RESUMO
Background: Police and mental health co-responder programs operate internationally and can be effective in providing timely and appropriate assessment, brief intervention, and referral services for people experiencing mental health crises. However, these models vary greatly, and little is known about how the design and implementation of these programs impacts their effectiveness. Method: This study was a qualitative, post hoc implementation determinant evaluation of mental health co-responder units in Brisbane, Australia, comprising of verbal or written interviews with police and mental health staff with an on-road role in the co-responder units, and their managers. The Consolidated Framework for Implementation Research was used to identify barriers and enablers to the program's implementation and effectiveness. Results: Participants (n = 30) from all groups felt strongly that the co-responder units are a substantial improvement over the usual police management of mental health crisis cases, and lead to better outcomes for consumers and the service. Enablers included an information-sharing agreement; the Mental Health Co-Responder (MHCORE) program's compatibility with existing police and mental health services; and the learning opportunity for both organizations. Barriers included cultural differences between the organizations, particularly risk-aversion to suicidality for police and a focus on least-restrictive practices for health; extensive documentation requirements for health; and a lack of specific mental health training for police. Conclusions: Using an evidence-based implementation science framework enabled identification of a broad range of contextual barriers and enablers to implementation of police mental health co-responder programs. Adapting the program to address these barriers and enablers during the planning, implementation, monitoring, and evaluation phases increases the likelihood of the service's effectiveness. These findings will inform the spread and scale of the co-responder program across Queensland, and will be relevant to police districts internationally considering implementing a co-responder program.
A large and rising proportion of calls to police relate to mental health crises, however police lack the resources, knowledge, training and supports to effectively address these crises. In Brisbane, Australia people in mental health crisis who are attended by police are routinely transported under an Emergency Examination Authority to a hospital emergency department. This is time-consuming for both the consumer and police, may be stressful or distressing for the consumer, and can put pressure on emergency departments. Co-responder programs team a senior mental health clinician with a senior police officer. There is evidence that a qualified and experienced mental health clinician providing people in mental health crisis with a timely assessment and brief intervention in the field, and where appropriate, referral to support services, leads to better outcomes for the consumer, reduced hospital transport, reduced time per case, and reduced overall service costs. Although many papers have been written evaluating the outcomes of these programs, few have considered factors that impact the implementation, effectiveness, and sustainment of co-responder teams. We used an implementation science approach based on the Consolidated Framework for Implementation Research to identify barriers to and enablers of mental health co-responder program implementation within an Australian metropolitan setting. Understanding these barriers and enablers allows a more streamlined, better-informed roll-out of future programs, and adaptation of existing models to improve effectiveness. The results will be relevant for any police district considering implementing a mental health co-responder program. They will also be used to inform the ongoing program.
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Hereditary equine dermal asthenia (HERDA) is an autosomal recessive skin disease that affects predominantly Quarter Horses and related breeds. Typical symptoms are easy bruising and hyperextensible skin on the back. The prognosis is guarded, as affected horses cannot be ridden normally and are often euthanised. In the Quarter Horse, HERDA is associated with a mutation in cyclophilin B (PPIB), an enzyme involved in triple helix formation of collagen. Here we describe the case of a Swiss Warmblood filly with symptoms of HERDA without PPIB-mutation and in which we also could exclude Ehlers-Danlos syndrome Type IV, VI, VIIA, VIIB and VIIC (dermatosparaxis type) as etiological diseases.
