Nutraceutical potential of Asparagopsis taxiformis (Delile) Trevisan extracts and assessment of a downstream purification strategy.

The main goal of the present work was to determine the nutraceutical potential of Asparagopsis taxiformis D. extracts from Madeira Archipelago south coast. Extraction methodologies consisted either/or in 72 hours stirring, at room temperature (M1), or 6 cycles of Soxhlet extraction (M2), both with re-extraction. Solvents used were distilled water, ethanol, methanol and ethyl acetate. M1 allowed to obtain the highest values for extraction yield (31.65 g.100g-1 dw) using water, whereas iodine content (3.37 g.100g-1 dw), TPC (1.71 g GAE.100g-1 dw) and chlorophyll a (45.96 mg.100g-1 dw) were obtained using ethanol, and TCC (36.23 mg.100g-1 dw) with methanol. Extracts that showed higher reduction activity in M1 were derived from ethanol extraction (1,908 mg AAE.100g-1 dw). Water and ethanol were the best solvents for higher DPPH scavenging activity in M2, both with same result (IC50 1.37 mg.mL-1). The lowest value of IC50 for chelating activity (1.57 mg.mL-1) was determined in M1, using ethyl acetate. The remaining residue was used to obtain other products, i.e. lipid extraction (M1, 2.05 g.100g-1 dw), carrageenans (M2, 21.18 g.100g-1 dw) and cellulose (M1, 23.81 g.100g-1 dw) with subsequent FTIR ATR analysis. Our results show that A. taxiformis is a valuable source of bioactive compounds. The M1 extraction methodology using ethanol is the most effective solvent to produce an iodine rich bioactive extract with potential of being used as a nutraceutical supplement. Also, we have demonstrated a possible downstream strategy that could be implemented for multiple compound extraction from A. taxiformis residue. This has a vital importance for future feasibility, when using this biomass as an industrial feedstock for multiple products production. Statistical analysis, using SPSS 24.0, was also performed and important correlations were found between assays and methods.

Modulation of the prostaglandin-endoperoxide synthase 2 gene expression by variant haplotypes: influence of the 3'-untranslated region

The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3′-untranslated region (3′-UTR). Different PR (rs689465, rs689466, rs20417) and 3′-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3′-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3′-UTR) were compared to respective parental PR sequences. The contribution of 3′-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3′-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).

Modulation of the prostaglandin-endoperoxide synthase 2 gene expression by variant haplotypes: influence of the 3'-untranslated region.

The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3'-untranslated region (3'-UTR). Different PR (rs689465, rs689466, rs20417) and 3'-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3'-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3'-UTR) were compared to respective parental PR sequences. The contribution of 3'-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3'-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).

Phenotypic and genotypic characterization of clinically relevant bacteria isolated from dental waste and waste workers' hands, mucosas and coats.

Infectious wastes are potential sources of pathogenic micro-organisms, which may represent a risk to the professionals who manage them. In this study, we aimed to characterize the infectious bacteria present in dental waste and waste workers. The dental waste produced over 24 h was collected and waste workers were sampled by swabbing. Isolate resistance profiles were characterized by Vitek® and PCR and biofilm formation by Congo Red agar, string test and microtitre assay. To assess similarity between the waste and the workers' samples, a random amplified polymorphic DNA test was used. Twenty-eight bacteria were identified as clinically relevant. The most frequent gene was blaTEM present in five Gram-negative micro-organisms, and one blaSHV in Klebsiella pneumoniae. All Pseudomonas aeruginosa were positive to extracellular polymeric substances formation, except one isolated from a worker. Klebsiella pneumoniae had negative results for the string test. Pseudomonas aeruginosa showed better adherence at 25°C after 48 h of incubation and K. pneumonia had the best biofilm formation at the same temperature, after 24 h. The similarity between P. aeruginosa recovered from dental waste and from workers was low, however, it is important to note that a pathogen was found on a worker's hands and that improvements in biosafety are required. SIGNIFICANCE AND IMPACT OF THE STUDY: Infectious dental waste can contain clinically relevant bacteria with important resistance and biofilm profiles. These micro-organisms could be transmitted to waste workers, other professionals and patients if the principles of biosafety measures are neglected. To our knowledge, no study has ever evaluated the microbial characterization and the potential contamination risk of dental infectious waste and waste handlers. The presence of clinically relevant bacteria in the hands and nasal mucosa of waste workers highlights the need for studies in this field to clarify the risk of these pathogens in dental healthcare services, and to stress the need for an efficient waste management.

