Pharmacologically targeting molecular motor promotes mitochondrial fission for anti-cancer

Mitochondrial shape rapidly changes by dynamic balance of fusion and fission to adjust to constantly changing energy demands of cancer cells. Mitochondrial dynamics balance is exactly regulated by molecular motor consisted of myosin and actin cytoskeleton proteins. Thus, targeting myosin-actin molecular motor is considered as a promising strategy for anti-cancer. In this study, we performed a proof-of-concept study with a natural-derived small-molecule J13 to test the feasibility of anti-cancer therapeutics

SMYD3-PARP16 axis accelerates unfolded protein response and mediates neointima formation

Neointimal hyperplasia after vascular injury is a representative complication of restenosis. Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) is involved in the pathogenesis of vascular intimal hyperplasia. PARP16, a member of the poly(ADP-ribose) polymerases family, is correlated with the nuclear envelope and the ER. Here, we found that PERK and IRE1

First karyotype report on Colocasia oresbia: a comparative cytogenetic study between two varieties / Primer reporte del cariotipo de Colocasia oresbia: un estudio citogenético comparativo entre dos variedades

ABSTRACT Karyotypes of two Colocasia oresbia botanical varieties from Bangladesh were analyzed and compared with orcein, chromomycin A3 (CMA) and 4´-6 diamidino-2-phenylindole (DAPI). Both varieties had 2n=2x=26 chromosomes (karyotypic formula: 20m+6sm) and a pair of satellites each. Total chromosome length was 144.18 ± 2.45 μm in C. oresbia var. oresbia and 133.02 ± 2.75 μm in C. oresbia var. stolonifera. The karyotype of Colocasia oresbia var. oresbia is 2A whereas that of C. oresbia var. stolonifera is 1A. Six CMA and four DAPI bands were observed in C. oresbia var. oresbia and eight CMA and six DAPI bands in C. oresbia var. stolonifera. However, in these two morphologically distinct C. oresbia varieties of two different ecological zones, the same somatic chromosome number, diversification in various karyotypic parameters and CMA/DAPI-banding patterns were observed. In addition to taxonomic characters, the studied karyotype features will contribute to the characterization of these two C. oresbia varieties and to establish a base for future research.

RESUMEN Se analizaron y compararon los cariotipos de dos variedades botánicas de Colocasia oresbia de Bangladesh con orceína, chromomicina A3 (CMA) y 4-6 diamidino-2-phenilindol (DAPI). Ambas variedades presentaron 2n=2x=26 cromosomas (fórmula cariotípica: 20m+6sm) y un par de satélites cada una. La longitud total de cromosomas fue 144,18 ± 2,45 μm en C. oresbia var. oresbia y 133.02 ± 2.75 μm en C. oresbia var. stolonifera. El cariotipo de Colocasia oresbia var. oresbia es 2ª, y 1ª el de C. oresbia var. stolonifera. Se observaron seis bandas CMA y cuatro DAPI en C. oresbia var. oresbia y ocho bandas CMA y seis DAPI en C. oresbia var. stolonifera. Sin embargo, en estas dos variedades morfológicamente distintivas de C. oresbia de dos zonas ecológicas diferentes se observó el mismo número cromosómico somático, diversificación en varios parámetros cariotípicos y en patrones de bandeo CMA/DAPI. En adición a los caracteres taxonómicos, las características de los cariotipos estudiados contribuirán a la caracterización de estas dos variedades de C. oresbia y a establecer una base para futuras investigaciones.

