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1.  Population Pharmacokinetic Modeling of the Unbound Levofloxacin Concentrations in Rat Plasma and Prostate Tissue Measured by Microdialysis 
Levofloxacin is a broad-spectrum fluoroquinolone used in the treatment of both acute and chronic bacterial prostatitis. Currently, the treatment of bacterial prostatitis is still difficult, especially due to the poor distribution of many antimicrobials into the prostate, thus preventing the drug to reach effective interstitial concentrations at the infection site. Newer fluoroquinolones show a greater penetration into the prostate. In the present study, we compared the unbound levofloxacin prostate concentrations measured by microdialysis to those in plasma after a 7-mg/kg intravenous bolus dose to Wistar rats. Plasma and dialysate samples were analyzed using a validated high-pressure liquid chromatography-fluorescence method. Both noncompartmental analysis (NCA) and population-based compartmental modeling (NONMEM 6) were performed. Unbound prostate tissue concentrations represented 78% of unbound plasma levels over a period of 12 h by comparing the extent of exposure (unbound AUC0–∞) of 6.4 and 4.8 h·μg/ml in plasma and tissue, respectively. A three-compartment model with simultaneous passive diffusion and saturable distribution kinetics from the prostate to the central compartment gave the best results in terms of curve fitting, precision of parameter estimates, and model stability. The following parameter values were estimated by the population model: V1 (0.38 liter; where V1 represents the volume of the central compartment), CL (0.22 liter/h), k12 (2.27 h−1), k21 (1.44 h−1), k13 (0.69 h−1), Vmax (7.19 μg/h), kM (0.35 μg/ml), V3/fuprostate (0.05 liter; where fuprostate represents the fraction unbound in the prostate), and k31 (3.67 h−1). The interindividual variability values for V1, CL, Vmax, and kM were 21, 37, 42, and 76%, respectively. Our results suggest that levofloxacin is likely to be substrate for efflux transporters in the prostate.
PMCID: PMC3910848  PMID: 24217697
2.  Improvement in metabolic parameters in obese subjects after 16 weeks on a Brazilian-staple calorie-restricted diet 
Nutrition Research and Practice  2014;8(4):410-416.
The standard pattern of Brazilian food consumption is based on the combination of rice and beans served together in the main meals. This study assessed the effects of Brazilian-staple calorie-restricted (BS-diet) dietary advice, with brown rice and beans, on metabolic parameters, body composition, and food intake in overweight/obese subjects.
Twentyseven subjects were randomly assigned to a conventional-type calorie-restricted diet (CT-diet) (n = 13) or a BS-diet (n = 14). Glucose metabolism, lipid profile, anthropometric and body composition parameters, and food intake were measured before and after 16 weeks. Paired t-tests/Wilcoxon tests were used for comparison of differences from baseline and unpaired t-tests/Mann-Whitney tests were used for comparison of differences between the groups.
After 16 weeks, both groups showed reductions in weight and waist circumference (P < 0.02), and the BS-diet group showed a decrease in body fat (P = 0.0001), and significant improvement in glucose metabolism (fasting plasma glucose, glucose and insulin areas under the curve, Cederholm index, and HOMA2-%β) (P ≤ 0.04) and lipid profile (cholesterol, triacylglycerol, LDL-c, VLDL-c, and cholesterol/HDL-c ratio) (P ≤ 0.05). In addition, the BS-diet group showed significant improvement in HOMA2-%β, compared to the CT-diet group (P = 0.03). The BS-diet group also showed a significant reduction in energy, lipids, carbohydrate, and cholesterol intake (P ≤ 0.04) and an increase in fiber intake (P ≤ 0.001), while the CT-diet group showed a significant reduction in intake of energy, macronutrients, PUFA, and cholesterol (P ≤ 0.002).
These results demonstrate the benefits of the BS-diet on metabolic parameters in obese subjects.
PMCID: PMC4122713  PMID: 25110561
Glycemic index; obesity; brown rice; beans
3.  Comparison of Fluconazole Renal Penetration Levels in Healthy and Candida albicans-Infected Wistar Rats 
Antimicrobial Agents and Chemotherapy  2012;56(11):5852-5857.
