Neisseria meningitidis PorA variable regions: rapid detection of P1.7 and P1.19 variants by PCR

AIM: Rapid characterization of variable region (VR)1 variants of the porA gene among invasive strains is crucial for outbreak management and epidemiology studies. Recent sequence analysis studies in Brazil showed that the VR1 P1.7 and P1.19 variants are highly prevalent, accounting for 68%, of the total number of VR1 variants characterized. The aim of this work is to develop a rapid polymerase chain reaction (PCR)-based method for genosubtyping Neisseria meningitidis by detection of porA variable regions P1.7 and P1.19. METHODS AND RESULTS: PCR primers for the detection of porA VR1 P1.7 and P1.19 were designed and tested using 198 clinical N. meningitidis isolates that had been previously evaluated by porA sequencing. All 50 strains with VR1 P1.7 and all 65 strains with VR1 P1.19 were positively identified by the respective VR-specific PCR and no false-positive reactions occurred. CONCLUSIONS: VR-specific PCR amplification accurately identified VR P1.7 and P1.19 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: To overcome the disadvantages of serosubtyping and sequencing for typing the porA VR1 segment of N. meningitidis, we developed a PCR-based method to rapidly and accurately detect VR1 P1.7 and P1.19 variants. This approach is highly specific and sensitive; moreover it may allow for genotype determination of culture-negative samples.     

Species distribution and antifungal susceptibility patterns of <Emphasis Type="Italic">Candida</Emphasis> spp. bloodstream isolates from a Brazilian tertiary care hospital

In this work, we collect data from surveys of bloodstream Candida isolates performed in Brazil from 1996 to 2004. Besides, we analyzed the species distribution of bloodstream Candida isolates together with potential risk factors for candidemia and the susceptibility profile of these isolates in patients from Hospital das Clínicas in Goiania city, Brazil. Blood samples were collected in the admission day and on every 7 days, in the intensive care unit (ICU) of a tertiary hospital. Candida isolates were identified by standard protocols that included germ tube formation, chlamydoconidia production on cornmeal agar and sugar fermentation and assimilation tests. Data of patients were recorded and analyzed according to age at the time of diagnosis, gender and presence of potential risk factors. Statistical analysis was used to determine if the time of hospital permanence increased Candida colonization in ICU patients’ blood. The antifungal susceptibility testing was performed by broth microdilution method according to document NCCLS/CLSI M27–A2. Among the 345 blood samples cultured, candidemia was recovered in 33 patients, which were isolated 51.5% of Candida non-albicans. Fungemia was associated with long-term hospitalization. Fluconazole, itraconzole, voriconazole and amphotericin B exhibited a potent activity against all isolates of Candida. Voriconazole MICs were much low for all isolates tested. This work confirms data of increase of Candida non-albicans species in bloodstream in ICU and shows that voriconazole in vitro activity was higher than those of itraconazole, fluconazole and amphotericin B.     

Single-step pathway for synthesis of glucosylglycerate in Persephonella marina

A single-step pathway for the synthesis of the compatible solute glucosylglycerate (GG) is proposed based on the activity of a recombinant glucosylglycerate synthase (Ggs) from Persephonella marina. The corresponding gene encoded a putative glycosyltransferase that was part of an operon-like structure which also contained the genes for glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP), the enzymes that lead to the synthesis of GG through the formation of glucosyl-3-phosphoglycerate. The putative glucosyltransferase gene was expressed in Escherichia coli, and the recombinant product catalyzed the synthesis of GG in one step from ADP-glucose and d-glycerate, with K(m) values at 70 degrees C of 1.5 and 2.2 mM, respectively. This glucosylglycerate synthase (Ggs) was also able to use GDP- and UDP-glucose as donors to form GG, but the efficiencies were lower. Maximal activity was observed at temperatures between 80 and 85 degrees C, and Mg(2+) or Ca(2+) was required for catalysis. Ggs activity was maximal and remained nearly constant at pH values between 5.5 and pH 8.0, and the half-lives for inactivation were 74 h at 85 degrees C and 8 min at 100 degrees C. This is the first report of an enzyme catalyzing the synthesis of GG in one step and of the existence of two pathways for GG synthesis in the same organism.     