Occurrence of microsporidians Glugea hertwigi and Pleistophora ladogensis, in smelt Osmerus eperlanus from two German rivers, North Sea coast.

Monthly samples of smelt Osmerus eperlanus (Linnaeus, 1758) were collected from July 1985 to May 1986, in the river Elbe (Germany), and examined for infections with microsporidians. Two microsporidians were found: Glugea hertwigi Weissenberg, 1911, infecting the digestive tract and Pleistophora ladogenis Voronin, 1978, infecting the skeletal musculature. G. hertwigi infection led to the formation of xenomas, whereas P. ladogensis was characterized by diffuse infections, with the production of macroscopic visible thread-like or oval-shaped infection foci. Development of G. hertwigi in the host cells showed characteristics typical of the genus Glugea. The ultrastructural development of P. ladogensis showed features typical of the genus Pleistophora, without evidence of the production of 2 types of spores. Host reaction consisted of inflammatory tissue surrounding some of the infection foci as well as phagocytosis of spores. G. hertwigi was only found in juvenile smelt (<10 cm in length), whereas P. ladogensis infected smelts from 6 to 26 cm in length. Prevalence increased with fish length to a maximum value of 9.6%. Seasonal fluctuations in prevalence of infection were also found, with the lowest value in the winter months (2.5% in January 1986) and the highest in summer (11.8% in July 1985). The differences in prevalence of infection with fish length and date of sampling were significant. Additionally, samples of smelt caught in April 1986 from the rivers Eider and Ems revealed infections with P. ladogensis in the first river system only.

Genetic Diversification and Dispersal of Taro (Colocasia esculenta (L.) Schott).

Taro (Colocasia esculenta (L.) Schott) is widely distributed in tropical and sub-tropical areas. However, its origin, diversification and dispersal remain unclear. While taro genetic diversity has been documented at the country and regional levels in Asia and the Pacific, few reports are available from Americas and Africa where it has been introduced through human migrations. We used eleven microsatellite markers to investigate the diversity and diversification of taro accessions from nineteen countries in Asia, the Pacific, Africa and America. The highest genetic diversity and number of private alleles were observed in Asian accessions, mainly from India. While taro has been diversified in Asia and the Pacific mostly via sexual reproduction, clonal reproduction with mutation appeared predominant in African and American countries investigated. Bayesian clustering revealed a first genetic group of diploids from the Asia-Pacific region and to a second diploid-triploid group mainly from India. Admixed cultivars between the two genetic pools were also found. In West Africa, most cultivars were found to have originated from India. Only one multi-locus lineage was assigned to the Asian pool, while cultivars in Madagascar originated from India and Indonesia. The South African cultivars shared lineages with Japan. The Caribbean Islands cultivars were found to have originated from the Pacific, while in Costa Rica they were from India or admixed between Indian and Asian groups. Taro dispersal in the different areas of Africa and America is thus discussed in the light of available records of voyages and settlements.

Pregnancy in Hystricomorpha: gestational age and embryonic-fetal development of agouti (Dasyprocta prymnolopha, Wagler 1831) estimated by ultrasonography.