The aims of this study were to evaluate free levels of fluconazole (FCZ) in the kidneys of healthy and Candida albicans-infected Wistar rats using microdialysis and to establish the relationship between free renal and total plasma levels under both conditions. Microdialysis recovery rates were determined in vitro by dialysis, and retrodialysis recovery rates were determined in vivo by retrodialysis. The recovery rate was around 50%, independent of the method, drug concentration, or condition (in vitro or in vivo) used. FCZ kidney penetration in healthy and infected rats was investigated after the administration of 10 mg/kg of body weight intravenously (i.v.) or 50 mg/kg orally (n = 6/group) and blood and microdialysate sample harvesting at predetermined time points up to 24 and 18 h, respectively. There were no statistical differences between the area under the free concentration-time curve (AUC0–∞) values in plasma and in tissue for either healthy or infected groups for the same dose regimen investigated. The antifungal tissue penetrations were similar for both doses and under all conditions investigated (ranging from 0.77 to 0.84). The unbound fraction of FCZ was concentration independent (86.0% ± 2.0%), allowing the prediction of free renal levels using pharmacokinetic parameters obtained from total plasma fitting. The results showed that free renal and free plasma levels are similar in healthy and systemically C. albicans-infected rats. Therefore, free plasma levels are a good surrogate to estimate free FCZ renal concentrations in systemic candidiasis and can be used to optimize dosing regimens for this drug.
PMCID: PMC3486536  PMID: 22948869
4.  Osmolality-Dependent Relocation of Penicillin-Binding Protein PBP2 to the Division Site in Caulobacter crescentus 
Journal of Bacteriology  2012;194(12):3116-3127.
The synthesis of the peptidoglycan cell wall is carefully regulated in time and space. In nature, this essential process occurs in cells that live in fluctuating environments. Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. The penicillin-binding protein PBP2, which is commonly branded as an essential cell elongation-specific transpeptidase, switches its localization from a dispersed, patchy pattern to an accumulation at the FtsZ ring location in response to osmotic upshifts as low as 40 mosmol/kg. This osmolality-dependent relocation to the division apparatus is initiated within less than a minute, while restoration to the patchy localization pattern is dependent on cell growth and takes 1 to 2 generations. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Consistent with its ecological distribution, C. crescentus displays a narrow range of osmotolerance, with an upper limit of 225 mosmol/kg in minimal medium. Collectively, our findings reveal an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation.
PMCID: PMC3370875  PMID: 22505677
5.  Association of Moderate Coffee Intake with Self-Reported Diabetes among Urban Brazilians 
Coffee has been associated with reductions in the risk of non-communicable chronic diseases (NCCD), including diabetes mellitus. Because differences in food habits are recognizable modifying factors in the epidemiology of diabetes, we studied the association of coffee consumption with type-2 diabetes in a sample of the adult population of the Federal District, Brazil. This cross-sectional study was conducted by telephone interview (n = 1,440). A multivariate analysis was run controlling for socio-behavioural variables, obesity and family antecedents of NCCD. A hierarchical linear regression model and a Poisson regression were used to verify association of type-2 diabetes and coffee intake. The independent variables which remained in the final model, following the hierarchical inclusion levels, were: first level—age and marital status; second level—diabetes and dyslipidaemias in antecedents; third level—cigarette smoking, supplement intake, body mass index; and fourth level—coffee intake (≤100 mL/d, 101 to 400 mL/day, and >400 mL/day). After adjusting hierarchically for the confounding variables, consumers of 100 to 400 mL of coffee/day had a 2.7% higher (p = 0.04) prevalence of not having diabetes than those who drank less than 100 mL of coffee/day. Compared to coffee intake of ≤100 mL/day, adults consuming >400 mL of coffee/day showed no statistically significant difference in the prevalence of diabetes. Thus, moderate coffee intake is favourably associated with self-reported type-2 diabetes in the studied population. This is the first study to show a relationship between coffee drinking and diabetes in a Brazilian population.
PMCID: PMC3166738  PMID: 21909302
coffee intake; diabetes mellitus; chlorogenic acids; caffeine; body mass index
6.  A 1.5 million–base pair inversion polymorphism in families with Williams-Beuren syndrome 
Nature genetics  2001;29(3):321-325.
