The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 μM) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells.     

Identification of nitrogen-fixing Paenibacillus from different plant rhizospheres and a novel nifH gene detected in the P. stellifer

A total of 534 isolates were selectively obtained from different plant rhizospheres based on their growth on nitrogen-free medium and their resistance to 80 degrees C for 15 min. Of the 534 isolates, 23 isolates had nifH gene and exhibited nitrogenase activities. Based on 16S rDNA sequence, G + C content assay and DNA-DNA hybridization, by the 23 isolates, which were divided into four monophyletic clusters, all belonged to the Paenibacillus genus. NifH gene deduced amino acid alignment analysis revealed that cluster I, including 15 isolates, showed the highest NifH identity with Paenibacillus genus; while cluster II identified as P stellifer by DNA-DNA hybridization was consistent with four uncultured bacterial clones. This study suggested that the nitrogen-fixing Paenibacillus were distributed in various ecosystems and prevalent in different plant rhizospheres. It was the first demonstration that nitrogen fixation existed in P. jamilae and P. stellifer. In eight isolates identified as P. stellfer species, a novel nifH gene was detected in Paenibacillus.     

The bactericidal activity of lectins from nitrogen-fixing bacilli

Lectins I and II isolated from the nitrogen-fixing soil bacterium Paenibacillus polymyxa 1460 were found to be able to suppress the growth of Rhizobium leguminosarum 252 and Bacillus subtilis 36 at nearly all the concentrations tested (from 1 to 10 micrograms/ml). Lectin I was also inhibitory to Azospirillum brasilense 245 and Erwinia carotovora subsp. citrulis 603, while lectin II exerted bactericidal activity against Xanthomonas campestris B-610 and B-611 and A. brasilense 245. The bacillar lectins incubated with Rhizobium and Azospirillum cells caused leakage of low-molecular-weight substances from the cells, presumably resulting from impairment of the membrane barrier function. We believe that one of the possible mechanisms of the bacterial growth inhibition by lectins is mediated by the lectin-specific receptors occurring on the bacterial membrane, whose interaction with the lectin molecules induces conformational alterations in the membrane and concurrent malfunction of the metabolism of bacterial cells.     

Are Alzheimer neurofibrillary tangles insoluble polymers?

The two methods currently available for the bulk isolation of Alzheimer tangles of paired helical filaments (PHF) are based on a brief treatment of a neuronal-enriched preparation with sodium dodecyl sulfate (SDS) (Method I) and on heating of whole brain homogenate with SDS and beta-mercaptoethanol (Method II). PHF were isolated from the same Alzheimer brain by these two methods, subjected to SDS-polyacrylamide gel electrophoresis and immuno-labelled with monoclonal antibodies to PHF after transferring from the gel to nitrocellulose paper. The PHF isolated by method I revealed the presence of 45 kilodalton to 62 kilodalton PHF polypeptides, whereas the PHF isolated by method II were excluded from the gel. However, PHF isolated by both methods were digested with proteinase-K, though the degradation of PHF of method I was considerably more rapid than that of PHF isolated by method II. These findings should establish that the solubility of PHF might depend on the methods employed for their isolation and that they might not be insoluble polymers of covalently crosslinked polypeptides which accumulate irreversibly in the brain of patients with Alzheimer disease.     

Development of multiplex PCR for fast detection of Paenibacillus Larvae in putrid masses and in isolated bacterial colonies

The present study was performed to develop a fast and sensitive multiplex polymerase chain reaction protocol for routine diagnostics of American foulbrood. A new approach for detection of Paenibacillus larvae in putrid masses was described. Forty five samples of putrid masses obtained from bee combs suspicious for American foulbrood, a reference strain Paenibacillus larvae (NBIMCC 8478), clinical isolates and 4 strains of closely related bacterial species were included in experiments. Bacterial colonies?? DNA was isolated by heat and centrifugation method (standard procedure) and with prepGem commercial kit. DNA from putrid masses was isolated by standard and modified procedure. Three pairs of primers specific for 16S rRNA and one pair specific for 35 kDa metalloproteinase genes of Paenibacillus larvae were tested as single pair and in different combinations as multiplex PCR. The sensitivity of the multiplex PCR protocol for putrid masses, developed in study was 100%, versus 45.2% for the standard protocol. The developed multiplex PCR protocol could be successfully used for rapid and specific detection of Paenibacillus larvae in both putrid masses and isolated bacterial colonies.     

