucleic acid was not significantly blocked by the antibiotic, suggesting that the stimulation of pyrimidine nucleoside incorporation is not due to the inhibition of de novo pyrimidine nucleotide synthesis. Net DNA and RNA syntheses, observed by [32P]phosphate uptake, were not significantly affected by cadeguomycin. The enzymatic activity of thymidine, deoxycytidine and uridine kinases was higher in cadeguomycin-treated cells than in untreated cells, suggesting that the enhancement of pyrimidine nucleoside uptake occurs in the phosphorylation process. The stimulatory activity of cadeguomycin of thymidine uptake was reversed by guanosine and deoxyguanosine, but not by adenosine and deoxyadenosine, suggesting that intracellular metabolism and/or action of cadeguomycin is related to that of guanosine and deoxyguanosine. The stimulation of pyrimidine nucleoside incorporation by cadeguomycin was also found with YAC-1 cells, but not with the other cell lines. The enhancement effect of the antibiotic seems to be not directly related to its cytotoxicity.

Cadeguomycin, a new nucleoside analog antibiotic, has been purified as colorless needle crystals by recycling preparative HPLC. The antibiotic, C12H14O7N4, mp 231 approximately 239 degrees C (dec.), FD-MS: m/z 326 (M+); is a weakly acidic substance, showing UV gamma H2Omax (epsilon) 232 (19677), 272 (6881) and 298 nm (7607), and IR nu KBrmax 1650 (C = O) and 3420 (NH or OH) cm-1. The UV spectrum is similar to other pyrrolo[2,3-d]pyrimidines. The structure of cadeguomycin, 2-amino-3,4-dihydro-4-oxo-7-beta-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine-5- carboxylic acid, has been elucidated by 1H NMR and 13C NMR in comparison with other pyrrolo[2,3-d]pyrimidines and their nucleosides.

Lactoquinomycin A (LQM-A), an antibiotic containing a quinone moiety in the molecule, inhibited biosyntheses of DNA, RNA and protein to a similar extent in doxorubicin-resistant mouse leukemia L5178Y cells at concentrations higher than 0.08 micrograms/ml. The antibiotic caused cell death in a short period of incubation and the degree of cell death correlated with that of the inhibition of macromolecular syntheses, suggesting that the inhibition of macromolecular syntheses was not a primary effect of LQM-A. LQM-A served as a good electron acceptor, when cytochrome c reductase was used as a quinone reductase. The treatment of the cells with LQM-A significantly reduced cellular NADH and ATP levels. The generation of superoxide radical by LQM-A in cell lysate was observed by reduction of nitro blue tetrazolium, and the production of hydroxyl radical was confirmed by electron spin resonance. The importance of radical formation for the cytotoxicity of LQM-A is discussed.

Streptomyces tanashiensis IM8442T was found to produce lactoquinomycin B, a novel antibiotic, together with lactoquinomycin A. Lactoquinomycin B was purified, and the physico-chemical and biological characteristics were studied. Lactoquinomycin B, C24H27NO9, mp 149-152 degrees C (dec), FD-MS m/z 473 (M+), is a basic substance, showing UV lambda MeOHmax (epsilon) 239 (15,100), 287 (3,450) and 369 nm (5,300), and IR nu CHCl3max 1790 (gamma-lactone), and 1700 and 1650 (quinone) cm-1. The structure of lactoquinomycin B was elucidated by 1H NMR and 13C NMR in comparison with those of lactoquinomycin A, indicating that B is a 4a,10a-epoxide derivative of A. Lactoquinomycin B displayed inhibitory activity against bacteria, particularly Gram-positive organisms, and cytotoxicity against human and murine tumor cell lines. LD50 for mice was ca. 40 mg/kg by iv route.