Thirty-one pregnant agoutis, between Days 9 and 103 of gestation (Day 1 = day of detection of sperm in the vaginal smear), underwent B-mode ultrasonography; gestational sac diameter (GSD), crown-rump length (CRL), embryonic-fetal diameter (EFD), and placenta diameter (PD) were measured. There were positive correlations (P < 0.05) between GSD and CRL (r = 0.98), GSD and PD (r = 0.88), CRL and PD (r = 0.86), days of gestation (DG) and CRL (r = 0.85), and DG and PD (r = 0.73). The gestational sac was first observed on Day 14. The embryo was first seen on Day 18 in 9/31 of pregnant agoutis and on Day 22 in 20/31 of pregnant agoutis. Heartbeats were detected from the Day 25 and placentas were observed in 100% of the animals from Day 25. Early limb bud and ossification of the fetal skull were identified on Days 27 (15/31) and 45 (24/31), respectively. Fetal orientation (head and body) was evident from Day 40, the stomach, liver and lungs were identified on Day 50, the kidneys were reliably seen only on Day 55, and the aorta and vena cava were seen on Day 70. The fetal bowel and the urinary bladder were the last structures to be observed (Day 85). Ultrasonography was effective for early pregnancy diagnosis in agouti and for obtaining information on embryonic and fetal structures that could be used to predict gestational age and birth, thereby contributing to their reproductive management in captivity.

Helminth parasites of the oceanic horse mackerel Trachurus picturatus Bowdich 1825 (Pisces: Carangidae) from Madeira Island, Atlantic Ocean, Portugal.

The helminth parasite fauna of the oceanic horse mackerel Trachurus picturatus Bowdich 1825, caught off the Madeira Islands was composed of six different taxa. Prevalence and abundance of larval Anisakis sp. (Nematoda: Anisakidae) and Nybelinia lingualis (Trypanorhyncha: Tentaculariidae), the most common parasite taxa, were 24.3%, 0.9 and 37.9%, 0.7, respectively. Bolbosoma vasculosum (Acanthocephala: Polymorphidae) and the monogeneans Heteraxinoides atlanticus (Monogenea: Heteraxinidae) and Pseudaxine trachuri (Monogenea: Gastrocotylidae) were comparatively rare. The depauperate helminth fauna of the oceanic horse mackerel at Madeira compared to other geographical regions of the north-eastern Atlantic, namely the Azores banks and the West African coast, may be attributed to the paucity of nutrients off oceanic islands and to a low density of the fish population.

In vitro selection of ertapenem and piperacillin/tazobactam-resistant strains of Bacteroides fragilis and analysis of their virulence in gnotobiotic mice.

Ertapenem and piperacillin/tazobactam are beta-lactam antibiotics with a broad spectrum of activity, used for the treatment of mixed infections, in which Bacteroides fragilis plays an important etiological role. The aim of this study was to select strains of B. fragilis resistant to these drugs and correlate the phenotype profiles of these lineages with changes in the virulence of the original bacterium. B. fragilis ATCC 25285, sensitive to the drugs listed, was used in this study. Strains resistant to these drugs were obtained by multi-step method and this condition was confirmed by comparing the time-kill curve of the original strain with those curves obtained from derived-resistant strains. To assess the virulence, germ-free mice were challenged intragastrically with the original strain or those derived-resistant. The mouse infection by the piperacillin/tazobactam-resistant B. fragilis strain produced increased levels of C-reactive protein, alkaline phosphatase and white blood cells and reduced platelet counts, what may indicate that acquisition of piperacillin/tazobactam resistance may enhance the pathogenic properties of these B. fragilis strains.

Isolation of clinically relevant fungal species from solid waste and environment of dental health services.

AIMS: This study was undertaken to detect, identify and determine antifungal susceptibility of yeast strains isolated from dental solid waste and to evaluate airborne fungi in the Brazilian dental health care environment and in the waste storage room. METHODS AND RESULTS: A group of 17 yeast strains were identified by macroscopic and microscopic characteristics, API 20C Aux system and Multiplex PCR. All 104 airborne fungal colonies were identified by macroscopic and microscopic morphology. The CLSI broth microdilution method was utilized as the susceptibility test. Candida parapsilosis was the prevailing yeast species recovered from waste, followed by Rhodotorula glutinis. Three strains of Candida guilliermondii presented minimal inhibitory concentration values considered to be susceptible dose dependent (2 µg ml(-1)) to voriconazole. Of all airborne fungal species, 69% were recovered from the waste storage room and 31% were recovered from the clinical/surgical environment. Most of them were identified as Cladosporium spp. CONCLUSIONS: These findings reinforce the potential risk of waste handling and point out the need for safe management to minimize the spread of these agents to the environment. Filamentous fungi isolation in almost all sampled environments indicates that a periodic monitoring of airborne microbiota in the dental health care service environment is required. SIGNIFICANCE AND IMPACT OF THE STUDY: The survival of yeast strains for 48 h suggests that dental waste should be carefully controlled and monitored.