Assuntos
Astenia/veterinária , Ciclofilinas/genética , Doenças dos Cavalos/genética , Dermatopatias/veterinária , Animais , Astenia/genética , Astenia/patologia , Colágeno/metabolismo , Síndrome de Ehlers-Danlos/genética , Feminino , Doenças dos Cavalos/patologia , Cavalos , Humanos , Mutação , Dermatopatias/genética , Dermatopatias/patologiaRESUMO
The re-emergence of necrotizing enteritis (NE) in Swiss pig breeding farms raised concern that, besides C. perfringens type C strains, additional C. perfringens toxinotypes might cause this disease. Therefore we retrospectively investigated the association of NE with C. perfringens type C or different C. perfringens toxinotypes. We evaluated pathological lesions, routine diagnostic bacteriology results, and multiplex real-time PCR analyses from DNA extracts of archived intestinal samples of 199 piglets from our diagnostic case load. 96.5% of NE cases and 100% of herds affected by NE were positive for C. perfringens type C genotypes. Animals without necrotizing enteritis revealed a significantly lower detection rate of type C genotypes. Non affected piglets showed a high prevalence for beta-2-toxin positive C. perfringens type A strains. Collectively, our data indicate that outbreaks of NE in piglets in Switzerland cannot be attributed to newly emerging pathogenic toxinotypes, but are due to a spread of pathogenic C. perfringens type C strains.
Assuntos
Infecções por Clostridium/veterinária , Clostridium perfringens/isolamento & purificação , Enterite/veterinária , Doenças dos Suínos/epidemiologia , Animais , Animais Recém-Nascidos , Toxinas Bacterianas/genética , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Clostridium perfringens/classificação , Clostridium perfringens/patogenicidade , DNA Bacteriano/análise , Surtos de Doenças/veterinária , Enterite/epidemiologia , Enterite/microbiologia , Enterotoxinas/genética , Feminino , Genótipo , Masculino , Necrose/epidemiologia , Necrose/microbiologia , Necrose/veterinária , Estudos Retrospectivos , Suínos , Doenças dos Suínos/microbiologia , Suíça/epidemiologiaRESUMO
Infection of canine footpads with canine distemper virus (CDV) can result in so-called hard pad disease characterized by footpad epidermal proliferation and hyperkeratosis. Cultured canine footpad keratinocytes (CFK) were inoculated with a virulent canine distemper virus strain (A75/17-CDV) to study the effects of CDV-infection on keratinocyte proliferation. Infection was analyzed by immunohistochemistry and in situ hybridization for CDV nucleoprotein (N-protein) antigen and mRNA. CDV caused a persistent, non-cytocidal infection with spread from single cells to infection of the confluent cell layer 7 days post infection (p.i.). Absolute cell numbers were significantly higher in infected cultures compared to control cultures from day 4 until day 6 p.i. Infected cultures contained significantly more total DNA on day 5 p.i. compared to controls. Immunohistochemical investigation of proliferation markers Ki67 and BrdU demonstrated a nearly two-fold increase in numbers of positive cells on day 5 p.i. compared to controls. These findings demonstrate that canine distemper virus infection of canine footpad keratinocytes in vitro was associated with proliferation.
Assuntos
Vírus da Cinomose Canina/patogenicidade , Cinomose/virologia , Doenças do Cão/virologia , Dermatoses do Pé/veterinária , Queratinócitos/virologia , Animais , Antígenos Virais/metabolismo , Proliferação de Células , Células Cultivadas , Cinomose/patologia , Doenças do Cão/patologia , Cães , Dermatoses do Pé/patologia , Dermatoses do Pé/virologia , Imuno-Histoquímica/veterinária , Hibridização In Situ/veterinária , Queratinócitos/citologia , Queratinócitos/fisiologia , Antígeno Ki-67/metabolismo , Nucleoproteínas/metabolismo , RNA ViralRESUMO