Williams-Beuren syndrome (WBS) is most often caused by hemizygous deletion of a 1.5-Mb interval encompassing at least 17 genes at 7q11.23 (refs. 1, 2). As with many other haploinsufficiency diseases, the mechanism underlying the WBS deletion is thought to be unequal meiotic recombination, probably mediated by the highly homologous DNA that flanks the commonly deleted region3. Here, we report the use of interphase fluorescence in situ hybridization (FISH) and pulsed-field gel electrophoresis (PFGE) to identify a genomic polymorphism in families with WBS, consisting of an inversion of the WBS region. We have observed that the inversion is hemizygous in 3 of 11 (27%) atypical affected individuals who show a subset of the WBS phenotypic spectrum but do not carry the typical WBS microdeletion. Two of these individuals also have a parent who carries the inversion. In addition, in 4 of 12 (33%) families with a proband carrying the WBS deletion, we observed the inversion exclusively in the parent transmitting the disease-related chromosome. These results suggest the presence of a newly identified genomic variant within the population that may be associated with the disease. It may result in predisposition to primarily WBS-causing microdeletions, but may also cause translocations and inversions.
PMCID: PMC2889916  PMID: 11685205 CAMSID: cams401
8.  Osteopoikilosis, short stature and mental retardation as key features of a new microdeletion syndrome on 12q14 
Journal of Medical Genetics  2007;44(4):264-268.
This report presents the detection of a heterozygous deletion at chromosome 12q14 in three unrelated patients with a similar phenotype consisting of mild mental retardation, failure to thrive in infancy, proportionate short stature and osteopoikilosis as the most characteristic features. In each case, this interstitial deletion was found using molecular karyotyping. The deletion occurred as a de novo event and varied between 3.44 and 6 megabases (Mb) in size with a 3.44 Mb common deleted region. The deleted interval was not flanked by low‐copy repeats or segmental duplications. It contains 13 RefSeq genes, including LEMD3, which was previously shown to be the causal gene for osteopoikilosis. The observation of osteopoikilosis lesions should facilitate recognition of this new microdeletion syndrome among children with failure to thrive, short stature and learning disabilities.
PMCID: PMC2598049  PMID: 17220210
osteopoikilosis; short stature; mental retardation;  HMGA2 ;  GRIP1
9.  Localization of PBP3 in Caulobacter crescentus is highly dynamic and largely relies on its functional transpeptidase domain 
Molecular microbiology  2008;70(3):634-651.
In rod-shaped bacteria, septal peptidoglycan synthesis involves the late recruitment of the ftsI gene product (PBP3 in Escherichia coli) to the FtsZ ring. We show that in Caulobacter crescentus, PBP3 accumulates at the new pole at the beginning of the cell cycle. Fluorescence recovery after photobleaching experiments reveal that polar PBP3 molecules are, constantly and independently of FtsZ, replaced by those present in the cellular pool, implying that polar PBP3 is not a remnant of the previous division. By the time cell constriction is initiated, all PBP3 polar accumulation has disappeared in favor of an FtsZ-dependent localization near midcell, consistent with PBP3 function in cell division. Kymograph analysis of time-lapse experiments shows that the recruitment of PBP3 to the FtsZ ring is progressive and initiated very early on, shortly after FtsZ ring formation and well before cell constriction starts. Accumulation of PBP3 near midcell is also highly dynamic with a rapid exchange of PBP3 molecules between midcell and cellular pools. Localization of PBP3 at both midcell and pole appears multifactorial, primarily requiring the catalytic site of PBP3. Collectively, our results suggest a role for PBP3 in pole morphogenesis and provide new insights into the process of peptidoglycan assembly during division.
PMCID: PMC2581642  PMID: 18786147
Bacterial cell wall; peptidoglycan; ftsI; FRAP; cell division
10.  Submicroscopic deletions of 11q24-25 in individuals without Jacobsen syndrome: re-examination of the critical region by high-resolution array-CGH 
Jacobsen syndrome is a rare contiguous gene disorder that results from a terminal deletion of the long arm of chromosome 11. It is typically characterized by intellectual disability, a variety of physical anomalies and a distinctive facial appearance. The 11q deletion has traditionally been identified by routine chromosome analysis. Array-based comparative genomic hybridization (array-CGH) has offered new opportunities to identify and refine chromosomal abnormalities in regions known to be associated with clinical syndromes.