LyeTx I, a potent antimicrobial peptide from the venom of the spider Lycosa erythrognatha.

LyeTx I, an antimicrobial peptide isolated from the venom of Lycosa erythrognatha, known as wolf spider, has been synthesised and its structural profile studied by using the CD and NMR techniques. LyeTx I has shown to be active against bacteria (Escherichia coli and Staphylococcus aureus) and fungi (Candida krusei and Cryptococcus neoformans) and able to alter the permeabilisation of L: -alpha-phosphatidylcholine-liposomes (POPC) in a dose-dependent manner. In POPC containing cholesterol or ergosterol, permeabilisation has either decreased about five times or remained unchanged, respectively. These results, along with the observed low haemolytic activity, indicated that antimicrobial membranes, rather than vertebrate membranes seem to be the preferential targets. However, the complexity of biological membranes compared to liposomes must be taken in account. Besides, other membrane components, such as proteins and even specific lipids, cannot be discarded to be important to the preferential action of the LyeTx I to the tested microorganisms. The secondary structure of LyeTx I shows a small random-coil region at the N-terminus followed by an alpha-helix that reached the amidated C-terminus, which might favour the peptide-membrane interaction. The high activity against bacteria together with the moderate activity against fungi and the low haemolytic activity have indicated LyeTx I as a good prototype for developing new antibiotic peptides.

Esophageal manometry in 28 systemic sclerosis Brazilian patients: findings and correlations.

Systemic sclerosis (SSc) is a multisystem disease of unknown etiology. Esophageal involvement affects 50-90% of patients and is characterized by abnormal motility and hypotonic lower esophageal sphincter. Data on the association of esophageal abnormalities and age, gender, SSc subset or duration, autoantibody profile, esophageal symptoms, and medication are lacking or conflicting. The aim of this study was the evaluation of these associations in Brazilian sclerodermic patients from the Rheumatology Division, Clinics Hospital, Federal University, Minas Gerais. They underwent medical records review, clinical interview, and esophageal manometry. The normal cutoff level for lower esophageal sphincter pressure was 14 mmHg. Abnormal peristalsis occurred when less than 80% of peristaltic waves were propagated. P-values less than 0.05 were considered significant. Twenty-eight patients were included: 71% were women. The population presented medium age and disease duration of 46 years and 12 years, respectively. Cutaneous diffuse SSc occurred in 39% and its limited form in 61%. Dysphagia, pyrosis, and regurgitation occurred, respectively, in 71%, 43%, and 61% of patients. Lower esophageal sphincter pressure and number of peristaltic waves-propagated medias were, respectively, 17.2 mmHg and 2.3. SSc-related manometric abnormalities were present in 86% of patients. Manometry revealed distal esophageal body hypomotility, hypotonic lower esophageal sphincter, or both, respectively, in 82%, 39%, and 36% of patients. One patient presented the manometric pattern of esophageal achalasia. Male patients more frequently presented hypotonic inferior esophageal sphincter. Manometric findings have had no relationship with the other variables. Nifedipine use did not influence manometric findings.

Purification and partial characterization of a bacteriocin produced by an oral Fusobacterium nucleatum isolate.

AIMS: To purify and partially characterize a bacteriocin produced by a Fusobacterium nucleatum strain. METHODS AND RESULTS: Following protein precipitation the effect of different treatments on a bacteriocin produced by a F. nucleatum strain named P12.2 isolated from a patient with periodontitis was evaluated. The antagonistic activity of the intracellular fraction obtained at 80% ammonium sulphate was preserved at pH values from 6.0 to 9.0 and showed to be sensitive to high temperatures and to treatment with proteases. The fraction was submitted to sequential steps of gel filtration, ion exchange, and reverse phase chromatography, and SDS-PAGE. Data obtained by mass spectrometry revealed that the molecular mass of the protein was 27,296 Da. CONCLUSIONS: For the first time a bacteriocin produced by a F. nucleatum strain was purified and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description on characterization of a bacteriocin produced by F. nucleatum. It is possible that the bacteriocin plays a role in the regulation of population levels of periodontopathic organisms.

Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom.

Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0-0.5M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H(2)O(2) production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l-leucine. The correlation between in vivo neutralization of lethal toxicity (ED(50)) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED(50) and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 (p<0.001) and 0.747 (p<0.001), respectively. The hemolytic and leishmanicidal (anti-Leishmania amazonensis) activity of LAAO was also determined.

Arterial supply of the penis in agoutis (Dasyprocta prymnolpha, Wagler, 1831).

The arterial vascularization of agoutis' penis (Dasyprocta prymnolopha) were analysed using ten male adults from 'Núcleo de Estudos e Preservação de Animais Silvestres da Universidade Federal do Piauí' (FUFPI/IBAMA n degrees 02/99). Among the total number of specimens, six animals had natural death and were members of the research collection of the Laboratory of Anatomy, and four were killed after anaesthesia. Stained bi-centrifugated-Cis-I-4 latex was injected in arterial vessels responsible for penis vascularization throughout the abdominal portion of aorta. The samples were fixed in 10% formaldehyde solution and arteries were dissected. The penile artery is originated as a branch of internal pudendal artery. At the level of ischiatic arch, the penile artery project two branches, the penile dorsal and the deep arteries; those arteries irrigates the penile dorsal surface and the corpus cavernosum penis. The penile dorsal arteries have an independent course up to the glans penis. Based on the conditions of this work a remarkable similarity regarding the distribution of vessels destined to the agouti penis when compared to other domestic, wild and lagomorph rodents as rabbits.

The influence of molecular oxygen exposure on the biology of Prevotella intermedia, with emphasis on its antibiotic susceptibility.

AIM: This study focuses on investigating the molecular and physiological characteristics of Prevotella intermedia after molecular oxygen exposure (MOE) and the effect on drug susceptibility patterns. METHODS AND RESULTS: Samples of P. intermedia were used as parent strains: ATCC25611 and four clinical isolates. Strains adapted to oxidative stress by MOS were obtained by the enrichment technique. Drug susceptibility was evaluated by minimal inhibitory concentrations (MIC) using agar dilution. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to evaluate the genetic diversity of all strains and physiological analyses were made by sodiumdodecylsulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis of crude, cell-free extracts. The genetic profile showed that lineages with altered MIC values were selected after MOE. Overall, we found significant decrease in drug susceptibility for the aero-strains against all tested antimicrobials (amoxicillin, amoxicillin+clavulanic acid, clindamycin, chloramphenicol, ertapenen and metronidazole). We also observed markedly different protein expression patterns between the parent and selected aero-strains. CONCLUSIONS: MOE induces changes in the genetic profile and protein expression patterns of P. intermedia that may also be linked to its drug resistance mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The effects of MOE on anaerobic bacterial physiology and behaviour may influence antimicrobial susceptibility patterns with potential consequences to antimicrobial chemotherapy.

Physiological alterations of a Fusobacterium nucleatum strain exposed to oxidative stress.

AIM: The purpose of this study was to investigate the effect of oxidative stress on physiological and genetic characteristics of Fusobacterium nucleatum and its interference on this microbial identification methods. METHODS AND RESULTS: Fus. nucleatum ssp. nucleatum ATCC 25586 (wt-strain) and an oxidative-stress-adapted strain derived from the wt-strain (aero-strain) were employed in the study. Cell-free crude protein extracts were obtained from both strains and differentially expressed proteins were identified by two-dimensional electrophoresis. Bacterium identification was performed by conventional biochemical tests, automated Rapid ID 32A system and specific PCR analysis. Genetic diversity between wt- and aero-strain was assessed by arbitrarily-primed (AP)-PCR. There were significant changes in the protein profile of aero-strain. The identification of the wt-strain was confirmed by all methods employed. Similar results were obtained for aero-strain when conventional biochemical tests and PCR were used. However, aero-strain was identified as Fusobacterium varium when submitted to Rapid ID 32A system. According to AP-PCR analysis, no significant genetic alteration was detected in aero-strain. CONCLUSIONS: The adaptive response of Fus. nucleatum to oxidative stress is associated with changes on its biology, which may lead to misidentification of the organism, according to the conventional identification methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxidative stress may act as a cause of adaptive response in Fus. nucleatum with consequences to its biology, such as alterations on biochemical and physiological profile.