Screening and evaluation of probiotics as a biocontrol agent against pathogenic Vibrios in marine aquaculture

AIMS: The present work aims at finding potential probionts from marine sources as a biocontrol agent against pathogenic Vibrio species in shrimp larval culture. METHODS AND RESULTS: A total of 109 bacterial strains were isolated from seawater, sediment and marine fish-gut samples, and were screened for their antagonistic activity against Vibrio species. Three strains (Q, Q1 and M) isolated from the marine sediment were found antagonistic against Vibrio strains. Based on 16S ribosomal DNA gene sequence analysis, the strain Q was identified as Paenibacillus spp. (EF012164); Q1 as Bacillus cereus (DQ915582); and the M as Paenibacillus polymyxa (DQ915580). Further, the two bacterial species, Paenibacillus spp. and B. cereus were challenged separately at two different concentrations of 10(4) and 10(5) CFU ml(-1) for probiotic activity in the postlarvae of Penaeus monodon against pathogenic Vibrio harveyi and Vibrio spp. CONCLUSIONS: The present study identified the probiotic activity of Paenibacillus spp., B. cereus and Pa. polymyxa against the pathogenic Vibrios in the postlarvae of P. monodon. SIGNIFICANCE AND IMPACT OF THE STUDY: In vivo study reveals that the marine bacterial species can be used as probionts against pathogenic Vibrios in shrimp larval culture practices.     

Purification and characterization of human pancreatic colipase.

Two colipases, named colipase I and colipase II, have been isolated from extracts of human pancreatic gland. The two proteins can be separated by ion-exchange chromatography, isoelectric focusing and slab technique gel electrophoresis. The result of this study indicates that the two colipases, both of which are glycoproteins, have identical amino acid compositions. The pI values were found to be 6.1 for colipase I and 5.8 for colipase II. The different colipases have also been found in human pancreatic juice. The N-terminal amino acid was glycine for both colipase I (gland) and colipase II (juice). Only minor differences were found between the colipases isolated from gland and juice, and colipase I from gland alone was examined in detail.     

Real-time PCR detection of Paenibacillus spp. in raw milk to predict shelf life performance of pasteurized fluid milk products

Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (C(T)) of 19.14 ± 0.54. While 14/17 Bacillus isolates showed no signal (C(T) > 40), 3 Bacillus isolates showed very weak positive signals (C(T) = 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 10(1) CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.     

Phylogeny of 16S rRNA and nifH genes and regulation of nitrogenase activity by oxygen and ammonium in the genus Paenibacillus

All Paenibacillus 16S rDNA sequences, except for that of Paenibacillus massiliensis T7, formed a coherent cluster, distinct from gram-positive nitrogen-fixing Clostridium pasteurianum and Heliobacterium chlorum. All Paenibacillus NifH sequences formed two main clusters. Cluster I encompassing the NifH sequences from most of members of Paenibacillus spp., such as Paenibacillus azotofixans NifH1 and NifH2, Paenibacillus polymyxa and Paenibacillus macerans. Cluster II including only P. azotofixans NifH3. Curiously, three copies of nifH genes of Paenibacillus sabine T27 clustered within P. azotofixans cluster I (NifH1 and NifH2). The effect of O₂ and ammonium on nitrogenase activity was studied with 14 different nitrogenfixing Paenibacillus strains. The optimal oxygen concentration level for all Paenibacillus strains is in the 0 to 0.05% range, similar to that for Klebsiella pneumoniae. In all Paenibacillus strains, the highest nitrogenase activity is obtained in the condition of 0?C0.1 mM NH₄Cl and the increase of NH₄Cl from 0.1 to 5 mM caused a rapid inhibition of nitrogenase activity. However, the inhibition was reversible in the presence of 200 mM NH₄Cl in some Paenibacillus strains. It is the first time to use almost all of the recognized nitrogen-fixing Paenibacilus spp. to investigate the phylogeny of 16S rRNA and nifH genes. The data that the inhibition of O₂ and ammonium on nitrogenase acitivity will provide a base for studying the molecular regulatory mechanism of nitrogen fixation in the genus Paenibacillus.     

Biochemical comparative studies between eye- and brain-derived growth factors

Two bovine brain-derived growth factors, BDGF I and BDGF II, were isolated using the same extraction procedure as previously described for eye-derived growth factors (EDGF). The hypothesis that these growth factors were identical to EDGF I and EDGF II, respectively, was supported by their similar molecular weights (16,000 and 15,000, respectively) and isoelectric points (9.0 and 5.0, respectively), their identical retention behavior on reverse-phase chromatography and their similar amino acid compositions. From studies on their binding properties to cell surfaces, competition between EDGF I and BDGF I as well as competition between EDGF II and BDGF II to the same receptor was observed. The amino terminal sequence of EDGF II (1-16) was shown to be identical to the amino acid residues (7-22) of the acidic FGF, strongly confirming our observations on the identity of the factors isolated from bovine brain and retina.     