3--Acetyl-11-keto- -boswellic acid ameliorated aberrant metabolic landscape and inhibited autophagy in glioblastoma

Glioblastoma is the most common and aggressive primary tumor in the central nervous system, accounting for 12%-15% of all brain tumors. 3--Acetyl-11-keto--boswellic acid (AKBA), one of the most active ingredients of gum resin from Birdw., was reported to inhibit the growth of glioblastoma cells and subcutaneous glioblastoma. However, whether AKBA has antitumor effects on orthotopic glioblastoma and the underlying mechanisms are still unclear. An orthotopic mouse model was used to evaluate the anti-glioblastoma effects of AKBA. The effects of AKBA on tumor growth were evaluated using MRI. The effects on the alteration of metabolic landscape were detected by MALDI-MSI. The underlying mechanisms of autophagy reducing by AKBA treatment were determined by immunoblotting and immunofluorescence, respectively. Transmission electron microscope was used to check morphology of cells treated by AKBA. Our results showed that AKBA (100 mg/kg) significantly inhibited the growth of orthotopic U87-MG gliomas. Results from MALDI-MSI showed that AKBA improved the metabolic profile of mice with glioblastoma, while immunoblot assays revealed that AKBA suppressed the expression of ATG5, p62, LC3B, p-ERK/ERK, and P53, and increased the ratio of p-mTOR/mTOR. Taken together, these results suggested that the antitumor effects of AKBA were related to the normalization of aberrant metabolism in the glioblastoma and the inhibition of autophagy. AKBA could be a promising chemotherapy drug for glioblastoma.

Neutrophil-mimicking therapeutic nanoparticles for targeted chemotherapy of pancreatic carcinoma

Due to the critical correlation between inflammation and carcinogenesis, a therapeutic candidate with anti-inflammatory activity may find application in cancer therapy. Here, we report the therapeutic efficacy of celastrol as a promising candidate compound for treatment of pancreatic carcinoma naïve neutrophil membrane-coated poly(ethylene glycol) methyl ether--poly(lactic--glycolic acid) (PEG-PLGA) nanoparticles. Neutrophil membrane-coated nanoparticles (NNPs) are well demonstrated to overcome the blood pancreas barrier to achieve pancreas-specific drug delivery . Using tumor-bearing mice xenograft model, NNPs showed selective accumulations at the tumor site following systemic administration as compared to nanoparticles without neutrophil membrane coating. In both orthotopic and ectopic tumor models, celastrol-loaded NNPs demonstrated greatly enhanced tumor inhibition which significantly prolonged the survival of tumor bearing mice and minimizing liver metastases. Overall, these results suggest that celastrol-loaded NNPs represent a viable and effective treatment option for pancreatic carcinoma.

A fluorometric method for direct detection of inorganic polyphosphate in enterohemorrhagic O157:H7 / 南方医科大学学报

OBJECTIVE@#To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7.@*METHODS@#The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃, and freezing at -20 ℃, all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two mutant strains.@*RESULTS@#At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was =1849+127.5 (R=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with deletion.@*CONCLUSIONS@#We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.

Screening of antiproliferative activity mediated through apoptosis pathway in human non-small lung cancer A-549 cells by active compounds present in medicinal plants

Objective:To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques.Methods:We investigated the antiproliferative effects of methanolic extracts from different parts of seven plants on A-549 (lung cancer) cells and primary cell culture (chick embryo fibroblast cells, as normal cells) using MTT assay and the potent plant was fractioned further. All these fractions were screened again for anti-proliferative activity. DNA fragmentation and DAPI staining were used to study apoptosis. Quantitative real-time was used to investigate the expression of apoptotic-related genes. LC-MS andResults:Methanolic extract of Vitex negundo (V. negundo) was selected as a potent fraction. Among all fractions screened, ethylacetate fraction of V. negundo was selected as the most potent antiproliferative fraction and phytochemical analysis of the extract revealed the presence of secondary metabolites. Ethaylacetate fraction of V. negundo was found to cause characteristic apoptotic morphological changes and generation of ROS in A-549 cells. Ethaylacetate fraction of V. negundo also induced apoptosis in A-549 which was supported by DNA fragmentation and DAPI staining. To investigate the molecular mechanism behind the cytotoxic effect of ethaylacetate fraction of V. negundo, quantitative real-time PCR was used to measure expression levels of p53, bax, bcl2, casp-3 and casp-9. Using LC-MS andConclusions:It is concluded from the present study that V. negundo is capable of triggering growth-inhibitive and apoptosis effects in A-549 cells, signifying that V. negundo may possesses anti-lung cancer activity.