Using the 1 Mb BAC array (Spectral Genomics), we screened 70 chromosomally normal children with idiopathic intellectual disability (ID) and congenital abnormalities, and identified five cases with submicroscopic abnormalities believed to contribute to their phenotypes. Here, we provide detailed molecular cytogenetic descriptions and clinical presentation of two unrelated subjects with de novo submicroscopic deletions within chromosome bands 11q24-25. In subject 1 the chromosome rearrangement consisted of a 6.18 Mb deletion (from 128.25–134.43 Mb) and an adjacent 5.04 Mb duplication (from 123.15–128.19 Mb), while in subject 2, a 4.74 Mb interstitial deletion was found (from 124.29–129.03 Mb). Higher resolution array analysis (385 K Nimblegen) was used to refine all breakpoints. Deletions of the 11q24-25 region are known to be associated with Jacobsen syndrome (JBS: OMIM 147791). However, neither of the subjects had the typical features of JBS (trigonocephaly, platelet disorder, heart abnormalities). Both subjects had ID, dysmorphic features and additional phenotypic abnormalities: subject 1 had a kidney abnormality, bilateral preauricular pits, pectus excavatum, mild to moderate conductive hearing loss and behavioral concerns; subject 2 had macrocephaly, an abnormal MRI with delayed myelination, fifth finger shortening and squaring of all fingertips, and sensorineural hearing loss.
Two individuals with ID who did not have the typical clinical features of Jacobsen syndrome were found to have deletions within the JBS region at 11q24-25. Their rearrangements facilitate the refinement of the JBS critical region and suggest that a) deletion of at least 3 of the 4 platelet function critical genes (ETS-1, FLI-1 and NFRKB and JAM3) is necessary for thrombocytopenia; b) one of the critical regions for heart abnormalities (conotruncal heart defects) may lie within 129.03 – 130.6 Mb; c) deletions of KCNJ1 and ADAMTS15 may contribute to the renal anomalies in Jacobsen Syndrome; d) the critical region for MRI abnormalities involves a region from 124.6 – 129.03 Mb. Our results reiterate the benefits of array-CGH for description of new phenotype/genotype associations and refinement of previously established ones.
PMCID: PMC2648978  PMID: 19000322
11.  The Timing of cotE Expression Affects Bacillus subtilis Spore Coat Morphology but Not Lysozyme Resistance▿  
Journal of Bacteriology  2006;189(6):2401-2410.
The synthesis of structural components and morphogenetic factors required for the assembly of the Bacillus subtilis spore coat is governed by a mother cell-specific transcriptional cascade. The first two temporal classes of gene expression, which involve RNA polymerase sigma σE factor and the ancillary regulators GerR and SpoIIID, are deployed prior to engulfment of the prespore by the mother cell. The two last classes rely on σK, whose activation follows engulfment completion, and GerE. The cotE gene codes for a morphogenetic protein essential for the assembly of the outer coat layer and spore resistance to lysozyme. cotE is expressed first from a σE-dependent promoter and, in a second stage, from a promoter that additionally requires SpoIIID and that remains active under σK control. CotE localizes prior to engulfment completion close to the surface of the developing spore, but formation of the outer coat is a late, σK-controlled event. We have transplanted cotE to progressively later classes of mother cell gene expression. This created an early class of mutants in which cotE is expressed prior to engulfment completion and a late class in which expression of cotE follows the complete engulfment of the prespore. Mutants of the early class assemble a nearly normal outer coat structure, whereas mutants of the late class do not. Hence, the early expression of CotE is essential for outer coat assembly. Surprisingly, however, all mutants were fully resistant to lysozyme. The results suggest that CotE has genetically separable functions in spore resistance to lysozyme and spore outer coat assembly.
PMCID: PMC1899386  PMID: 17172339
12.  Interaction between Coat Morphogenetic Proteins SafA and SpoVID▿  
Journal of Bacteriology  2006;188(22):7731-7741.
Morphogenetic proteins such as SpoVID and SafA govern assembly of the Bacillus subtilis endospore coat by guiding the various protein structural components to the surface of the developing spore. Previously, a screen for peptides able to interact with SpoVID led to the identification of a PYYH motif present in the C-terminal half of the SafA protein and to the subsequent demonstration that SpoVID and SafA directly interact. spoVID and safA spores show deficiencies in coat assembly and are lysozyme susceptible. Both proteins, orthologs of which are found in all Bacillus species, have LysM domains for peptidoglycan binding and localize to the cortex-coat interface. Here, we show that the interaction between SafA and SpoVID involves the PYYH motif (region B) but also a 13-amino-acid region (region A) just downstream of the N-terminal LysM domain of SafA. We show that deletion of region B does not block the interaction of SafA with SpoVID, nor does it bring about spore susceptibility to lysozyme. Nevertheless, it appears to reduce the interaction and affects the complex. In contrast, lesions in region A impaired the interaction of SafA with SpoVID in vitro and, while not affecting the accumulation of SafA in vivo, interfered with the localization of SafA around the developing spore, causing aberrant assembly of the coat and lysozyme sensitivity. A peptide corresponding to region A interacts with SpoVID, suggesting that residues within this region directly contact SpoVID. Since region A is highly conserved among SafA orthologs, this motif may be an important determinant of coat assembly in the group of Bacillus spore formers.