Polyisoprenylated benzophenones in cuban propolis; biological activity of nemorosone

The Copey tree (Clusia rosea) has a large distribution in Cuba and its floral resin is a rich source of polyisoprenylated benzophenones. To determine the presence of these natural products, we carried out a study by HPLC of 21 propolis samples produced by honey bees (Apis mellifera) from different provinces of Cuba. Nemorosone resulted to be the most abundant polyisoprenylated benzophenone and the mixture of xanthochymol and guttiferone E was also observed, but in minor proportion. We studied the biological activity of the pure natural product nemorosone and its methyl derivatives. We found that nemorosone has cytotoxic activity against epitheloid carcinoma (HeLa), epidermoid carcinoma (Hep-2), prostate cancer (PC-3) and central nervous system cancer (U251). It also exhibited antioxidant capacity. Methylated nemorosone exhibited less biological activity than the natural product.     

Purification of annexin I and annexin II from human placental membranes by high-performance liquid chromatography.

Annexin I and annexin II were extracted from human placental membranes with ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) and purified by high-performance liquid chromatography by measuring their ability to inhibit phospholipase A2 activity in vitro. Neither protein was capable of binding to a DEAE-5PW HPLC column at neutral pH; however, they were resolved through binding to a Mono S column and passage through size-exclusion HPLC columns. Annexin I and its covalently linked dimer (36 and 66 kDa, respectively, by sodium dodecyl sulfate (SDS)-gel electrophoresis) reacted in one-dimensional immunoblots with monoclonal antibodies to annexin I and calpactin II, and with monoclonal and polyclonal antibodies to lipocortin I, confirming that annexin I, calpactin II, and lipocortin I are the same or closely related proteins. Milligram amounts of monomeric annexin I containing negligible amounts of the cross-linked dimeric annexin I were selectively isolated from placental membranes by using buffers containing the sulfhydryl reagent iodoacetic acid. Milligram amounts of cross-linked annexin I were selectively isolated when placental membranes were initially treated with buffers that did not contain iodoacetic acid and then extracted with Triton X-100, suggesting that sulfhydryl-dependent transglutaminase activity contributes to the selective isolation of this protein. A third phospholipase A2-inhibitory protein (35 kDa by SDS-gel electrophoresis) that reacted in immunoblots with monoclonal antibodies to calpactin I and annexin II, indicating their similar identity, was isolated. The procedure employed allows the rapid purification of annexins I and II in milligram amounts from placental membranes within 2 days.     

Comparison of the cytotoxic effects of enantiopure PPAPs, including nemorosone and clusianone

The synthesis of an unnatural polyprenylated acylphloroglucinol (PPAP), regioisomeric with nemorosone and clusianone, has been accomplished. The separated enantiomers of this new PPAP, along with those of nemorosone and clusianone, have been screened for activity against HeLa (cervix carcinoma), MIA-PaCa-2 (pancreatic carcinoma), and MCF7 (mamma carcinoma) cancer cell lines. All of the isomers examined gave surprisingly similar results in the screens.     

Genome sequence of Paenibacillus sp. strain Aloe-11, an endophytic bacterium with broad antimicrobial activity and intestinal colonization ability

Paenibacillus sp. strain Aloe-11, a Gram-positive, spore-forming, facultatively anaerobic bacterium isolated from the root of Aloe chinensis in the southwest region of China, has excellent antibiotic activity and intestine colonization ability. Here, we present the 5.8-Mb draft genome sequence of Paenibacillus sp. strain Aloe-11.     

Salt induced responses of chloroplast activities in species of differing salt tolerance. Photosynthetic electron transport in Aster tripolium and Pisum sativum

A comparative study was made of the effects of high concentrations of NaCl, KCl and MgCl₂ on two electron transport reactions of thylakoids isolated from a mesophyte, Pisum sativum and a halophyte, Aster tripolium . The rate of photosystem I mediated electron transport from reduced N, N, N', N'-tetramethyl- p -phenylenediamine (TMPD) to methyl viologen was determined polarographically, and photosystem II mediated electron flow from water to 2,6-dichlorophenolindophenol (DCPIP) was monitored spectrophotometrically. The response of photosystem II to increasing in vitro salt concentrations was similar for thylakoids isolated from both A. tripolium and P. sativum , but differences in the response of photosystem I to salinity changes were observed for the two species. Increasing NaCl, KCl and MgCl₂ concentrations produced similar patterns of response of photosystem I activity in P. sativum thylakoids, whilst for A. tripolium KCl induced a completely different response pattern compared to NaCl and MgCl₂. The salinity of the culture medium in which A. tripolium was grown also had an effect on both the absolute in vitro activities of photosystems I and II and their response to changes in salt concentration of the reaction media.     

