Serine/threonine protein kinase PrkA of the human pathogen Listeriamonocytogenes: Biochemical characterization and identification of interacting partners through proteomic approaches

Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human disease. The analysis of the proteins coded by the L. monocytogenes genome reveals the presence of two eukaryotic-type Ser/Thr-kinases (lmo1820 and lmo0618) and a Ser/Thrphosphatase (lmo1821). Protein phosphorylation regulates enzyme activities and protein interactions participating in physiological and pathophysiological processes in bacterial diseases. However in the case of L. monocytogenes there is scarce information about biochemical properties of these enzymes, as well as the physiological processes that they modulate. In the present work the catalytic domain of the protein coded by lmo1820 was produced as a functional His6-tagged Ser/Thr-kinase, and was denominated PrkA. PrkA was able to autophosphorylate specific Thr residues within its activation loop sequence. A similar autophosphorylation pattern was previously reported for Ser/Thr-kinases from related prokaryotes, whose role in kinase activity and substrate recruitment was demonstrated. We studied the kinase interactome using affinity chromatography and proteomic approaches. We identified 62 proteins that interact, either directly or indirectly, with the catalytic domain of PrkA, including proteins that participate in carbohydrates metabolism, cell wall metabolism and protein synthesis. Our results suggest that PrkA could be involved in the regulation of a variety of fundamental biological processes.Agencia Nacional de Investigación e Innovació

Detection of Histoplasma capsulatum Antigen in Panamanian Patients with Disseminated Histoplasmosis and AIDS ᰔ

Histoplasmosis is a common endemic mycosis in the Americas, often causing severe disease in patients with AIDS. Antigen detection has become an important method for rapid diagnosis of histoplasmosis in the United States but not in Central or South America. Isolates from patients in the United States are predominantly found to be class 2 isolates when typed using the nuclear gene YPS3, while isolates from Latin America are predominantly typed as class 5 or class 6. Whether infection with these Latin American genotypes produces positive results in the Histoplasma antigen assay has not been reported. In this study, we have compared the sensitivity of antigen detection for AIDS patients from Panama who had progressive disseminated histoplasmosis to that for those in the United States. Antigenuria was detected in the MVista Histoplasma antigen enzyme immunoassay (EIA) in 95.2% of Panamanian cases versus 100% of U.S. cases. Antigenemia was detected in 94.7% of the Panamanian cases versus 92% of the U.S. cases. Two clinical isolates from Panama were typed using YPS3 and were found to be restriction fragment length polymorphism class 6. We conclude that the MVista Histoplasma antigen EIA is a sensitive method for diagnosis of histoplasmosis in Panama. Progressive disseminated histoplasmosis (PDH) is a common and serious opportunistic disease among patients with AIDS in the United States and Latin America. In Panama, PDH occurred in 8% of hospitalized patients from 1997 to 2003 and was fatal in 12% of cases (6). The clinical picture was similar to that reported elsewhere and usually included fever, weight loss, and respiratory symptoms, often in association with diarrhea, as well as hepatosplenomegaly, hepatic enzyme elevation, and pancytopenia. Skin lesions were noted in 17% of the Panamanian cases (6), lower than the observed rate for cases from Brazil (66%) but higher than that for cases from the United States (ϳ3%) (7). Disease in patients in Mexico and Latin America has been described as being caused by isolates of Histoplasma capsulatum var. capsulatum that exhibit distinct genetic profiles in comparison to those from the United States (3, 8, 9). Keath et al. (9) first described typing based upon restriction fragment length polymorphism (RFLP) in the YPS3 gene. They showed that Panamanian strains were typed as YPS3 class 3, 5, or 6, while the North American strains were predominantly class 2. Later, Kasuga et al. used the DNA sequence substitution rates in four independent protein-coding genes to identify at least eight different genotypes, or clades (8). North American class 2 was the predominant genotype in the United States and Latin American group A in Latin America. Latin American group B was found only in Columbia and Argentina. Based on only four isolates from Panama included in the study by Kasuga et al., Panamanian strains included Latin American group A and a lone lineage, designated H81. The diagnosis of PDH relies on demonstration of the organism in clinical specimens or detection of antigen in body fluids (2). Histopathology may be falsely negative in up to 50% of patients, caused by sampling error, paucity of organisms, or inexperience of the pathologist. Culture may require several weeks to isolate and identify the organism and may be falsely negative for 15% of patients (13). Antigenuria can be detected in 95% of cases in patients with AIDS in the United States (4, 13) but has not been evaluated in Latin America. Whether genetic differences in Latin American isolates would affect the sensitivity for diagnosis by antigen detection is unknown. The prevalence of PDH in AIDS patients from Panama offered the opportunity to address this question. MATERIALS AND METHODS Case definition. The definition included the presence of AIDS based upon a serologic test positive for human immunodeficiency virus infection, a CD4 cell count below 200 cells/l and/or a previous AIDS defining condition according to CDC classification, and PDH proven by culture or histopathology. Methods. We collected samples of serum and urine from Panamanian patients with AIDS and clinical suspicion of PDH who were admitted to the AIDS ward of the Arnulfo Arias Madrid Hospital in Panama City, Panama, from December 2005 through November 2006. All of the samples were taken before the start of antifungal therapy, referred to as baseline, and then stored frozen at Ϫ20°C. Samples were shipped in batches to MiraVista Diagnostics on three occasions during the course of the study, according to International Air Transport Association regulations. We included 21 patients with PDH in the study. The study was approved by the institution's review board and followed guidelines for clinical research. All patients read and signed an informed-consent form before the samples were taken. The investigators also completed a case report form for each patient with demographic and clinically relevant data. For comparison, paired serum and urine specimens collected at enrollment or week 1 or 2 of treatment from 65 AIDS patients in the United States with PD