Screening of antiproliferative activity mediated through apoptosis pathway in human non-small lung cancer A-549 cells by active compounds present in medicinal plants

Objective: To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques. Methods: We investigated the antiproliferative effects of methanolic extracts from different parts of seven plants on A-549 (lung cancer) cells and primary cell culture (chick embryo fibroblast cells, as normal cells) using MTT assay and the potent plant was fractioned further. All these fractions were screened again for anti-proliferative activity. DNA fragmentation and DAPI staining were used to study apoptosis. Quantitative real-time was used to investigate the expression of apoptotic-related genes. LC-MS and

Inhalable oridonin-loaded poly(lactic--glycolic)acid large porous microparticles fortreatment of primary non-small cell lung cancer

Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. Traditional chemotherapy for this disease leads to serious side effects. Here we prepared an inhalable oridonin-loaded poly(lactic--glycolic)acid (PLGA) large porous microparticle (LPMP) fortreatment of NSCLC with the emulsion/solvent evaporation/freeze-drying method. The LPMPs were smooth spheres with many internal pores. Despite a geometric diameter of ~10 µm, the aerodynamic diameter of the spheres was only 2.72 µm, leading to highly efficient lung deposition.studies showed that most of oridonin was released after 1 h, whereas the alveolar macrophage uptake of LPMPs occurred after 8 h, so that most of oridonin would enter the surroundings without undergoing phagocytosis. Rat primary NSCLC models were built and administered with saline, oridonin powder, gemcitabine, and oridonin-loaded LPMPsairway, respectively. The LPMPs showed strong anticancer effects. Oridonin showed strong angiogenesis inhibition and apoptosis. Relevant mechanisms are thought to include oridonin-induced mitochondrial dysfunction accompanied by low mitochondrial membrane potentials, downregulation of BCL-2 expressions, upregulation of expressions of BAX, caspase-3 and caspase-9. The oridonin-loaded PLGA LPMPs showed high anti-NSCLC effects after pulmonary delivery. In conclusion, LPMPs are promising dry powder inhalations fortreatment of lung cancer.

Heterochromatin base pair composition and diversification in holocentric chromosomes of kissing bugs (Hemiptera, Reduviidae)

The subfamily Triatominae (Hemiptera, Reduviidae) includes 150 species of blood-sucking insects, vectors of Chagas disease or American trypanosomiasis. Karyotypic information reveals a striking stability in the number of autosomes. However, this group shows substantial variability in genome size, the amount and distribution of C-heterochromatin, and the chromosome positions of 45S rDNA clusters. Here, we analysed the karyotypes of 41 species from six different genera with C-fluorescence banding in order to evaluate the base-pair richness of heterochromatic regions. Our results show a high heterogeneity in the fluorescent staining of the heterochromatin in both autosomes and sex chromosomes, never reported before within an insect subfamily with holocentric chromosomes. This technique allows a clear discrimination of the heterochromatic regions classified as similar by C-banding, constituting a new chromosome marker with taxonomic and evolutionary significance. The diverse fluorescent patterns are likely due to the amplification of different repeated sequences, reflecting an unusual dynamic rearrangement in the genomes of this subfamily. Further, we discuss the evolution of these repeated sequences in both autosomes and sex chromosomes in species of Triatominae.

Biofilm formation by Rhodococcus equi and putative association with macrolide resistance / Formação de biofilme por Rhodococcus equi e possível associação com resistência a macrolídeos

Rhodococcus equi is a facultative intracellular pathogen, which cause severe pyogranulomatous pneumonia in foals and tuberculosis-like lesions in humans. Its ability to form biofilm was described in strains isolated from chronic diseases associated to treatment failures in humans. This study aimed to verify the biofilm formation by 113 R. equi isolated from equine samples (clinical and fecal) using two different methods (biofilm-culturing with and without additional glucose and epifluorescence microscopy). We also aimed to determine the efficacy of azithromycin, clarithromycin and erythromycin on R. equi in established biofilm. We found 80.5% (26/41) and 63% (58/72) biofilm-positive isolates, in fecal and clinical samples, respectively. The additional glucose increased the biofilm formation by R. equi fecal samples, but not by clinical samples. The antimicrobials tested herein were not able to eradicate R. equi in biofilm even at higher concentrations. This is the first study showing the biofilm formation by R. equi isolated from equine samples. Our findings indicate that R. equi biofilm-producers may be more resistant to the antimicrobials evaluated. Further studies are warranted to test this hypothesis...