Lipids of axenically-cultured Giardia lamblia trophozoites were compared with those of cells undergoing in vitro encystation. Although the lipid composition of the organisms grossly resembled those of low-bile or high-bile culture media, differences were clearly detected. Encysting trophozoites incubated in a high-bile medium for 24 h had a higher concentration of unsaturated fatty acids in the total cellular lipids than did nonencysting trophozoites. The organism, but not the medium, contained linoleate and linolenate, suggesting that G. lamblia desaturates oleate. The presence of a fatty acid desaturase activity in the organism was demonstrated by the conversion of a radiolabeled monounsaturated fatty acid (oleate) to radiolabeled polyunsaturated fatty acids. Triglycerides, a common form of storage lipids, were unusually low in G. lamblia, but steryl esters (which can also serve as reserves) were abundant. Steryl esters increased during encystation of G. lamblia. The changes observed in G. lamblia lipids (increased fatty acid unsaturation and the accumulation of storage lipids) are consistent with parasite differentiation into a cyst stage that is able to survive outside the host at reduced temperatures and reduced available nutrient resources. This study also demonstrated that G. lamblia not only has the capacity to de novo synthesize isoprenoid lipids (ubiquinone, prenylated proteins), but it can also metabolize fatty acids by the addition of double bonds.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Giardia lamblia/metabolismo , Metabolismo dos Lipídeos , Animais , Bile/metabolismo , Meios de Cultura/química , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Giardia lamblia/enzimologia , Giardia lamblia/crescimento & desenvolvimento , Lipídeos/química , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Esteróis/metabolismoRESUMO
The new species Saccharomyces turicensis sp. nov. isolated from different kefyr grains is described. Although its morphological properties differ, its physiological characteristics come close to those of Saccharomyces bayanus Saccardo and Saccharomyces pastorianus Reess ex E. C. Hansen. However, electrophoretic karyotyping and restriction fragment length polymorphism of the internal transcribed spacer region yield clear differences. Sequences (270 nucleotides) of the D2 domain at the 5'-terminal end of the large subunit ribosomal RNA gene reveal 98.0% identity with Saccharomyces exiguus. Since strains of a particular yeast species usually show less than 1% substitution in the D2 domain, the yeast in question is considered to be a new species. The name Saccharomyces turicensis is proposed indicating the place Zürich (Turicum in Latin) where the yeast had been isolated.
Assuntos
Saccharomyces/genética , Iogurte/microbiologia , DNA Ribossômico/genética , Cariotipagem , Filogenia , RNA Fúngico/genética , RNA Ribossômico 5,8S/genética , Saccharomyces/classificação , Saccharomyces/crescimento & desenvolvimentoRESUMO
Dichloran 18% glycerol (DG18) agar was originally developed to enumerate xerophilic foodborne moulds. However, some laboratories are using DG18 agar as a general medium to enumerate foodborne moulds and yeasts. A collaborative study, with the participation of seven laboratories, was undertaken to compare DG18 agar with dichloran rose bengal chloramphenicol (DRBC) agar, tryptone glucose yeast extract chloramphenicol (TGYC) agar, and plate count agar supplemented with chloramphenicol (PCAC) for enumerating 14 species of common food spoilage yeasts. Comparison of the mean values of populations of all yeasts recovered on each medium revealed no significant differences among DRBC agar, PCAC, and TGYC agar, while each of these media supported the development of significantly (P < or = 0.05) higher numbers of colonies than DG18 agar. However, differences were only 0.08 to 0.10 log10 cfu/ml, making the practical significance questionable. The overall coefficient of variation (CV) for within laboratory repeatability was 1.71%, while the CV for reproducibility of counts obtained among laboratories was 6.96%. Compared to DRBC agar, TGYC agar, and PCAC, yeast colonies were smaller on DG18 agar. Growth of Brettanomyces anomalus, Cryptococcus albidus, and Rhodotorula mucilaginosa was particularly retarded or inhibited on DG18 agar. Based on the performance of media in supporting colony development and ease of counting colonies, the use of DG18 agar as a general enumeration medium for foodborne yeasts cannot be recommended.