Ischemic Preconditioning in the Animal Kidney, a Systematic Review and Meta-Analysis

Ischemic preconditioning (IPC) is a potent renoprotective strategy which has not yet been translated successfully into clinical practice, in spite of promising results in animal studies. We performed a unique systematic review and meta-analysis of animal studies to identify factors modifying IPC efficacy in renal ischemia/reperfusion injury (IRI), in order to enhance the design of future (clinical) studies. An electronic literature search for animal studies on IPC in renal IRI yielded fifty-eight studies which met our inclusion criteria. We extracted data for serum creatinine, blood urea nitrogen and histological renal damage, as well as study quality indicators. Meta-analysis showed that IPC reduces serum creatinine (SMD 1.54 [95%CI 1.16, 1.93]), blood urea nitrogen (SMD 1.42 [95% CI 0.97, 1.87]) and histological renal damage (SMD 1.12 [95% CI 0.89, 1.35]) after IRI as compared to controls. Factors influencing IPC efficacy were the window of protection (<24 h = early vs. ≥24 h = late) and animal species (rat vs. mouse). No difference in efficacy between local and remote IPC was observed. In conclusion, our findings show that IPC effectively reduces renal damage after IRI, with higher efficacy in the late window of protection. However, there is a large gap in study data concerning the optimal window of protection, and IPC efficacy may differ per animal species. Moreover, current clinical trials on RIPC may not be optimally designed, and our findings identify a need for further standardization of animal experiments

Roles of Microglial Phagocytosis and Inflammatory Mediators in the Pathophysiology of Sleep Disorders

Sleep serves crucial learning and memory functions in both nervous and immune systems. Microglia are brain immune cells that actively maintain health through their crucial physiological roles exerted across the lifespan, including phagocytosis of cellular debris and orchestration of neuroinflammation. The past decade has witnessed an explosive growth of microglial research. Considering the recent developments in the field of microglia and sleep, we examine their possible impact on various pathological conditions associated with a gain, disruption, or loss of sleep in this focused mini-review. While there are extensive studies of microglial implication in a variety of neuropsychiatric and neurodegenerative diseases, less is known regarding their roles in sleep disorders. It is timely to stimulate new research in this emergent and rapidly growing field of investigation.Peer reviewe