Rhodococcus equi é um patógeno intracelular facultativo, o qual causa pneumonia piogranulosa severa em potros e lesões semelhantes à tuberculose em humanos. A sua capacidade de formar biofilme foi descrita em cepas humanas, isoladas a partir de doenças crônicas associadas a falhas de tratamento. Este estudo teve como objetivo verificar a formação de biofilme por 113 cepas de R. equi, isoladas a partir de amostras de equinos (clínicas e fecais), utilizando-se dois diferentes métodos (biofilme em cultura - com e sem adição de glicose - e microscopia de epifluorescência). Além disso, buscou-se determinar a eficácia da azitromicina, claritromicina e eritromicina sobre biofilme consolidado de R. equi. Verificou-se 80,5% (26/41) e 63% dos isolados (58/72) positivos para formação de biofilme, em amostras fecais e clínicas, respectivamente. A adição de glicose amentou a formação de biofilme em amostras fecais, mas não em amostras clínicas. Os antimicrobianos aqui testados não foram capazes de erradicar R. equi em biofilme consolidado, mesmo em concentrações elevadas. Este é o primeiro estudo a demonstrar a formação de biofilme por cepas de R. equi isoladas a partir de amostras de equinos. Os resultados indicam que os isolados de R. equi produtores de biofilme podem ser mais resistentes aos antimicrobianos avaliados. Estudos adicionais são necessários para testar essa hipótese...

Anticancer Activity of Oscillatoria Terebriformis Cyanobacteria in Human Lung Cancer Cell Line A549.

Purpose: To evaluate the anti-cancer properties of the cyanobacterial extract of Oscillatoria terebriformis Methods: The extract was tested in Human lung cancer cell lines and examined for its effect on cell viability, nuclear morphology and sub-G1 formation. Cell viability was determined by micro culture tetrazolium technique (MTT), nuclear morphology investigated using 4’-6-diamidino-2-phenylindole (DAPI) staining technique, and apoptosis assay using DNA fragmentation. Results: The results showed decreasing cell viability in a concentration-dependent manner. Altered cell morphology after treatment with the extract demonstrated that cells experienced apoptosis. Conclusion: The data demonstrate that Oscillatoria Terebriformis extract induced apoptosis in Human lung cancer A549 cells, and therefore, has a potential as an anti-cancer agent.

Comparative cytogenetics in astyanax (characiformes: characidae) with focus on the cytotaxonomy of the group

Astyanax is a diverse group of Neotropical fishes, whose different forms occupy different environments. This great diversity is also reflected on cytogenetic aspects and molecular markers, which have repeatedly been demonstrated by cytogenetic studies. In order to characterize the karyotype of species of this genus, six species were studied: Astyanax altiparanae, A.argyrimarginatus, A. elachylepis, A. xavante, and two new species provisionally called Astyanax sp. and A. aff. bimaculatus. A detailed cytogenetic study based on conventional staining with Giemsa, AgNORs, C-banding, base-specific fluorochromes, and FISH using ribosomal genes 18S and 5S was conducted, aiming to understand some of the chromosomal mechanisms associated with the high diversification that characterizes this group and culminated with the establishment of these species. The results showed 2n = 50 chromosomes for five species and a karyotype with 52 chromosomes in Astyanax sp. Small variations in the macrostructure of the karyotypes were identified, which were quite relevant when analyzed by classical banding, fluorochromes, and FISH methods. These differences among Astyanax spp. (2n = 50) are largely due to changes in the amount and types of heterochromatic blocks. Astyanax sp (2n = 52), in addition to variations due to heterochromatic blocks, has its origin possibly by events of centric fission in a pair of chromosomes followed by minor rearrangements.These results show an interesting karyotypic diversity in Astyanax and indicate the need of a review of the group referred as A. aff. bimaculatus and the description of Astyanax sp., including the possibility of inclusion of this unit in another genus.

