Cloning and analysis of the sfrB (sex factor repression) gene of Escherichia coli K-12.

The sfrB gene of Escherichia coli K-12 and the rfaH gene of Salmonella typhimurium LT2 are homologous, controlling expression of the tra operon of F and the rfa genes for lipopolysaccharide synthesis. We have determined a restriction map of the 19-kilobase ColE1 plasmid pLC14-28 which carries the sfrB gene of E. coli. After partial Sau3A digestion of pLC14-28, we cloned a 2.5-kilobase DNA fragment into the BamHI site of pBR322 to form pKZ17. pKZ17 complemented mutants of the sfrB gene of E. coli and the rfaH gene of S. typhimurium for defects of both the F tra operon and the rfa genes. pKZ17 in minicells determines an 18-kilodalton protein not determined by pBR322. A Tn5 insertion into the sfrB gene causes loss of complementing activity and loss of the 18-kilodalton protein in minicells, indicating that this protein is the sfrB gene product. These data indicate that the sfrB gene product is a regulatory element, since the single gene product elicits the expression of genes for many products for F expression and lipopolysaccharide synthesis.     

Accumulation of incomplete metabolic side products of lipid A in Salmonella typhimurium during inhibition of 3-deoxy-D-manno-octulosonate incorporation by a new class of antibacterial agents

A new class of antibacterial agents for Gram-negative bacteria, rationally designed to inhibit the incorporation of 3-deoxy-D-manno-octulosonate into lipopolysaccharide (LPS), was recently reported. In Salmonella typhimurium, where the lipid A species are well characterised, it was previously demonstrated that the addition of a compound which inhibits the enzyme 3-deoxy-manno-octulosonate cytidylytransferase (CMP-KDO synthetase; EC 2.7.7.38) leads to rapid accumulation of lipid A derivatives. The major lipid A species, IVA (O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1-6)-2-amino-2-deoxy-alpha-D - glucose, acylated at positions 2, 3, 2', 3' with beta-hydroxymyristoyl groups and bearing phosphates at positions 1 and 4'), was shown to be converted mainly to LPS by pulse-chase experiments in the absence of inhibitor. Labelled precursor (IVA) was also chased to other more polar lipid A derivatives. During chase in the presence of inhibitor, there was no conversion to LPS, while the major lipid A species was converted to the same polar lipid A derivatives as in chase without inhibitor. Our data indicate that despite the accumulation of several species of lipid A derivatives during inhibition of LPS synthesis, only IVA is destined for synthesis of mature LPS when LPS synthesis resumes. The more polar lipid A derivatives would thus represent aberrant side reaction products which occur when the pathway is inhibited.     

Drug release properties of polyethylene-glycol-treated ciprofloxacin-Indion 234 complexes

The polyethylene glycol (PEG) treatment of ciprofloxacin-Indion 234 complex was aimed to retard rapid ion exchange drug release at gastric pH. Ciprofloxacin loading on Indion 234 was performed in a batch process, and the amount of K⁺ in Indion 234 displaced by drug with time was studied as equilibrium constant K_DM. Drug-resin complex (DRC) was treated with aqueous PEG solution (0.5%–2% wt/vol) of different molecular weights (MWs) for 2 to 30 minutes. The PEG-treated ciprofloxacin-Indion 234 complex was evaluated for particle size, water absorption time, and drug release at gastric pH. During drug loading on Indion 234, the equilibrium constant (K_DM) increased rapidly up to 20 minutes with efficient drug loading. Increased time of immersion of the drug resinate in PEG solutions significantly retained higher size particles upon dehydration. The larger DRC particles showed longer water absorption times owing to compromised hydrating power. The untreated DRC showed insignificant drug release in deionized water; while at gastric pH, ciprofloxacin release was complete in 90 minutes. A trend of increased residual particle size, proportionate increase in water absorption time, and hence the retardation of release with time of immersion was evident in PEG-treated DRC. The time of immersion of DRC in PEG-treated DRC. The time of immersion of DRC in PEG solution had predominant release retardant effect, while the effect of molecular weight of PEG was insignificant. Thus, PEG treatment of DRC successfully retards ciprofloxacin ion exchange release in acidic pH.     