Assuntos
Meios de Cultura , Microbiologia de Alimentos , Leveduras/crescimento & desenvolvimento , Ágar , Compostos de Anilina/metabolismo , Técnicas Bacteriológicas , Cloranfenicol/metabolismo , Contagem de Colônia Microbiana , Corantes Fluorescentes/química , Fungicidas Industriais/metabolismo , Glicerol/metabolismo , Reprodutibilidade dos Testes , Rosa Bengala/química , Leveduras/isolamento & purificaçãoRESUMO
Pneumocystis carinii is the paradigm of opportunistic infections in immunocompromised mammals. Prior to the acquired immunodeficiency syndrome (AIDS) pandemic and the use of immunosuppressive therapy in organ transplant and cancer patients, P. carinii was regarded as a curiosity, rarely observed clinically. Interest in this organism exploded when it was identified as the agent of P. carinii pneumonia (PcP), the direct cause of death among many AIDS patients. Aggressive prophylaxis has decreased the number of acute PcP cases, but it remains among the most prevalent opportunistic infections found within this patient population. The taxonomic assignment of P. carinii has long been argued; molecular genetics data now demonstrate that it is a fungus. Several antimycotic drugs are targeted against ergosterol or its biosynthesis, but these are not as effective against PcP as they are against other fungal infections. This can now be explained in part by the identification of the sterols of P. carinii. The organism lacks ergosterol but contains distinct C28 and C29 delta7 24-alkylsterols. Also, 24-methylenelanost-8-en-3beta-ol (C31) and pneumocysterol, (24Z)-ethylidenelanost-8-en-3beta-ol (C32) were recently identified in organisms infecting humans. Together, the delta7 24-alkylsterols and pneumocysterol are regarded as signature lipids of the pathogen that can be useful for the diagnosis of PcP, since no other lung pathogen is known to contain them. Cholesterol (C27), the dominant sterol component in P. carinii, is probably totally scavenged from the host. De novo synthesis of sterols has been demonstrated by the presence of lovastatin-sensitive 3-hydroxy-3-methylglutaryl-CoA reductase activity, the incorporation of radiolabeled mevalonate and squalene into P. carinii sterols, and the reduction in cellular ATP in cells treated with inhibitors of enzymes in sterol biosynthesis.
Assuntos
Pneumocystis/química , Esteróis/análise , Animais , Cromatografia Gasosa , Humanos , Pulmão/microbiologia , Pneumocystis/classificação , Pneumocystis/isolamento & purificação , Pneumonia por Pneumocystis/microbiologia , RatosRESUMO
An animal model of tinnitus was developed to study chronic salicylate-induced tinnitus in rats. Novel features of the model included oral dosing of salicylate, test stimuli that included a range of pure tones and silence, and assessment of tinnitus for several months. Experimental subjects were given sodium salicylate in their drinking water while control subjects received normal tap water. Subjects were conditioned to press a lever for food in the presence of continuous white noise. At random intervals, offset of the noise was paired with a noxious stimulus, resulting in cessation of lever pressing during the silent test periods. At other randomly scheduled intervals, a test tone was substituted for the white noise, unpaired with noxious stimuli. When the test stimuli were pure tones, the salicylate-treated subjects suppressed less than the control subjects. One explanation for this result is that the experimental subjects' sensations of tones were noisier than those of the controls because experimental subjects were experiencing tinnitus.
Assuntos
Comportamento Animal , Modelos Animais de Doenças , Zumbido/psicologia , Estimulação Acústica , Animais , Limiar Auditivo , Condicionamento Operante , Masculino , Discriminação da Altura Tonal , Ratos , Ratos Long-Evans , Salicilato de Sódio , Zumbido/induzido quimicamenteRESUMO
Several lipids and macromolecular lipoconjugates of Leishmania spp. have now been well characterized; however, the glycolipids of L. donovani have not been thoroughly examined. In the present study, 3 neutral and 3 phosphorylated glycolipids were detected in promastigote forms of the organism grown in a chemically defined medium. The fatty acid and sugar compositions of these glycolipids, isolated and purified by adsorption column chromatography and thin-layer chromatographic procedures, were identified and quantified by gas-liquid chromatography and mass spectrometry. Myristate (14:0), palmitate (16:0), palmitoleate (16:1), stearate (18:0), oleate (18:1), and linoleate (18:2) were the major fatty acids in all 6 glycolipids. Arabinose, mannose, glucose, and galactose were detected in the glycolipids. The biochemical nature of these lipids suggested that the major components in the isolated preparations of the 6 glycolipids are diacylglycerophospholipids, distinct from the major precursors of macromolecular lipoconjugates such as the lipid anchors of cell surface antigens that have been reported. These appear to be terminal products of lipid biosynthesis in this parasite.