Bone mineral density in familial amyloid polyneuropathy and in other neuromuscular disorders.

Neuromuscular diseases are a known risk factor for immobilization-induced osteoporosis. The aim of the study was to analyse bone mineral density (BMD) in patients with familial amyloid polyneuropathy (FAP) type I (Val30 Met) and to compare them with a population of patients with other neuromuscular disorders. We studied 24, ambulatory, neuromuscular patients, all men and premenopausal women. We included 12 FAP patients (GI) and 12 patients with other disorders (GII). Clinical data included age, sex, height, weight, alcohol intake, smoking, calcium intake, physical activity and history of fractures. Serum and urinary calcium, osteocalcin, bone alkaline phosphatase, parathyroid hormone, thyroid stimulating hormone and urinary N-telopeptide cross-linked type 1 collagen were determined in all patients. Bone mineral density of lumbar spine, hip and wrist were determined by dual energy X-ray absorptiometry scan. No statistical differences were found in clinical or analytic data between the two groups, except for body mass index and calciuria, which were lower in GI. In GI, 54.5% were osteoporotic, against 23.1% in GII (P = 0.04). Bone mineral density was lower in GI when compared with GII, and tended to decrease with disease duration. Decreased BMI and the early autonomic involvement in GI probably explain the results. The prevention and early treatment of osteoporosis, in FAP patients should be considered a priority.

[Effect of gamma-aminobutyric shunt metabolites on NAD-, NADP-isocitrate dehydrogenases, and aconitate hydratase activities in higher plants]. / Vliianie metabolitov shunta gamma-aminomaslianoi kisloty na aktivnost' NAD- i NADF-isotsitratdegidrogenaz i akonitatgidratazy vysshikh rastenii.

We studied the influence of metabolites of gamma-aminobutyric shunt of the tricarboxylic acid cycle on the activities of aconitate hydratase (EC.4.2.1.3) as well as NAD- and NADP-specific isocitrate dehydrogenases (EC.1.1.1.41 and EC.1.1.1.42, respectively) using purified enzyme preparations from pea leaves (Pisum sativum L.) and maize scutellum (Zea mays L.). gamma-Aminobutyric acid and succinate proved to have no significant effect on these enzymes, while 0.1-0.2 mM glutamate considerably activated NADP isocitrate dehydrogenase from the both sources, particularly, at unsaturating concentration of the substrate. Succinic semialdehyde stimulated the activities of aconitate hydratase and NADP isocitrate dehydrogenase. The obtained data points to a similar pattern of the effect of intermediates of gamma-aminobutyric shunt on the studied enzymatic activities for both photosynthetic tissues (pea leaves) and those with acidifying, transport, and digestive functions (maize scutellum). However, the absence of pronounced control effects of most metabolites on the studied enzymes allows us to assign them to a relatively inert pool of metabolites.

Cytokine production in response to endodontic infection in germ-free mice.

This study evaluated the cytokine profiles (type 1 or type 2) that are triggered by and modulate endodontic periapical infections in the root canal system of germ-free mice. Microorganisms isolated from two patients with pulpal necrosis were inoculated into two groups of experimental animals: group I (Gemella morbillorum) and group II (Bifidobacterium adolescentis, Fusobacterium nucleatum and Clostridium butyricum). In vitro, G. morbillorum induced type 1 cytokine synthesis, while the modulation processed in vivo seemed to have the opposite effect, with a reduction in the basal levels of IL-12 and IFN-gamma, IL-4-independent down-modulation. In vitro, microorganisms from group II, in poly-infection, induced a reduction of type 1 cytokine levels from day 10 to day 20, which seemed to be modulated via IL-4. In vivo, however, a predominance of the immune response to one species over the others occurred.