Astyanax é um grupo bastante diverso de peixes neotropicais cujas diferentes formas ocupam distintos ambientes. Esta grande variabilidade também se reflete em aspectos citogenéticos e moleculares, que têm sido repetidamente demonstrados por meio de estudos citogenéticos. A fim de caracterizar o cariótipo de representantes deste gênero, seis espécies foram estudadas: Astyanax altiparanae, A. elachylepis, A. xavante, A. argyrimarginatus e duas espécies novas provisoriamente citadas como Astyanax sp. e A. aff. bimaculatus. Um estudo citogenético detalhado com base na coloração convencional com Giemsa, AgNORs, banda C, fluorocromos base-específicos, e FISH com sondas para genes ribossomais 18S e 5S foi realizado com o objetivo de compreender alguns dos mecanismos cromossômicos associados com a alta diversificação que caracteriza este grupo de peixes e que culminou com o estabelecimento dessas espécies. Os resultados revelaram 2n = 50 cromossomos para cinco espécies e 2n = 52 cromossomos para Astyanax sp. Pequenas variações na macroestrutura dos cariótipos foram identificadas e se mostraram relevantes quando analisadas com base nos bandamentos clássicos, coloração por fluorocromos base-específicos e FISH com sondas de DNA 18S e 5S. Esssa diversidade cariotípica detectada indica a necessidade de uma revisão taxonômica no grupo de indivíduos aqui referidos como A. aff. bimaculatus, inclusive com a descrição de Astyanax sp., incluindo a possibilidade de inserção dessa unidade em outro gênero distinto de Astyanax.

Comparison of the marker effects of two different fluorescent dyes in labeling endogenous neural stem cells in the central nervous system / 解放军医学杂志

Objective To observe the marker effects of two different fluorescent dyes, DIL and DAPI, in labeling endogenous neural stem cells (ENSCs) in rat central nervous system. Methods Thirty-six Sprague-Dawley rats were randomized into staining groups, comprising DIL group and DAPI group, and the corresponding control groups, including DMSO group for DIL group and PBS group for DAPI group. 0.2% DIL 10μl or 10μg/ml DAPI 10μl was stereotactically injected into the lateral ventricle of rats of DIL group or DAPI group, while DMSO or PBS 10μl was introduced into that of DMSO group or PBS group. Neurological severity score (NSS) was determined 2 hours and 24 hours respectively after the operation. Rats were sacrificed at day 1, 3, 7 after the injection. Serial coronal sections of the brain and spinal cord were carried out on a cryostat, and then they were observed under a confocal microscope. The fluorescence intensity of the targeted area, which highlighted by labeled ependymal cells, in the brain and spinal cord of cervical vertebrae, thoracic vertebrae and lumbar vertebrae were semi-quantified. Fluorescence intensity of each section was measured in triplicate, and a mean value was obtained. Statistical analysis was performed on 3 data sets, randomly selected from sections of brain and spinal cord obtained at day 1, 3, 7. Results Two hours after DIL injection, the rats showed no evident neurological defect. NSS value was very low, and there was no significant difference compared with the DMSO group (P>0.05). Twenty-four hours later, normal neurological function recovered in all the rats. Red fluorescence could be seen in the cytoplasm of ependymal cells in the lateral ventricle and each spinal cord segment at day 1 after the DIL injection, and it did not disappear until the 7th day. Nuclei of DAPI-labeled lateral ventricle cells were blue, with clear nuclear morphology. Choroid plexus cells of the ventricle were also labeled. However, there was no blue fluorescence in the medulla oblongata or any segment ofthe spinal cord. The picture at day 3 and day 7 was similar to that of day 1. No significant difference was found between fluorescence intensity in DIL or DAPI stained cells(P>0.05) at any time point. Conclusions DIL may serve as a marker of the cytoplasm of ependymal cells in the brain ventricle and spinal central canal. DAPI, which is often used in the nuclear staining, can label the ENSCs in the brain ventricle. Intraventricular injection of fluorescent dye is a relatively safety procedure.

