Epidemiological study of indirect blood pressure measured using oscillometry in clinically healthy cats at initial evaluation.

We investigated the clinical characteristics of healthy cats in accordance with the target organ damage (TOD) risk category, on the basis of systolic blood pressure (SBP). This prospective multi-center study included 137 healthy cats. Indirect blood pressure was measured using an oscillometric technique. The median SBP in all cats was 147 mmHg (interquartile range: 134-158). On the basis of the TOD risk category, 57.7, 19.7, 21.9, and 0.7% of the cats were classified into categories I-IV, respectively. Age, sex, and body weight did not affect the SBP. This study provides basic information on the distribution of TOD risk categories in clinically healthy cats.

The discovery and characterization of K-563, a novel inhibitor of the Keap1/Nrf2 pathway produced by Streptomyces sp.

Keap1/Nrf2 pathway regulates the antioxidant stress response, detoxification response, and energy metabolism. Previous reports found that aberrant Keap1/Nrf2 pathway activation due to Kelch-like ECH-associated protein 1 (Keap1) mutations or Nuclear factor E2-related factor 2 (Nrf2) mutations induced resistance of cancer cells to chemotherapy and accelerated cell growth via the supply of nutrients. Therefore, Keap1/Nrf2 pathway activation is associated with a poor prognosis in many cancers. These previous findings suggested that inhibition of Keap1/Nrf2 pathway could be a target for anti-cancer therapies. To discover a small-molecule Keap1/Nrf2 pathway inhibitor, we conducted high-throughput screening in Keap1 mutant human lung cancer A549 cells using a transcriptional reporter assay. Through this screening, we identified the novel Keap1/Nrf2 pathway inhibitor K-563, which was isolated from actinomycete Streptomyces sp. K-563 suppressed the expression of Keap1/Nrf2 pathway downstream target genes or the downstream target protein, which induced suppression of GSH production, and activated reactive oxygen species production in A549 cells. K-563 also inhibited the expression of downstream target genes in other Keap1- or Nrf2-mutated cancer cells. Furthermore, K-563 exerted anti-proliferative activities in these mutated cancer cells. These in vitro analyses showed that K-563 was able to inhibit cell growth in Keap1- or Nrf2-mutated cancer cells by Keap1/Nrf2 pathway inhibition. K-563 also exerted synergistic combinational effects with lung cancer chemotherapeutic agents. An in vivo study in mice xenotransplanted with A549 cells to further explore the therapeutic potential of K-563 revealed that it also inhibited Keap1/Nrf2 pathway in lung cancer tumors. K-563, a novel Keap1/Nrf2 pathway inhibitor, may be a lead compound for development as an anti-cancer agent.

Diagnostic utility of cardiac troponin I in cats with hypertrophic cardiomyopathy.

BACKGROUND: Cardiac troponin I (cTnI) is useful for assessing hypertrophic cardiomyopathy (HCM) in cats. OBJECTIVE: To measure plasma cTnI concentrations in healthy cats and evaluate the clinical utility of cTnI in determining the severity of HCM. ANIMALS: Clinically healthy cats (n = 88) and cats with HCM (n = 93). METHODS: Multicenter prospective study. Cats with HCM, including hypertrophic obstructive cardiomyopathy at various stages, were diagnosed using echocardiography. Plasma cTnI concentrations were analyzed by a commercial laboratory. Receiver-operating characteristic curve analysis was used to evaluate the accuracy of plasma cTnI concentrations to detect HCM. RESULTS: The median cTnI concentration was 0.027 ng/mL (interquartile range, 0.012-0.048 ng/mL) in healthy cats. Concentrations were significantly higher in diseased cats than in healthy controls, and concentrations were significantly higher in cats with heart failure than in asymptomatic cats. A plasma cTnI concentration of 0.163 ng/mL had a sensitivity of 62.0% and specificity of 100% when used to distinguish normal cats from asymptomatic HCM cats without left atrial dilatation. A cutoff of 0.234 ng/mL had high sensitivity (95.0%) and specificity (77.8%) for assessing heart failure. The areas under the receiver-operating characteristic curves were 0.85 and 0.93, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased cTnI concentrations reflect the severity of HCM. If other causes of cardiac injury are ruled out, plasma cTnI concentration may be useful for predicting the severity of HCM in cats.