Crystallo-co-agglomeration: A novel technique to obtain ibuprofen-paracetamol agglomerates

The purpose of this research was to obtain directly compressible agglomerates of ibuprofen-paracetamol containing a desired ratio of drugs using a crystallo-co-agglomeration technique. Crystallo-co-agglomeration is an extension of the spherical crystallization technique, which enables simultaneous crystallization and agglomeration of 2 or more drugs or crystallization of a drug and its simultaneous agglomeration with another drug or excipient. Dichloromethane (DCM)-water system containing polyethylene glycol (PEG) 6000, polyvinyl pyrollidone, and ethylcellulose was used as the crystallization system. DCM acted as a good solvent for ibuprofen and bridging liquid for agglomeration. The process was performed at pH 5, considering the low solubility of ibuprofen and the stability of paracetamol. Loss of paracetamol was reduced by maintaining a low process temperature and by the addition of dextrose as a solubility suppressant. The agglomerates were characterized by differential scanning calorimetry, powder x-ray diffraction (PXRD), and scanning electron microscopy and were evaluated for tableting properties. The spherical agglomerates contained an ibuprofen-paracetamol ratio in the range of 1.23 to 1.36. Micromeritic, mechanical, and compressional properties of the agglomerates were affected by incorporated polymer. The PXRD data showed reduction in intensities owing to dilution and reduced crystallinity. Thermal data showed interaction between components at higher temperature. Ethylcellulose imparted mechanical strength to the agglomerates as well as compacts. The agglomerates containing PEG have better comparessibility but drug release in the initial stages was affected owing to asperity melting, yielding harder compacts. The agglomeration and properties of agglomerates were influenced by the nature of polymer.     

Preparation and characterization of flurbiprofen beads by melt solidification technique

Amelt solidification technique has been developed to obtain sustained-release waxy beads of flurbiprofen. Low glass transition temperature (t _g) and shear-induced crystallization of flurbiprofen made it a suitable candidate for melt solidification technique. The process involved emulsification and solidification of flurbiprofen-cetyl alcohol melt at significantly low temperature (5°C). The effect of variables, namely, the amount of cetyl alcohol and the speed of agitation, was studied using 3² factorial design. The technique and the beads were evaluated on the basis of process and desired yield, surface topography, Fourier-transform infrared (FT-IR), differential scanning calorimetry (DSC), particle size distribution, crushing strength, and drug release. Average values for process and desired yields were 97% wt/wt and 26% wt/wt, respectively. No interaction was observed between drug and excipient. Multiple regression analysis was carried out, and response surfaces were obtained. A curvilinear relationship was observed between percentage of desired yield and the amount of cetyl alcohol. Linear decrease in crushing strength was observed with increase in the amount of cetyl alcohol. Drug released from the beads followed zero order kinetics. Burst release was shown to a greater extent in beads containing a lower amount of cetyl alcohol. Response surfaces of time required for certain percentage of drug (t _D%) showed that after critical concentration of about 20% of cetyl alcohol (400 mg/batch), no significant release retardant effect was observed.     

Identification of human sperm peptide sequence involved in egg binding for immunocontraception

Development of a vaccine based on sperm antigens represents a promising approach to contraception. The sperm-zona pellucida (ZP) interaction constitutes the most important event in the fertilization process, and the molecular sequences involved at this site may provide the most attractive candidates for immunocontraception. In the present study, using the phase peptide display technique, a novel dodecamer sequence, designated as YLP(12), was identified that is involved in sperm-ZP recognition/binding. The synthetic 12-mer peptide based on this sequence and its monovalent Fab' antibodies specifically and significantly (P < 0.05) inhibited human sperm-ZP binding. In Western blot and immunoprecipitation procedures, the YLP(12) peptide recognized the ZP3 component of solubilized human ZP proteins. In the Western blot procedure involving 10 different human tissue extracts, the anti-YLP(12) Fab' antibodies recognized a protein band of approximately 72 +/- 2 kDa only in the testis lane. The peptide sequence was localized on the acrosomal region of the human sperm cell. These findings indicate that the novel testis-specific 12-mer YLP(12) that is present in the acrosomal region and is involved in human sperm-ZP interaction may find applications in contraceptive vaccine development, as well as in diagnosis and treatment of male infertility mediated through sperm dysfunction.