Comparison of the marker effects of two different fluorescent dyes in labeling endogenous neural stem cells in the central nervous system / 解放军医学杂志

Objective To observe the marker effects of two different fluorescent dyes, DIL and DAPI, in labeling endogenous neural stem cells (ENSCs) in rat central nervous system. Methods Thirty-six Sprague-Dawley rats were randomized into staining groups, comprising DIL group and DAPI group, and the corresponding control groups, including DMSO group for DIL group and PBS group for DAPI group. 0.2% DIL 10μl or 10μg/ml DAPI 10μl was stereotactically injected into the lateral ventricle of rats of DIL group or DAPI group, while DMSO or PBS 10μl was introduced into that of DMSO group or PBS group. Neurological severity score (NSS) was determined 2 hours and 24 hours respectively after the operation. Rats were sacrificed at day 1, 3, 7 after the injection. Serial coronal sections of the brain and spinal cord were carried out on a cryostat, and then they were observed under a confocal microscope. The fluorescence intensity of the targeted area, which highlighted by labeled ependymal cells, in the brain and spinal cord of cervical vertebrae, thoracic vertebrae and lumbar vertebrae were semi-quantified. Fluorescence intensity of each section was measured in triplicate, and a mean value was obtained. Statistical analysis was performed on 3 data sets, randomly selected from sections of brain and spinal cord obtained at day 1, 3, 7. Results Two hours after DIL injection, the rats showed no evident neurological defect. NSS value was very low, and there was no significant difference compared with the DMSO group (P>0.05). Twenty-four hours later, normal neurological function recovered in all the rats. Red fluorescence could be seen in the cytoplasm of ependymal cells in the lateral ventricle and each spinal cord segment at day 1 after the DIL injection, and it did not disappear until the 7th day. Nuclei of DAPI-labeled lateral ventricle cells were blue, with clear nuclear morphology. Choroid plexus cells of the ventricle were also labeled. However, there was no blue fluorescence in the medulla oblongata or any segment ofthe spinal cord. The picture at day 3 and day 7 was similar to that of day 1. No significant difference was found between fluorescence intensity in DIL or DAPI stained cells(P>0.05) at any time point. Conclusions DIL may serve as a marker of the cytoplasm of ependymal cells in the brain ventricle and spinal central canal. DAPI, which is often used in the nuclear staining, can label the ENSCs in the brain ventricle. Intraventricular injection of fluorescent dye is a relatively safety procedure.

Genome re-assignment of Arachis trinitensis (Sect. Arachis, Leguminosae) and its implications for the genetic origin of cultivated peanut

The karyotype structure of Arachis trinitensis was studied by conventional Feulgen staining, CMA/DAPI banding and rDNA loci detection by fluorescence in situ hybridization (FISH) in order to establish its genome status and test the hypothesis that this species is a genome donor of cultivated peanut. Conventional staining revealed that the karyotype lacked the small "A chromosomes" characteristic of the A genome. In agreement with this, chromosomal banding showed that none of the chromosomes had the large centromeric bands expected for A chromosomes. FISH revealed one pair each of 5S and 45S rDNA loci, located in different medium-sized metacentric chromosomes. Collectively, these results suggest that A. trinitensis should be removed from the A genome and be considered as a B or non-A genome species. The pattern of heterochromatic bands and rDNA loci of A. trinitensis differ markedly from any of the complements of A. hypogaea, suggesting that the former species is unlikely to be one of the wild diploid progenitors of the latter.