Assuntos
Ácidos Graxos/análise , Glicolipídeos/química , Leishmania donovani/química , Monossacarídeos/análise , Animais , Cromatografia em Camada Fina , Meios de Cultura , Cromatografia Gasosa-Espectrometria de Massas , Éteres de Glicerila/análise , Éteres de Glicerila/metabolismo , Glicolipídeos/isolamento & purificação , Glicolipídeos/metabolismo , Hidrólise , Leishmania donovani/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética , FosforilaçãoRESUMO
We describe the establishment and characterisation of equine keratinocyte cultures with maintenance of a high proliferative capacity up to the second passage. Improved attachment and growth were obtained by seeding primary cells on equine feeder layers. Subcultured keratinocytes showed optimal growth when seeded on collagen type I. The proliferation rate of cells on this substrate exceeded that seen for cells seeded on equine feeder layers. By immunohistochemistry, epithelial origin and state of differentiation of the equine keratinocytes were determined. They expressed keratin and desmoplakin I/II, but lacked keratin 10. Electron microscopy revealed typical features of cultured keratinocytes. Purity of keratinocyte cultures was determined by vimentin staining. This is the first report on the establishment of equine keratinocytes derived from lip epithelium. It forms the basis to study equine keratinocyte biology and the pathogenesis of epidermal diseases. Since wound healing represents a severe problem in equine dermatology, our data may be essential for the establishment of new and improved therapy.
Assuntos
Queratinócitos/fisiologia , Pele/citologia , Animais , Diferenciação Celular , Divisão Celular , Células Cultivadas , Colágeno , Cavalos , Imuno-Histoquímica/veterinária , Queratinócitos/ultraestrutura , Microscopia Eletrônica/veterináriaRESUMO
Immunisation of sows using Clostridium perfringens type C toxoid vaccines is recommended to prevent necrotising enteritis (NE) on pig breeding farms. Absence of disease, however, oftentimes leads to the false assumption of pathogens being eradicated. The prevalence of C perfringens type C was determined by PCR in faecal samples of piglets and sows in three Swiss pig breeding farms two to four years after implementation of a vaccination programme following disease outbreaks. C perfringens type C could still be detected several years after an outbreak despite absence of NE. In-herd prevalence of the pathogens varied significantly between the farms and was also lower compared with a farm which experienced a recent outbreak. In conclusion, C perfringens type C can be detected on once-affected farms, even in the absence of NE for several years.
Assuntos
Vacinas Bacterianas/administração & dosagem , Infecções por Clostridium/veterinária , Clostridium perfringens/isolamento & purificação , Surtos de Doenças/veterinária , Doenças dos Suínos/epidemiologia , Animais , Animais Recém-Nascidos , Vacinas Bacterianas/imunologia , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Surtos de Doenças/prevenção & controle , Enterite/epidemiologia , Enterite/microbiologia , Enterite/prevenção & controle , Enterite/veterinária , Enterotoxinas/genética , Fezes/microbiologia , Feminino , Masculino , Necrose/epidemiologia , Necrose/microbiologia , Necrose/prevenção & controle , Necrose/veterinária , Prevalência , Suínos , Doenças dos Suínos/microbiologia , Doenças dos Suínos/prevenção & controleRESUMO
Clostridium perfringens type C causes fatal necrotizing enteritis in different mammalian hosts, most commonly in newborn piglets. Human cases are rare, but the disease, also called pigbel, was endemic in the Highlands of Papua New Guinea. Lesions in piglets and humans are very similar and characterized by segmental necro-hemorrhagic enteritis in acute cases and fibrino-necrotizing enteritis in subacute cases. Histologically, deep mucosal necrosis accompanied by vascular thrombosis and necrosis was consistently reported in naturally affected pigs and humans. This suggests common pathogenetic mechanisms. Previous in vitro studies using primary porcine aortic endothelial cells suggested that beta-toxin (CPB) induced endothelial damage contributes to the pathogenesis of C. perfringens type C enteritis in pigs. In the present study we investigated toxic effects of CPB on cultured primary human macro- and microvascular endothelial cells. In vitro, these cells were highly sensitive to CPB and reacted with similar cytopathic and cytotoxic effects as porcine endothelial cells. Our results indicate that porcine and human cell culture based in vitro models represent valuable tools to investigate the pathogenesis of this bacterial disease in animals and humans.
