Structure of CARB-4 and AER-1 CarbenicillinHydrolyzing β-Lactamases

We determined the nucleotide sequences of bla_CARB-4 encoding CARB-4 and deduced a polypeptide of 288 amino acids. The gene was characterized as a variant of group 2c carbenicillin-hydrolyzing β-lactamases such as PSE-4, PSE-1, and CARB-3. The level of DNA homology between the bla genes for these β-lactamases varied from 98.7 to 99.9%, while that between these genes and bla_CARB-4 encoding CARB-4 was 86.3%. The bla_CARB-4 gene was acquired from some other source because it has a G+C content of 39.1%, compared to a G+C content of 67% for typical Pseudomonas aeruginosa genes. DNA sequencing revealed that bla_AER-1 shared 60.8% DNA identity with bla_PSE-3 encoding PSE-3. The deduced AER-1 β-lactamase peptide was compared to class A, B, C, and D enzymes and had 57.6% identity with PSE-3, including an STHK tetrad at the active site. For CARB-4 and AER-1, conserved canonical amino acid boxes typical of class A β-lactamases were identified in a multiple alignment. Analysis of the DNA sequences flanking bla_CARB-4 and bla_AER-1 confirmed the importance of gene cassettes acquired via integrons in bla gene distribution.Penicilloyl serine transferases, routinely called β-lactamases, cleave the cyclic amide bond of β-lactam antibiotics via the formation of a serine ester-linked penicilloyl enzyme giving a product devoid of antibacterial activity (46). A close inspection of databases indicated that in the last 3 years, a collection of at least 150 DNA sequences from plasmid-mediated and chromosomal bla genes has been acquired. Analysis of deduced peptides confirmed that most have conserved motifs typical of serine active-site enzymes that are divided into three major classes (classes A, C, and D) on the basis of a level of amino acid sequence identity of more than 20% between members in each class (10).In 1969, a β-lactamase was found in Pseudomonas aeruginosa Dalgleish, it was noticed to be “markedly active against carbenicillin,” and the enzyme was named PSE-4 (13, 32). As other β-lactamase enzymes were found, it was noticed that some β-lactamases have better activities than others against carbenicillin. All β-lactamases except class C enzymes hydrolyze carbenicillin at very different levels; class C enzymes hydrolyse it poorly. Genes encoding enzymes similar to PSE-4 were subsequently discovered in other bacterial species and are now known to be part of multidrug resistance transposons (24). In addition to the four original β-lactamases called PSE-1, PSE-2, PSE-3, and PSE-4, a plethora of plasmid-mediated enzymes capable of hydrolyzing carbenicillin at a high rate, such as LCR-1 (10), AER-1 (16), CARB-3 (22), NPS-1 (26), CARB-5 (35), and CARB-4 (36), were identified; but these enzymes have subtle differences in their biochemical properties and in their substrate profiles (7). The amino acid sequences of PSE-1 (17), PSE-2 (18), PSE-3 (8), PSE-4 (4), and CARB-3 (23) have been compared to those of other class A and class D enzymes, and it has been confirmed that PSE-2 (OXA-10) is a class D enzyme (10).The relationship of CARB-4 to other plasmid-mediated β-lactamases has been tested by determining the neutralization of benzylpenicillin-hydrolyzing activity with antisera prepared against purified the TEM-1, OXA-4, and CARB-3 β-lactamases (36). Antisera prepared with CARB-3 antigen inactivated the CARB-4 β-lactamase as well as the PSE-1, PSE-4, and CARB-3 enzymes (22, 36). The bla_CARB-3 and bla_CARB-4 genes are localized within transposons Tn1413 (7 kb) and Tn1408 (25 kb), respectively; these mobile elements were from plasmids isolated from bacterial strains of distinct origins (24, 27, 47).Unusual β-lactamases such as a metalloenzyme have been reported in Aeromonas hydrophila, a water-borne, gram-negative rod known to be highly resistant to β-lactam antibiotics, including carbenicillin (42). A carbenicillin-hydrolyzing β-lactamase has been discovered in an isolate of A. hydrophila from blood (16). The substrate profile of the AER-1 enzyme resembled those of plasmid-mediated carbenicillin-hydrolyzing enzymes, but it had a different isoelectric point (pI 5.9) and molecular mass (29 kDa); these values are reminiscent of those for BRO-1 (pI 5.45), PSE-1 (pI 5.7), CARB-3 (pI 5.75), and CARB-5 (pI 5.35). The gene coding for AER-1 is part of the Ω7711 unit which is IncP mobilizable but RecA dependent and which inserts only between purC and guaB at a specific site in the Escherichia coli chromosome (16). The Ω7711 unit cotransfers resistance to the antibiotics chloramphenicol, streptomycin, and sulfonamide; the transfer of multidrug resistance and insertion at a unique site are properties analogous to those of Tn7.In the study described in this report, we have focused on a carbenicillin-hydrolyzing enzyme identified from a clinical isolate, P. aeruginosa p83372, containing the pUD12 plasmid and producing CARB-4. This enzyme has an acidic isoelectric point (pI 4.3) and hydrolyzes carbenicillin very efficiently (36). We also present the nucleotide sequences of bla_AER-1 and bla_CARB-4, including flanking sequences containing integrons that explain their distribution and presence in different mobile genetic elements (15). We compared the deduced AER-1 and CARB-4 polypeptides with those of other group 2c enzymes (7) via a multiple alignment.     

