Long-term effects of passive integrated transponder (PIT) tagging on the kelp grouper Epinephelus bruneus.

The effects of passive integrated transponder (PIT) tagging on the growth and survival of kelp grouper Epinephelus bruneus was examined over 1 year. The overall average mass gain for the 1 year period of the abdominal cavity tagged group was higher than the dorsal muscle tagged group. No significant differences were found in overall specific growth rate and food conversion between control and treatment groups.

Phage diversity in a methanogenic digester.

It has been shown that phages are present in natural and engineered ecosystems and influence the structure and performance of prokaryotic communities. However, little has been known about phages occurring in anaerobic ecosystems, including those in methanogenic digesters for waste treatment. This study investigated phages produced in an upflow anaerobic sludge blanket methanogenic digester treating brewery wastes. Phage-like particles (PLPs) in the influent and effluent of the digester were concentrated and purified by sequential filtration and quantified and characterized by transmission electron microscopy (TEM), fluorescence assay, and field inversion gel electrophoresis (FIGE). Results indicate that numbers of PLPs in the effluent of the digester exceeded 1 x 10(9) L-1 and at least 10 times greater than those in the influent, suggesting that substantial amounts of PLPs were produced in the digester. A production rate of the PLPs was estimated at least 5.2 x 10(7) PLPs day-1 L-1. TEM and FIGE showed that a variety of phages were produced in the digester, including those affiliated with Siphoviridae, Myoviridae, and Cystoviridae.

Solubility of (+/-)-ibuprofen and S (+)-ibuprofen in the presence of cosolvents and cyclodextrins.

Aqueous solubility is an important parameter for the development of liquid formulations and in the determination of bioavailability of oral dosage forms. Ibuprofen (IB), a nonsteroidal anti-inflammatory drug, is a chiral molecule and is currently used clinically as a racemate (racIB). However, the S form of ibuprofen or S(+)-ibuprofen (SIB) is the biologically active isomer and is primarily responsible for the antiinflammatory activity. Phase solubility studies were carried out to compare the saturation solubilities of racIB and SIB in the presence of common pharmaceutical solvents such as glycerol, sorbitol solution, propylene glycol (PG), and polyethylene glycol (PEG 300) over the range of 20% to 80% v/v in aqueous based systems. The solubilities of the two compounds were also compared in the presence of cyclodextrins such as beta cyclodextrin (CD), hydroxypropyl beta cyclodextrin (HPCD), and beta cyclodextrin sulfobutyl ether sodium salt (CDSB) over the range of 5% to 25% w/v. Solubility determinations were carried at 25 degrees C and 37 degrees C. Cosolvents exponentially increased the solubility of both SIB and racIB, especially in the presence of PG and PEG 300. Glycerol was not very effective in increasing the aqueous solubilities of both compounds, whereas sorbitol solution had a minimal effect on their solubility. PG and PEG 300 increased the solubility of SIB by 400-fold and 1500-fold, respectively, whereas the rise in solubility for racIB was 193-fold and 700-fold, respectively, at 25 degrees C for the highest concentration of the cosolvents used (80% v/v). Of the two compounds studied, higher equilibrium solubilities were observed for SIB as compared with racIB. The derivatized cyclodextrins increased the aqueous solubility of racIB and SIB in a concentration-dependent manner giving AL type of phase diagrams. The phase solubility diagrams indicated the formation of soluble inclusion complexes between the drugs and HPCD and CDSB, which was of 1:1 stoichiometry. The addition of underivatized CD reduced the solubility of racIB and SIB via the formation of an insoluble complex. The S form formed more stable complexes with HPCD and CDSB as compared with raclB. The solubilization process is discussed in terms of solvent polarity and differential solid-state structure of raclB and SIB. The thermodynamic parameters for the solubilization process are presented.

Controlled-release matrix tablets of ibuprofen using cellulose ethers and carrageenans: effect of formulation factors on dissolution rates.