Assuntos
Infecções por Clostridium/microbiologia , Infecções por Clostridium/veterinária , Clostridium perfringens , Células Endoteliais/patologia , Doenças dos Suínos/microbiologia , Animais , Toxinas Bacterianas/metabolismo , Células Cultivadas , Infecções por Clostridium/patologia , Células Endoteliais/microbiologia , Enterite/microbiologia , Enterite/patologia , Enterotoxinas/metabolismo , Humanos , Necrose/patologia , Papua Nova Guiné , Sus scrofa , Suínos , Doenças dos Suínos/patologiaRESUMO
Beta-toxin (CPB) is known to be the major virulence factor of Clostridium perfringens type C strains, which cause necrotizing enteritis in pigs, sheep, goats, calves, and humans. The exact mode of action, in particular the cellular targets of CPB in the intestine of naturally affected species, is however still not resolved. To investigate localization of CPB in naturally occurring necrotizing enteritis, we evaluated 52 piglets with spontaneously acquired C. perfringens type C enteritis and 14 control animals by immunohistochemistry. Our results consistently revealed binding of CPB to vascular endothelial cells in peracute to acute lesions of necrotizing enteritis. Subacute cases, in contrast, demonstrated reduced or no CPB staining at the endothelium, mainly due to widespread vascular necrosis. From these results we conclude, that the pathogenesis of C. perfringens type C induced necrotizing enteritis involves binding of CPB to endothelial cells in the small intestine during the early phase of the disease. Thus, by targeting endothelial cells, CPB might specifically induce vascular necrosis, hemorrhage and subsequent hypoxic tissue necrosis.
Assuntos
Toxinas Bacterianas/toxicidade , Células Endoteliais/efeitos dos fármacos , Enterocolite Necrosante/microbiologia , Doenças dos Suínos/microbiologia , Animais , Imuno-Histoquímica , Inflamação , Intestinos/irrigação sanguínea , Intestinos/patologia , Ligação Proteica , SuínosRESUMO
The biochemical characterizations of lipophosphoglycans from various Leishmania species reported by other workers may or may not contain several types of lipophosphoglycan molecules. This is the first report in which a specific lipophosphoglycan has been defined by both its antigenic and electrophoretic properties. Furthermore, a purification procedure for this specific lipophosphoglycan is described and some biochemical characterizations are presented. Phospholipase C and the so-called phosphatidylinositol-specific phospholipase C of Bacillus cereus convert the amphipathic form of the lipophosphoglycan antigen to the hydrophilic form. Under equivalent incubation conditions, other phospholipases tested were not effective in conversion of the amphipathic to the hydrophilic form. Since the amphipathic form is present in conditioned media, antigen shedding cannot be explained by phospholipase C digestion of the amphipathic form, which would result in the release of only the hydrophilic form into the medium. Both the pellet and the supernatant fractions of conditioned media contained both forms of the antigen and did not differ in the relative amounts of the two. This observation rules out membrane blebbing as the major mechanism for the release of the amphipathic form.
Assuntos
Antígenos de Protozoários/metabolismo , Glicoesfingolipídeos/metabolismo , Leishmania donovani/metabolismo , Animais , Glicoesfingolipídeos/isolamento & purificação , Leishmania donovani/imunologia , Fosfolipases Tipo CRESUMO
The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4',6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37 degrees C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G + C content of S. cerevisiae and P. carinii. A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism's chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P. carini carinii isolated from infected rat lungs are haploid organisms.