Characterization of two different mucolipin-like genes from Leishmania major

Here, we report the existence of two different mucolipin-like genes in Leishmania parasites. The Leishmania major mucolipin-like A and B genes (lmmlA and lmmlB) encode two proteins of 776 and 590 amino acids, respectively, and may be classified among the mucolipins family [transient receptors potential mucolipin (TRPML)] because (1) they include a large region that exhibits significant similarities with specific domains of ion transport proteins and transient receptors potential (TRP) channels, (2) they contain at least 173 residues that display significant homologies with conserved regions of different mucolipins from several species, and (3) as TRPMLs, they include six predicted transmembrane domains. Gene expression analysis reveals that lmmlB is upregulated in metacyclics and amastigotes relative to procyclics, while lmmlA is constitutively expressed in the three Leishmania developmental stages. These genes could constitute potential drug targets.     

Evaluation of nutritional value,characteristics, functional properties of <Emphasis Type="Italic">Cymodocea nodosa</Emphasis> and its benefits on health diseases

Background

Nutritional fact study has prime importance to make the species edible and commercially viable to the food consumers. This is the first report that indicates the chemical characterization, functional, antioxidant and antihypertensive properties of Cymodocea nodosa to evaluate its nutritional status.

Methods

Physico-chemical determination was determined by colorimetric and spectroscopic analysis. The functional and texture properties were evaluated since a desirable texture should be retained. Bioactive substances were determined by liquid chromatography-high resolution electrospray ionization mass spectrometry HPLC-DAD-ESI/MS2 analysis. Health benefit of this plant was highlighting by the antioxidant and antihypertensive potentials.

Results

Results showed that the seagrass powder was characterized by a high content of fibers (56.4%), the fatty acids profile was dominated by the oleic acid, which represents about 62.0% of the total fatty acids and the functional properties proved important values of swelling capacity (6.71?±?0.2) and water holding capacity (12.26?±?0.25), that were comparable to those of some foodstuffs. Finally, the physico-chemical analysis shows the wealth in phenolic compounds, that could be explained by the high antioxidant and antihypertensive ability which was concentration dependent.

Conclusion

The results from this study suggested that this marine plant could be utilized as a healthy food item for human consumption.

     

Molecular Study of Dirofilaria immitis and Dirofilaria repens in Dogs from Tunisia

Dirofilaria immitis and Dirofilaria repens are mosquito‐borne nematodes which infect primarily dogs as their main definitive hosts. They cause cardiopulmonary (D. immitis) or cutaneous (D. repens) dirofilariasis in canids and other carnivores and can accidentally be transmitted to humans where they can induce a variety of clinical outcomes depending on organ localization. Dirofilaria spp. infection in dogs was assessed using molecular methods (PCR and sequencing) to identify the different Dirofilaria species occurring in 200 dogs from Northern and Central Tunisia. The overall molecular prevalence of Dirofilaria spp. was 17.5% (35/200). The prevalence of D. immitis (14.5%) was significantly higher than for D. repens (3%). Molecular prevalence of D. immitis was significantly higher in suburban compared to urban and rural regions. There was no difference in molecular prevalence of D. immitis or D. repens according to the dogs’ (sex or use). Dirofilaria immitis amplicons (accession numbers KR676386 ) fall into the same clade with D. immitis from China, India and Taiwan. Comparison of the partial sequences of D. repensITS2 rDNA gene ( KR676387 ) revealed 99.6% similarity with D. repens reported in dogs from USA. It had also 97.6% similarity with D. repens from mosquitoes in Czech Republic. High dog parasite burdens should motivate both medical doctors and veterinarians to consider these frequent infections.     

Chemically modified carbon-based electrodes for the determination of paracetamol in drugs and biological samples

Paracetamol is a non-steroidal, anti-inflammatory drug widely used in pharmaceutical applications for its sturdy, antipyretic and analgesic action. However, an overdose of paracetamol can cause fulminant hepatic necrosis and other toxic effects. Thus, the development of advantageous analytical tools to detect and determine paracetamol is required. Due to simplicity, higher sensitivity and selectivity as well as costefficiency, electrochemical sensors were fully investigated in last decades. This review describes the advancements made in the development of electrochemical sensors for the paracetamol detection and quantification in pharmaceutical and biological samples. The progress made in electrochemical sensors for the selective detection of paracetamol in the last 10 years was examined, with a special focus on highly innovative features introduced by nanotechnology. As the literature is rather extensive, we tried to simplify this work by summarizing and grouping electrochemical sensors according to the by which manner their substrates were chemically modified and the analytical performances obtained.