PMCID: PMC1636312  PMID: 16950916
13.  A Gene Encoding a Holin-Like Protein Involved in Spore Morphogenesis and Spore Germination in Bacillus subtilis 
Journal of Bacteriology  2005;187(18):6443-6453.
We report here studies of expression and functional analysis of a Bacillus subtilis gene, ywcE, which codes for a product with features of a holin. Primer extension analysis of ywcE transcription revealed that a single transcript accumulated from the onset of sporulation onwards, produced from a σA-type promoter bearing the TG dinucleotide motif of “extended” −10 promoters. No primer extension product was detected in vivo during growth. However, specific runoff products were produced in vitro from the ywcE promoter by purified σA-containing RNA polymerase (EσA), and the in vivo and in vitro transcription start sites were identical. These results suggested that utilization of the ywcE promoter by EσA during growth was subjected to repression. Studies with a lacZ fusion revealed that the transition-state regulator AbrB repressed the transcription of ywcE during growth. This repression was reversed at the onset of sporulation in a Spo0A-dependent manner, but Spo0A did not appear to contribute otherwise to ywcE transcription. We found ywcE to be required for proper spore morphogenesis. Spores of the ywcE mutant showed a reduced outer coat which lacked the characteristic striated pattern, and the outer coat failed to attach to the underlying inner coat. The mutant spores also accumulated reduced levels of dipicolinic acid. ywcE was also found to be important for spore germination.
PMCID: PMC1236627  PMID: 16159778
14.  Assembly of an Oxalate Decarboxylase Produced under σK Control into the Bacillus subtilis Spore Coat 
Journal of Bacteriology  2004;186(5):1462-1474.
Over 30 polypeptides are synthesized at various times during sporulation in Bacillus subtilis, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat. The coat consists of three main layers, an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-dense striated outer layer. The product of the B. subtilis oxdD gene was previously shown to have oxalate decarboxylase activity when it was produced in Escherichia coli and to be a spore constituent. In this study, we found that OxdD specifically associates with the spore coat structure, and in this paper we describe regulation of its synthesis and assembly. We found that transcription of oxdD is induced during sporulation as a monocistronic unit under the control of σK and is negatively regulated by GerE. We also found that localization of a functional OxdD-green fluorescent protein (GFP) at the surface of the developing spore depends on the SafA morphogenetic protein, which localizes at the interface between the spore cortex and coat layers. OxdD-GFP localizes around the developing spore in a cotE mutant, which does not assemble the spore outer coat layer, but it does not persist in spores produced by the mutant. Together, the data suggest that OxdD-GFP is targeted to the interior layers of the coat. Additionally, we found that expression of a multicopy allele of oxdD resulted in production of spores with increased levels of OxdD that were able to degrade oxalate but were sensitive to lysozyme.
PMCID: PMC344410  PMID: 14973022
15.  Alternative Translation Initiation Produces a Short Form of a Spore Coat Protein in Bacillus subtilis 
Journal of Bacteriology  2001;183(6):2032-2040.
During endospore formation in Bacillus subtilis, over two dozen polypeptides are localized to the developing spore and coordinately assembled into a thick multilayered structure called the spore coat. Assembly of the coat is initiated by the expression of morphogenetic proteins SpoIVA, CotE, and SpoVID. These morphogenetic proteins appear to guide the assembly of other proteins into the spore coat. For example, SpoVID forms a complex with the SafA protein, which is incorporated into the coat during the early stages of development. At least two forms of SafA are found in the mature spore coat: a full-length form and a shorter form (SafA-C30) that begins with a methionine encoded by codon 164 of safA. In this study, we present evidence that the expression of SafA-C30 arises from translation initiation at codon 164. We found only a single transcript driving expression of SafA. A stop codon engineered just upstream of a predicted ribosome-binding site near codon M164 abolished formation of full-length SafA, but not SafA-C30. The same effect was observed with an alanine substitution at codon 1 of SafA. Accumulation of SafA-C30 was blocked by substitution of an alanine codon at codon 164, but not by a substitution at a nearby methionine at codon 161. We found that overproduction of SafA-C30 interfered with the activation of late mother cell-specific transcription and caused a strong sporulation block.
PMCID: PMC95099  PMID: 11222602

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