Assuntos
DNA Fúngico/análise , Haploidia , Pulmão/microbiologia , Pneumocystis/crescimento & desenvolvimento , Pneumocystis/genética , Pneumonia por Pneumocystis/microbiologia , Corantes de Rosanilina , Animais , Núcleo Celular/genética , Corantes , Citofotometria , DNA Fúngico/genética , Corantes Fluorescentes , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Indóis , Estágios do Ciclo de Vida , Complexos Multienzimáticos/metabolismo , Peptídeo Hidrolases/metabolismo , Pneumonia por Pneumocystis/genética , Ratos , Ratos Endogâmicos Lew , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de TempoRESUMO
Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was > 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (< 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated > 99.5% purity. The total non-P. carinii protein in the final preparation (< 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 10(8)-10(9) organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80-95%.
Assuntos
Lipídeos/análise , Pneumocystis/ultraestrutura , Animais , Colesterol/análise , Pulmão/microbiologia , Pulmão/patologia , Pulmão/ultraestrutura , Microscopia Eletrônica , Fosfolipídeos/análise , Pneumocystis/química , Pneumocystis/isolamento & purificação , Infecções por Pneumocystis/patologia , Proteolipídeos/análise , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/análise , Ratos , Ratos Endogâmicos LewRESUMO
The lipids of purified preparations of Pneumocystis carinii carinii freshly isolated from infected rats were analyzed and compared with those of whole lungs from normal and methylprednisolone-immunosuppressed uninfected rats. In this study, the neutral lipid fraction was examined in detail; the relative concentrations of individual classes making up this fraction were quantified. Of particular interest was the nature of the organism's ubiquinone (coenzyme Q, CoQ) fraction because atovaquone, a hydroxynaphtho-quinone (566C80) analog of ubiquinone, is efficacious in the treatment of P. carinii pneumonia. The ubiquinone concentration in both P. carinii and lung tissues was relatively low compared to that present in rat heart and liver tissues. Two homologs were identified in the organism: CoQ10 was the predominant homolog with lesser amounts of CoQ9 present. In contrast, the lungs of normal and immunosuppressed uninfected rats had CoQ9 and lesser amounts of CoQ8, but no detectable CoQ10. Furthermore, radiolabeled mevalonic acid was incorporated in vitro into the ubiquinone fraction of P. carinii indicating that the organism has the de novo branch of the isoprenoid biosynthetic pathway leading to polyprenyl formation. Hence, it was concluded that CoQ10 (if not both CoQ10 and CoQ9) in P. carinii was not scavenged from the host but was synthesized by the organism. Although lung tissues contained substantial free fatty acids, the organism was enriched in these lipids. The high concentration of free fatty acids and relatively low level of triglycerides in P. carinii suggest that fatty acids may represent major carbon sources for ATP production by the organism.
Assuntos
Lipídeos/análise , Pneumocystis/química , Ubiquinona/análogos & derivados , Animais , Coenzimas , Lipídeos/biossíntese , Pulmão/microbiologia , Estrutura Molecular , Pneumocystis/isolamento & purificação , Pneumocystis/metabolismo , Pneumonia por Pneumocystis/microbiologia , Ratos , Ubiquinona/análise , Ubiquinona/biossínteseRESUMO
The molecular and structural characteristics of intercellular adhesion were investigated in a squamous cell carcinoma (SCCA) cell line, originally derived from an oral tumor with an invasive growth pattern. The expression of adherens junction and desmosomal components were compared with that of cultured normal oral keratinocytes. Lack of membrane association in interdesmosomal areas, the disorganization of the actin cytoskeleton and the faster cell disassembly upon E-cadherin antibody binding in SCCA cells indicated decreased functional adherens junctions. These observations were supported by a significant reduction in E-, N-, and P-cadherin protein expression. In contrast, the level of desmosomal cadherin proteins, desmoglein 1/2 and desmocollin 2, were substantially upregulated and accompanied, ultrastructurally, by increased number and size of desmosomes. Since tumor invasion suppressor capacity has been proposed for desmosomal cadherins, we investigated the in vivo invasion potential of these SCCA cells by introducing them into SCID mice. Tumors developed, but with a benign phenotype. Based on these results, we hypothesize that the benign behavior of this SCCA cell line is a consequence of overexpressed desmosomal cadherins. This SCCA cell line, therefore, represents an excellent model system to further investigate the regulation and tumor invasion suppressor potential of desmosomal adhesion molecules.