Radioiodination of new EGFR inhibitors as potential SPECT agents for molecular imaging of breast cancer

In our search for the development of novel SPECT radioligands for EGFR positive tumours, new potentially irreversible tyrosine kinase (TK) inhibitors are being explored. The radioiodination of N-{4-[(3-chloro-4-fluorophenyl) amino]quinazoline-6-yl}-3-bromopropionamide, a novel EGFR-TK inhibitor synthesised in our laboratory, was accomplished via halogen exchange. Purification by RP-HPLC gave [125I]-N-{4-[(3-chloro-4-fluorophenyl)amino]quinazoline-6-yl}-3-iodopropionamide with a radiochemical purity higher than 95% and a high specific activity. In vitro studies indicate that both iodinated quinazoline and its bromo precursor inhibit A431 cell growth and also possess higher potency than the parent quinazoline to inhibit the EGFR autophosphorylation. In vivo stability studies suggest metabolization of the radioiodinated quinazoline indicating a short biological half-life. The in vitro results point out that these quinazoline derivatives could be promising candidates for SPECT imaging of EGFR positive tumours provided that they are selectively modified in order to achieve better in vivo radiochemical stability.     

Conjugated linoleic acid protects against age-associated bone loss in C57BL/6 female mice

Osteoporosis is one of the major causes of morbidity in the elderly. Inflammation exerts a significant influence on bone turnover, inducing the chronic form of osteoporosis. Dietary nutrition has the capacity to modulate inflammatory response. Therefore, nutritional strategies and lifestyle changes may prevent age-related osteoporosis, thereby improving the quality of life of the elderly population. Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Hence, this study was undertaken to examine the effect of CLA on bone mineral density (BMD) in middle-aged C57BL/6 female mice. After 10 weeks on diet, CLA-fed mice (14 months) maintained a higher BMD in different bone regions than corn oil (CO)-fed mice. The increased BMD was accompanied by a decreased activity of proinflammatory cytokines (such as tumor necrosis factor alpha, interleukin-6 and the receptor activator of NF-kappaB ligand) and decreased osteoclast function. Furthermore, a significant decrease in fat mass and an increase in muscle mass were also observed in CLA-fed mice compared to CO-fed mice. In conclusion, these findings suggest that CLA may prevent the loss of bone and muscle mass by modulating markers of inflammation and osteoclastogenic factors.     

Natural mortality factors of Leucoptera coffeella (Lepidoptera: Lyonetiidae) on Coffea arabica

The natural mortality of the coffee leafminer, Leucoptera coffeella (Guérin-Méneville) was investigated in three strata of coffee plant canopy for three seasons through construction and analysis of ecological life tables. Mortality of the leafminer was similar on all thirds of the canopy. Total mortality of immature stages was 95%, with 38.5, 43.8 and 12.7% occurring during egg, larval, and pupal stages. Rainfall killed 39.3% of eggs and larvae, and together with egg inviability (16.3%) and Vespidae (11.3%), were the highest mortality factors. Six wasp parasitoids caused 8% of larval mortality. Egg and larva were the critical stages. Variation in mortality was primarily associated with egg inviability, rainfall, and parasitism by Horismenus sp. (Hymenoptera: Eulophidae). Physiological disturbances during molting and metamorphosis also contributed for fluctuations in mortality of the leafminer. Tactics of integrated pest management to enhance natural mortality of the leafminer while conserving or augmenting the action of natural enemies are discussed.     