Functional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts

Phycobiliproteins obtained by dissociation of phycobilisomes were reassociated in vitro with intact thylakoids or isolated photosystems I and II preparations obtained from cyanophytes (prokaryotes) or green algae (eukaryotes) to form bound phycobilisome complexes. Energy transfer from Fremyella diplosiphon phycobiliproteins to chlorophyll a of reaction centers I and II was measured in: complexes containing intact thylakoids of the cyanophytes F. diplosiphon or Anacystis nidulans and the eukaryotic algae Euglena gracilis and mutants of Chlamydomonas reinhardtii; complexes containing isolated photosystem II particles of A. nidulans or C. reinhardtii; and complexes containing reaction center I of F. diplosiphon or C. reinhardtii. Energy transfer from phycoerythrin to chlorophyll a of photosystem II could be demonstrated in complexes containing phycobilisomes bound to cyanophyte thylakoids or isolated photosystem II particles of A. nidulans or C. reinhardtii. Bound phycobilisomes did not transfer energy to photosystem II within green algae thylakoids containing altered forms of light-harvesting chlorophyll a/b-protein complex (LHC) II antenna, reduced amounts of LHC II, or chlorophyll b, or chlorophyll b-less mutants, nor to chlorophyll a of photosystem I of intact thylakoids or isolated reaction centers. We conclude that phycobilisomes can form a specific and functional association with photosystem II particles of both cyanophytes and eukaryotic thylakoids. This interaction appears to be hindered by the presence of LHC II antenna in the eukaryotic thylakoids.     

Short communication: Construction of a cloning vector for Paenibacillus polymyxa and P. azotofixans

A cloning vector that could replicate in Paenibacillus polymyxa, P. azotofixans and Bacillus subtilis was constructed using two Staphylococcus aureus plasmids. The recombinant plasmid confers chloramphenicol and erythromycin resistance and contains unique restriction sites for PvuII and BclI. The stability of pRJ45 was analysed.     

The Bactericidal Activity of Lectins from Nitrogen-Fixing Bacilli

Lectins I and II isolated from the nitrogen-fixing soil bacterium Paenibacillus polymyxa 1460 were found to be able to suppress the growth of Rhizobium leguminosarum 252 and Bacillus subtilis 36 at nearly all the concentrations tested (from 1 to 10 g/ml). Lectin I was also inhibitory to Azospirillum brasilense 245 and Erwinia carotovora subsp. citrulis 603, while lectin II exerted bactericidal activity against Xanthomonas campestris B-610 and B-611 and A. brasilense 245. The bacillar lectins incubated with Rhizobiumand Azospirillum cells caused leakage of low-molecular-weight substances from the cells, presumably resulting from impairment of the membrane barrier function. We believe that one of the possible mechanisms of the bacterial growth inhibition by lectins is mediated by the lectin-specific receptors occurring on the bacterial membrane, whose interaction with the lectin molecules induces conformational alterations in the membrane and concurrent malfunction of the metabolism of bacterial cells.     

Immunological Studies on Dermatophytes IV. Chemical Structures and Serological Reactivities of Polysaccharides from Microsporum praecox, Trichophyton ferrugineum, Trichophyton sabouraudii, and Trichophyton tonsurans

The chemical structures and serological specificities of polysaccharides isolated from four species of dermatophytes, Microsporum praecox, Trichophyton ferrugineum, T. sabouraudii, and T. tonsurans, were investigated. Each of these species yielded a mixture of crude polysaccharides which could be separated into three water-soluble, neutral polysaccharides free of nitrogen. These were grouped as galactomannan I, galactomannan II, and glucan. The galactomannans I were quite similar in chemical structure. When measured by complement fixation, their serological cross-reactivities were similar with rabbit antisera to each of these species except T. sabouraudii. The differences in their relative reactivities with this antiserum could be correlated with differences in structure and specificity of this antiserum for galactofuranose end groups. The galactomannans II differed both in chemical structure and in their serological reactivities with antisera to each of these species. The galactomannan II from T. ferrugineum differed most in chemical structure and was the least reactive serologically. The glucans also differed in both structure and serological reactivities.