The study was conducted to investigate the effects of carrageenans, and cellulose ethers on the drug release rates of ibuprofen controlled-release tablet matrices prepared by direct compression. Polymer blends containing carrageenans or cellulose ethers were used for the formulation and the effect of varying the polymer concentration on the release of the drug was studied. Other factors such as changes in surface topography of the matrices due to hydration were observed using a cryogenic scanning electron microscopy technique. Multiple regression analysis was used to predict the time for 50% release (t50) as a function of the concentration of the polymers used. Most of the formulations showed linear release profiles (r(2)>or=0.96-0.99) and sustained the release of ibuprofen over 12-16 h. The highest t50 (9.3 h) was for the formulation that contained a blend of 1:2 ratio of Viscarin and HPMC, while the lowest (3 h) was for the matrices that contained a 2:1 ratio of methylcellulose and Gelcarin. The majority of the matrix tablets that contained 10% polymer disintegrated prematurely. Of all the polymer blends that were investigated, the combination of Viscarin and HPMC gave almost linear release profiles over the entire range of concentration that was studied. The least effective combination was methylcellulose in combination with HPMC. Most of the formulations released ibuprofen by an anomalous (non-Fickian) transport mechanism, except those matrices that contained methylcellulose and Gelcarin (in a 1:1 and 1:2 ratio), which showed zero-order release.

Effects of formulation variables and characterization of guaifenesin wax microspheres for controlled release.

Sustained-release wax microspheres of guaifenesin, a highly water-soluble drug, were prepared by the hydrophobic congealable disperse method using a salting-out procedure. The effects of formulation variables on the loading efficiency, particle properties, and in-vitro drug release from the microspheres were determined. The type of dispersant, the amount of wetting agent, and initial stirring time used affected the loading efficiency, while the volume of external phase and emulsification speed affected the particle size of the microspheres to a greater extent. The crystal properties of the drug in the wax matrix and the morphology of the microspheres were studied by differential scanning calorimetry (DSC), powder x-ray diffraction (XRD), and scanning electron microscopy (SEM). The DSC thermograms of the microspheres showed that the drug lost its crystallinity during the microencapsulation process, which was further confirmed by the XRD data. The electron micrographs of the drug-loaded microspheres showed well-formed spherical particles with a rough exterior.

Production of alternatives to fuel oil from organic waste by the alkane-producing bacterium, Vibrio furnissii M1.

AIMS: We investigated the production of alternatives to fuel oil through the bacterial metabolism of organic waste. The availability for this purpose of various sources of organic waste for hydrocarbon production by the alkane-producing bacterium, Vibrio furnissii M1, was examined. METHODS AND RESULTS: We screened 17 authentic compounds which can generally be found in organic waste for their hydrocarbon production. Carbon (3 mmol) in a 50-ml culture with acetic acid, lactic acid, butyric acid, succinic acid, malic acid, pentanoic acid, hexanoic acid glucose, xylose, starch or sucrose yielded 10-27 mg of alkanes or alkenes. The chain length of these alkanes or alkenes varied according to the culture from C14 to C27. Varying the ratio of carbon to nitrogen in the culture had no effect on the hydrocarbon production. Crude blackstrap molasses were also converted into alkanes with a conversion ratio of 20% (half of that in an authentic sucrose medium) of the total carbon consumption. CONCLUSIONS: V. furnissii M1 could produce hydrocarbons corresponding to kerosene or light oil from volatile fatty acids and sugars. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on bacterial hydrocarbon production from organic waste.

Two-phase melt systems of ibuprofen for enhanced membrane permeation.