Phytosterols: applications and recovery methods

Phytosterols, or plant sterols, are compounds that occur naturally and bear close structural resemblance to cholesterol, but have different side-chain configurations. Phytosterols are relevant in pharmaceuticals (production of therapeutic steroids), nutrition (anti-cholesterol additives in functional foods, anti-cancer properties), and cosmetics (creams, lipstick). Phytosterols can be obtained from vegetable oils or from industrial wastes, which gives an added value to the latter. Considerable efforts have been recently dedicated to the development of efficient processes for phytosterol isolation from natural sources. The present work aims to summarize information on the applications of phytosterols and to review recent approaches, mainly from the industry, for the large-scale recovery of phytosterols.     

2-Styrylchromones: novel strong scavengers of reactive oxygen and nitrogen species

2-Styrylchromones are a small group of naturally occurring chromones, vinylogues of flavones (2-phenylchromones). Natural and synthetic 2-styrylchromones have been tested in different biological systems, showing activities with potential therapeutic applications. In particular, the potential and hitherto understudied antioxidant behavior of these compounds has been raised as a matter of interest. Thus the present work consisted in the study of the in vitro scavenging activities for reactive oxygen species (ROS) and reactive nitrogen species (RNS) of various 2-styrylchromone derivatives and structurally similar flavonoids. Some of the studied 2-styrylchromones proved to be extremely efficient scavengers of the different ROS and RNS, showing, in some cases, IC(50)s under 1 microM. The hydroxylation pattern of 2-styrylchromones, especially in the B-ring but also in the A ring, modulates the activity of these compounds, the catecholic derivatives being the most effective scavengers. The styryl pattern also contributes to their observed outstanding antioxidant activity. In conclusion, the scavenging activities for ROS/RNS of 2-styrylchromone derivatives, here shown for the first time, provide novel and most promising compounds to be applied as antioxidants.     

Brazilian green propolis on Helicobacter pylori infection. a pilot clinical study

Recent in vitro studies suggest that propolis and some of its phenolic components are able to inhibit Helicobacter pylori growth. To date, there are no clinical studies. AIMS: To evaluate the effect of Brazilian green propolis on H. pylori-infected individuals. PATIENTS AND METHODS: Eighteen (11 females, 7 males, mean age 47 years) participants were included. Before treatment, all participants were submitted to gastroscopy, and H. pylori infection was confirmed by histology, urease test, and (13)C-urea breath test (UBT). Participants with UBT showing a delta over baseline (DOB) value higher than 4 per thousand were considered positive for H. pylori infection. Twenty drops from an alcoholic preparation of Brazilian green propolis were administered three times a day for 7 days. Clinical evaluation and UBT were performed at 1-3 days and at 40 days after the end of therapy to evaluate H. pylori suppression or eradication, respectively. RESULTS: All participants took all medication and completed the study. Eighty-three percent of the subjects did not succeed in suppressing or eradicating H. pylori. Two participants reached partial suppression after treatment, but became positive again at UBT performed 40 days after treatment. Another participant presented negative at UBT 40 days after treatment, not confirmed by a second UBT performed 100 days after treatment. CONCLUSIONS: Brazilian green propolis used in popular dose showed minimal effect on H. pylori infection. Larger studies with longer duration, larger dose, and different frequency of administration of propolis extract should be undertaken to define its role on H. pylori therapy.     

Cutaneous and meningeal sporotrichosis in a HIV patient

A male patient with HIV and past history of tuberculosis and suspected neurotoxoplasmosis was admitted to the hospital with vomiting and small nodules through all his body. Few of the nodules were found forming chains of enlarged lymphatic vessels, especially on lesions located on the limbs. Some of the nodules were ulcerated with a serosanguineous discharge. Collected samples from ulcerated and the nodular lesions showed the presence of Sporothrix schenckii in culture. Although all hemocultures were negative, a spinal fluid collected from this patient and cultures from the cutaneous lesions were both positive for S. schenckii. The patient showed improvement after treatment with Amphotericin B. Sadly, he later died of complications not related to the S. schenckii infection. This case of disseminated sporotrichosis is a remainder that in patients with immunological disorders exotic forms of this fungal clinical entity could be expected.