Relationship between indirect blood pressure and various stages of chronic kidney disease in cats.

Chronic kidney disease (CKD) is a common cause of secondary systemic hypertension in cats. We investigated the relationship between indirect blood pressure and the prevalence of systemic hypertension in various CKD stages in cats. Client-owned cats (24 control cats and 77 cats with CKD) were included. Biochemical examinations of plasma were conducted by a commercial laboratory. Diseased cats were divided into two groups based on the International Renal Interest Society (IRIS) guidelines (II and III-IV). Indirect blood pressure was measured using an oscillometric technique. Severe hypertension was diagnosed if systolic blood pressure (SBP) was ≥180 mmHg. Indirect blood pressures were significantly higher in IRIS stage III-IV than in the control cats. Of 77 cats with CKD, 25 (32.5%) had severe hypertension. The frequency of severe hypertension increased with an increase in IRIS stage; 0% in the controls, 27.6% in the IRIS stage II, and 47.4% in the IRIS stage III-IV, respectively. The indirect SBP was weakly correlated with urea nitrogen (r=0.27) and creatinine (r=0.23) concentrations in plasma. Binary logistic regression analysis showed that if plasma creatinine concentration is >3.7 mg/dl, cats with CKD had an increased risk for developing severe hypertension (P<0.001). Our results suggest that indirect blood pressure was correlated with the severity of CKD, and the prevalence of severe hypertension increased in cats with severe CKD. The risk of severe hypertension may be high in cats with severe CKD.

Naphthacemycins, novel circumventors of ß-lactam resistance in MRSA, produced by Streptomyces sp. KB-3346-5. I. The taxonomy of the producing strain, and the fermentation, isolation and antibacterial activities.

Screening for circumventors of ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) led us to find 17 novel antibiotics, naphthacemycins A1-A11, B1-B4 and C1-C2. The naphthacemycins were isolated from a cultured broth of Streptomyces sp. KB-3346-5 by repeated silica gel column chromatography and HPLC. Naphthacemycins enhanced imipenem activity 100-500 times against MRSA at 0.5 µg ml-1, and naphthacemycins A4-A11 themselves showed MIC50 values of 1-4 µg ml-1 against 22 MRSA strains.

The synthesis and evaluation of the antiproliferative activity of deacidified GEX1A analogues.

GEX1A/herboxidiene (1) is a natural product isolated from Streptomyces sp. and has been reported to target the pre-mRNA splicing process. Although 1 was shown to have antitumor activity in vivo, weight loss was observed in mice when 1 was consecutively administered. We assumed that the carboxylic acid moiety was one of the causes of this toxicity. In this study, a series of amide, carbamate and urea analogues of 1 were synthesized and their antiproliferative activity was evaluated in vitro. The synthesis of urea analogues featured Curtius rearrangement following amine treatment with the one-pot procedure from 1. Furthermore, a structure-activity relationship study of the urea analogues revealed that the pharmacologically preferable basic side chains were acceptable and that compound 9g was equipotent to parent 1. These basic urea analogues would be promising leads for the development of novel antitumor agents.

Simplifungin and Valsafungins, Antifungal Antibiotics of Fungal Origin.

The targets of antifungal antibiotics in clinical use are more limited than those of antibacterial antibiotics. Therefore, new antifungal antibiotics with different mechanisms of action are desired. In the course of our screening for antifungal antibiotics of microbial origins, new antifungal antibiotics, simplifungin (1) and valsafungins A (2) and B (3), were isolated from cultures of the fungal strains Simplicillium minatense FKI-4981 and Valsaceae sp. FKH-53, respectively. The structures of 1 to 3 including their absolute stereochemistries were elucidated using various spectral analyses including NMR and collision-induced dissociation (CID)-MS/MS as well as chemical approaches including modifications to the Mosher's method. They were structurally related to myriocin. They inhibited the growth of yeast-like and zygomycetous fungi with MICs ranging between 0.125 and 8.0 µg/mL. An examination of their mechanisms of action by the newly established assay using LC-MS revealed that 1 and 2 inhibited serine palmitoyltransferase activity, which is involved in sphingolipid biosynthesis, with IC50 values of 224 and 24 nM, respectively.

Paraphaeosphaeride D and berkleasmin F, new circumventors of arbekacin resistance in MRSA, produced by Paraphaeosphaeria sp. TR-022.