Quantitative and Qualitative Estimation of Bacteria Contaminating Human Hairs

Human hairs have been known to be easily contaminated with microorganisms. This study was performed in order to measure what bacterial species and how much microorganisms contaminate human hairs in specific place. Virgin human hairs were left at 6 positions in inside corner and beside window in a laboratory for 7 days. The number of viable bacterial cells, which were determined by most probable number method, contaminating the human hairs was measured at a maximum of 10(6)/g hair and a minimum of 10(3)/g hair in inside corner and maximum of 10(6)/g hair and a minimum of 10(3)/g hair beside window. The bacterial cells-contaminating human hairs were observed via fluorescence light microscopy after 4',6-diamino-2-phenylindole (DAPI) staining. The bacterial community contaminating human hairs was analyzed via the thermal gradient gel electrophoresis (TGGE) technique, based on the diversity of the 16S-rDNA variable region. In total, approximately 20 bacterial species were detected from 12 groups of hair samples. In this study, general experimental methods-fluorescence staining, TGGE and MPN-were combined to develop new method for observation and estimation of bacteria contaminating human hairs.

Quantitative and Qualitative Estimation of Bacteria Contaminating Human Hairs

Human hairs have been known to be easily contaminated with microorganisms. This study was performed in order to measure what bacterial species and how much microorganisms contaminate human hairs in specific place. Virgin human hairs were left at 6 positions in inside corner and beside window in a laboratory for 7 days. The number of viable bacterial cells, which were determined by most probable number method, contaminating the human hairs was measured at a maximum of 10(6)/g hair and a minimum of 10(3)/g hair in inside corner and maximum of 10(6)/g hair and a minimum of 10(3)/g hair beside window. The bacterial cells-contaminating human hairs were observed via fluorescence light microscopy after 4',6-diamino-2-phenylindole (DAPI) staining. The bacterial community contaminating human hairs was analyzed via the thermal gradient gel electrophoresis (TGGE) technique, based on the diversity of the 16S-rDNA variable region. In total, approximately 20 bacterial species were detected from 12 groups of hair samples. In this study, general experimental methods-fluorescence staining, TGGE and MPN-were combined to develop new method for observation and estimation of bacteria contaminating human hairs.

The karyotype of Nothoscordum arenarium Herter (Gilliesioideae, Alliaceae): a populational and cytomolecular analysis

The genus Nothoscordum Kunth comprises approximately 20 species native to South America. Karyologically, the genus is remarkable for its large chromosomes and Robertsonian translocations. Variation in chromosome number has been recorded in a few polyploid species and it is unknown among diploids. This study presents the chromosome number and morphology of 53 individuals of seven populations of N. arenarium Herter (2n = 10). In addition, karyotype analyses after C-banding, staining with CMA and DAPI, and in situ hybridization with 5S and 45S rDNA probes were performed in six individuals from one population. All individuals exhibited 2n = 10 (6M + 4A), except for one tetraploid (2n = 20, 12M + 8A) and one triploid (2n = 15, 9M + 6A) plant. C-banding revealed the presence of CMA+/DAPI- heterochromatin in the short arm and in the proximal region of the long arm of all acrocentric chromosomes. The 45S rDNA sites co-localized with the CMA+ regions of the acrocentrics short arms, while the 5S rDNA probe only hybridized with the subterminal region of a pair of metacentric chromosomes. A change in the pattern of CMA bands and rDNA sites was observed in only one individual bearing a reciprocal translocation involving the long arm of a metacentric and the long arm of an acrocentric chromosome. These data suggest that, despite isolated cases of polyploidy and translocation, the karyotype of N. arenarium is very stable and the karyotypic instability described for other species may be associated with their polyploid condition.

Characterization of the Arachis (Leguminosae) D genome using fluorescence in situ hybridization (FISH) chromosome markers and total genome DNA hybridization

Chromosome markers were developed for Arachis glandulifera using fluorescence in situ hybridization (FISH) of the 5S and 45S rRNA genes and heterochromatic 4'-6-diamidino-2-phenylindole (DAPI) positive bands. We used chromosome landmarks identified by these markers to construct the first Arachis species ideogram in which all the homologous chromosomes were precisely identified. The comparison of this ideogram with those published for other Arachis species revealed very poor homeologies with all A and B genome taxa, supporting the special genome constitution (D genome) of A. glandulifera. Genomic affinities were further investigated by dot blot hybridization of biotinylated A. glandulifera total DNA to DNA from several Arachis species, the results indicating that the D genome is positioned between the A and B genomes.