Inhibition of hydrolytic enzyme activities and plant pathogen growth by invertase inhibitors

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, alpha-L-arabinofuranosidase and beta-glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.     

Direct visualization by confocal fluorescent microscopy of the permeation of myristoylated peptides through the cell membrane

To study the permeability through the cellular membrane of synthetic peptides containing an hydrophobic moiety, we used a 13-mer myristoylated peptide labeled with a N-terminal fluorescent probe. After 2 h of incubation, the subcellular distribution was analyzed in intact chromaffin cells by confocal fluorescent microscopy. Our results demonstrate that myristoylated peptides diffuse into intact cells, showing an heterogeneous distribution, but they do not reach the cellular nucleous, at least during the time range used.     

3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine, a new prodrug of zidovudine. Synthesis, solid state characterization, and anti HIV-1 activity

Synthesis, solid state characterization and anti HIV-1 activity of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (2), a new prodrug of zidovudine (AZT, 1), are described. Two solid forms of 2 prepared by crystallization from ethyl acetate-petroleum ether (form alpha) and from a melt sample of form alpha (amorphous form) were characterized by X-ray diffractometry, infrared spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA) techniques. The novel nucleoside exhibited antiviral activity against standard and resistant strain panels of HIV-1 as well as cytotoxicity similar to that of AZT.     

Poly(alpha-alkyl gamma-glutamate)s of microbial origin. 2. On the microstructure and crystal structure of poly(alpha-ethyl gamma-glutamate)s

The stereochemical microstructure and crystalline structure of nearly racemic poly(alpha-ethyl gamma,DL-glutamate) obtained by esterification of biosynthetic poly(gamma-glutamic acid) were examined by NMR, DSC, and powder X-ray diffraction. The two enantiomerically pure poly(alpha-ethyl gamma-glutamate)s, as well as the racemic stereocopolymers with random and alternating microstructure, were prepared by chemical synthesis and studied in parallel to help in the interpretation of the data. The (13)C NMR analysis revealed that biosynthetic poly(alpha-ethyl gamma,DL-glutamate) consists of a block stereocopolymer accompanied by minor amounts of a mixture of the two optically pure homopolymers. The polymer is crystalline, with a degree of crystallinity and crystal structure essentially similar to those displayed by the optically pure polymers but clearly different from the alternating copolymer. Conversely, the racemic stereocopolymer with a random microstructure prepared by chemical synthesis is amorphous. The crystal structure of the racemic mixture of the D- and L-homopolymers seems to be very close to that of the biosynthetic stereocopolymer, although some indications suggesting the existence of a stereocomplex were found.     

Blend uniformity analysis using stream sampling and near infrared spectroscopy

A near infrared spectroscopic method was developed to determine drug content in a 20% (wt/wt) ibuprofen and spray-dried hydous lactose blend. A blending profile was obtained after blending for 0.5, 1, 3, 5, 10, and 20 minutes. Stream sampling was used to collect about 20 blend samples at each of the blending times from a laboratory scale V-blender. The samples collected were used to develop a near infrared calibration model. The calibration model was then used to determine the drug content of unknown samples from 2 validation blends. The validation blends were not included in the calibration model; they were used to evaluate the effectiveness of the calibration model. A total of 45 samples from the 2 validation blends were predicted by the near infrared calibration model and then analyzed by a validated UV spectrophotometric method. The root mean square error of prediction for the first validation blend was 5.69 mg/g and 3.30 mg/g for the samples from the second blend. A paired t test at the 95% confidence level did not indicate any differences between the drug content predicted by the near infrared spectroscopy (NIRS) method and the validated UV method for the 2 blends. The results show that the NIRS method could be developed while the blending profile is generated and used to thoroughly characterize a new formulation during development by analyzing a large number of samples. The new formulation could be transferred to a manufacturing plant with an NIRS method to facilitate blend uniformity analysis.     

Protein origami: therapeutic rescue of misfolded gene products

Receptor mutations that elicit loss of function are sometimes equated with defects that ablate receptor-ligand binding or receptor-effector interactions. Similarly, mutationally defective enzymes and ion channels are often viewed as compromised in substrate or ion recognition, respectively. Recent observations, however, suggest that an alternate mechanism may be surprisingly common, namely, that mutations in structural genes may not interfere with the inherent functionality of the affected protein, but nevertheless cause disease by preventing the cell's trafficking machinery from placing the affected protein at the appropriate subcellular compartment (e.g., at the cell membrane). Accordingly, therapies may be devised to ensure the placement of receptors (or other proteins) at locations where they can support cell function.     

Micellar electrokinetic capillary chromatography for the determination of fluoxetine and its metabolite norfluoxetine in biological fluids

A micellar electrokinetic capillary chromatography (MEKC) for determining fluoxetine and its metabolite (norfluoxetine) is proposed. Optimal conditions for the quantitative separation were investigated. A background electrolyte solution consisting of 5 mM phosphate buffer adjusted to pH 12.3 and 40 mM of 1-decanesulfonic acid sodium salt (DSS), hydrodynamic injection and 25 kV of separation voltage were used. Good linearity and precision were obtained for both compounds. Detection limits of 0.2 mg/l for fluoxetine and norfluoxetine were obtained. The developed method is rapid and it has been applied to determine fluoxetine and its metabolite in human serum and urine. The samples were purified and enriched by means of extraction-preconcentration step with a preconditioned C18 cartridge and eluting the compounds with methanol.     

Opioid analgetics retention-pharmacologic activity models using biopartitioning micellar chromatography

Opioids are drugs used in medicine for pain control. In this paper, retention-pharmacokinetics and retention-pharmacodynamics relationships of opioids are proposed and statistically validated. These models are based on the compound retention in the biopartitioning micellar chromatography system (BMC), a new methodology which has successfully been used to develop QRAR models for many other families of compounds. The obtained results are compared to the traditional QSAR models using lipophilicity data. The adequacy of QRAR models is due to the fact that the characteristics of the compounds such as the hydrophobicity, electronic charge and steric effects determine both their retention in BMC and their pharmacokinetic and pharmacodynamic behavior.     

Markovian chemicals "in silico" design (MARCH-INSIDE), a promising approach for computer aided molecular design II: experimental and theoretical assessment of a novel method for virtual screening of fasciolicides

A novel method for in silico selection of fluckicidal drugs is introduced. Two QSARs that permit us to discriminate between fasciolicide and non-fasciolicide drugs (the first) and to outline some conclusions about the possible mechanism of action of a chemical (the second) are performed. The first model correctly classified 93.85% of compounds in the training series and 89.5% of the compounds in the predicting one. This model correctly classified 87.7, 93.8, 92.2 and 93.9% of compounds in leave- n-out cross validation procedures when n takes values from 2 to until 6. The model seems to be stable in around 92% of good classification in leave- n-out cross validation analysis when n>6. The second model correctly classified 70% of non-fasciolicide compounds, 85.71% of beta-tubulin inhibitors and 100% of proton ionophores in the training set. This model recognizes as proton ionophores 100% of any nitrosalicylanilides in the predicting series. Both models have a low p-level <0.05. Finally, the experimental assay of six organic chemicals by an in vivo test permit us to carry out an assessment of the model with a fairly good 100% agreement between experiment and theoretical prediction.