Technologies of sleep research

Sleep is investigated in many different ways, many different species and under many different circumstances. Modern sleep research is a multidisciplinary venture. Therefore, this review cannot give a complete overview of all techniques used in sleep research and sleep medicine. What it will try to do is to give an overview of widely applied techniques and exciting new developments. Electroencephalography has been the backbone of sleep research and sleep medicine since its first application in the 1930s. The electroencephalogram is still used but now combined with many different techniques monitoring body and brain temperature, changes in brain and blood chemistry, or changes in brain functioning. Animal research has been very important for progress in sleep research and sleep medicine. It provides opportunities to investigate the sleeping brain in ways not possible in healthy volunteers. Progress in genomics has brought new insights in sleep regulation, the best example being the discovery of hypocretin/orexin deficiency as the cause of narcolepsy. Gene manipulation holds great promise for the future since it is possible not only to investigate the functions of different genes under normal conditions, but also to mimic human pathology in much greater detail

Avaliação longitudinal de uma rotina clínica para prevenção e tratamento de cárie oclusal

Com o melhor entendimento da etiopatogenia da doença cárie, programas preventivos não-invasivos de baixo custo e uso restrito de selantes têm sido sugeridos. O presente estudo tem como objetivo avaliar uma rotina clínica para prevenção e controle de lesões cariosas na superfície oclusal de primeiros molares permanentes (1°MP). Foram selecionadas 39 crianças, de ambos os sexos, na faixa etária de 5 a 7 anos, que procuraram atendimento odontológico na Universidade Luterana do Brasil, Canoas - RS. Todas as crianças incluídas no estudo apresentavam atividade de cárie e os quatro 1° MP erupcionados hígidos ou com lesão cariosa não-cavitada, sendo excluídos os que apresentavam cavidade de cárie, selante ou restaurações. O programa de atendimento foi baseado no controle da atividade de cárie, de acordo com as necessidades individuais de cada paciente, sem a utilização de selantes oclusais. Após 24 meses, 98% das superfícies oclusais diagnosticadas como hígidas no exame inicial permaneceram nessa condição clínica; e 91% das manchas brancas ativas evidenciadas no exame inicial tornaram-se inativas. Desta forma, verificou-se um aumento na proporção de lesões inativas e uma redução na proporção de lesõs ativas durante o período de acompanhamento longitudinal. Assim, os resultados evidenciam que programas de tratamento baseados no controle da atividade de cárie são capazes de prevenir e controlar lesões cariosas em superícies oclusais de 1° MP sem a utilização de selantes oclusais

Cytokine mRNA induction by interleukin-1β or tumor necrosis factor α in vitro and in vivo

Hypothalamic and cortical mRNA levels for cytokines such as interleukin-1β (IL1β), tumor necrosis factor alpha (TNFα), nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are impacted by systemic treatments of IL1β and TNFα. To investigate the time course of the effects of IL1β and TNFα on hypothalamic and cortical cytokine gene expression, we measured mRNA levels for IL1β, TNFα, interleukin-6 (IL-6), interleukin-10 (IL-10), IL1 receptor 1, BDNF, NGF, and glutamate decarboxylase-67 in vitro using hypothalamic and cortical primary cultures. IL1β and TNFα mRNA levels increased significantly in a dose-dependent fashion after exposure to either IL1β or TNFα. IL1β increased IL1β mRNA in both the hypothalamic and cortical cultures after 2–6 h while TNFα mRNA increased significantly within 30 min and continued to rise up to 2–6 h. Most of the other mRNAs showed significant changes independent of dose in vitro. In vivo, intracerebroventricular (icv) injection of IL1β or TNFα also significantly increased IL1β, TNFα and IL6 mRNA levels in the hypothalamus and cortex. IL1β icv, but not TNFα, increased NGF mRNA levels in both these areas. Results support the hypothesis that centrally active doses of IL1β and TNFα enhance their own mRNA levels as well as affect mRNA levels for other neuronal growth factors