Two new compounds, designated paraphaeosphaeride D (1) and berkleasmin F (2) together with a previously known compound, berkleasmin A (3), isolated from a culture broth of the fungus Paraphaeosphaeria sp. TR-022, proved to be new circumventors of arbekacin (ABK) resistance in methicillin-resistant Staphylococcus aureus (MRSA). The structures of 1 and 2 were elucidated by spectroscopic analyses, including various NMR experiments. All compounds showed 10-100 times ABK circumvention activities using the paper disc method and reduced the MIC values of ABK against MRSA from 16 µg ml(-1) to 4 µg ml(-1) (fourfold) using the agar dilution method. These new compounds might be promising lead compounds for developing circumventors of ABK resistance in MRSA.

Habiterpenol, a novel abrogator of bleomycin-induced G2 arrest in Jurkat cells, produced by Phytohabitans suffuscus 3787_5.

A small molecule named habiterpenol produced by actinomycete Phytohabitans suffuscus 3787_5 was found to abrogate bleomycin-induced G2 arrest in Jurkat cells. Habiterpenol showed no cytotoxic effect on Jurkat cells even at 273 µM; however, the compound inhibited bleomycin-induced G2 arrest in Jurkat cells with an IC50 value of 3.55 µM, while it showed no effect on colchicine-induced M arrest even at 273 µM. These results indicated that habiterpenol selectively abrogated bleomycin-induced G2 arrest in Jurkat cells.

A molecular screening approach to identify and characterize inhibitors of glioblastoma stem cells.

Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types.

Conformational significance of EH21A1-A4, phenolic derivatives of geldanamycin, for Hsp90 inhibitory activity.

Hsp90 is an attractive chemotherapeutic target because it is essential to maturation of multiple oncogenes. We describe the conformational significance of EH21A1-A4, phenolic derivatives of geldanamycin isolated from Streptomyces sp. Their native free structures are similar to the active form of geldanamycin bound to Hsp90 protein. Their conformational character is a probable reason for their high-affinity binding. Lack of toxic benzoquinone in EH21A1-A4 also adds to their potential as lead compounds for anti-tumor drugs.

MPC1001 and its analogues: new antitumor agents from the fungus Cladorrhinum species.

[structure: see text] Eight new compounds, MPC1001 and MPC1001B-H, were isolated from the fungus Cladorrhinum sp. KY4922. Multiple NMR experiments and CD data revealed MPC1001 to be an O-methyl derivative of emestrin, a 15-membered antifungal antibiotic containing a unique epidithiodioxopiperazine skeleton. Other compounds were elucidated to be structurally related novel analogues. MPC1001 and the analogues exerted potent antiproliferative activities against a human tumor cell line.

THE FRESHWATER CYANOBACTERIUM PLANKTOTHRIX SP. FP1: MOLECULAR IDENTIFICATION AND DETECTION OF PARALYTIC SHELLFISH POISONING TOXINS.

A filamentous cyanobacterium, belonging to the Order of Oscillatoriales, was found to be responsible for a toxic algal bloom in Lake Varese, Italy, during the summer of 1997. Morphological characters, as well as near complete 16S rRNA gene sequencing, revealed that the dominant species of the bloom was most closely related to the genus Planktothrix. In addition, genetic analysis of the phycocyanin operon of Planktothrix sp. FP1 revealed a novel primary structure, previously undescribed within the cyanobacteria, which was used as a genetic marker for rapid detection and identification of this toxic strain. The occurrence of saxitoxin (STX), a principal toxin in paralytic shellfish poisoning (PSP), was confirmed in the natural bloom sample by both pre-column and post-column derivatization high-performance liquid chromatography (HPLC) analyses, and eventually by liquid chromatography/mass spectrometry (LC/MS). The toxicity of this field sample was also revealed by electrophysiological assays in which the extract inhibited 90% of the voltage-dependent Na+ current in human neuroblastoma cells at the STX concentration of 80 nM. The cultured strain showed a lower physiologic activity than the bloom sample (67% blockage of Na+ current at a toxin concentration of 200 nM), and STX was detected only by pre-column HPLC, indicating the presence of a compound structurally close to STX. Chemical and molecular genetic analyses performed here add Planktothrix sp. FP1 to the growing list of diverse cyanobacterial species capable of synthesizing STX and its related compounds.