A novel method to convert S- and racemic (RS-) ibuprofen (Ibu) into an oily state at ambient temperature (25 degrees C) was developed. Using menthol and aqueous isopropanol (IPA) as melting point depressing agents, the two-phase melt systems (TMSs) of Ibu consisting of a homogeneous oily phase and a homogeneous aqueous phase were obtained. In TMS with a high S-Ibu: menthol ratio and a low IPA content, the oily phase primarily consisted of Ibu and menthol, whereas the majority of the aqueous phase was IPA and buffer. Using this method, the S-Ibu concentration in the oily phase reached as high as 70% (w/w). The compositional phase diagram was obtained using a titration method to study the relationship between the melting states of the solid components and system composition. S- and RS-Ibu showed different phase diagrams, and the maximum concentration of S-Ibu measured in the oily phase of TMS was much greater than that of RS-Ibu. The permeation study of a series of TMS and non-TMS systems showed that S-Ibu penetrated through shed snake skin faster than RS-Ibu, and the contents of IPA and menthol significantly affected the permeation rates of ibuprofen across shed snake skin, which may be attributed to the higher lipophilicity, and thus, higher solubility of S-Ibu in the skin than RS-Ibu. Such results support the use of S-Ibu TMS for topical formulation development.

Microencapsulation of a hydrophilic drug into a hydrophobic matrix using a salting-out procedure. I: Development and optimization of the process using factorial design.

The congealable disperse phase method for preparing sustained release microspheres involves an emulsification process using water as the external phase and molten hydrophobic wax as the disperse phase into which the drug is loaded. Attempts to entrap highly water-soluble drugs using this process have often resulted in low loading efficiency as the drugs partition into the external water phase during emulsification and are lost. A novel method employing salts and wetting agents was developed to improve the loading efficiency of the highly water-soluble drug, guaifenesin, using this method. The drug/wax ratio (D/W) and the presence of salts and wetting agents greatly influenced microsphere properties. To optimize the process for drug loading efficiency and release rate, three different D/Ws, salts and wetting agents were chosen and a full 3(3) factorial design experiment was performed. Any significant differences among the levels of the variables and their individual and joint effects on entrapment efficiency and T50 (time for 50% drug release) were determined. Entrapment efficiencies in the range 35.1-86.3% were obtained for the various factor-level combinations of the variables. Particle size was in the range 140-385 microm and T50 was 0.59-2.72 h for the microspheres obtained. The D/W and type of salt used significantly affected drug entrapment and T50, while the nature of wetting agent was not significant at p < 0.05. The microspheres prepared using 1:4 D/W showed the highest entrapment efficiency and slowest drug release.

Intranasal delivery of PEGylated salmon calcitonins: hypocalcemic effects in rats.

To evaluate the hypocalcemic effect of polyethylene gtycol-conjugated salmon calcitonins (PEG-sCT) in rats, mono-PEGylated sCTs (mono-PEG-sCTs) and unmodified sCT were administered via the intranasal route and serum calcium levels were measured by colorimetric assay using o-cresolphthalein. Mono-PEG-sCTs were prepared with different sizes of succinimidyl succinate monomethoxy PEG molecules (PEG2K), PEG5K, PEG12K) and characterized by HPLC and MALDI-TOF mass spectrometry. Nasal instillation of mono-PEG2K-sCT at a dose of 2 IU/kg resulted in sustained reduction in serum calcium levels over 8 hr, with a maximum reduction (% maxd) of 13% after 6 hr of application. Whereas unmodified sCT showed a transient decrease in serum calcium levels with the maximum reduction (5%) observed after 30 min of administration. The overall reductions in serum calcium levels expressed as the net change in AUC relative to control in 8 hr were 11.9 +/- 0.2, 4.6 +/- 0.7, and 2.6 +/- 0.7% for mono-PEG2K-, mono-PEG5K-, and mono-PEG12K-sCT, respectively, compared to 3.2 +/- 0.6% for unmodified sCT. The relative bioavailability of nasally administered 2 IU/kg of mono-PEG2K-sCT was approximately 4-fold higher than nasally administrated unmodified sCT, and the absolute bioavailability was approximately 91% of intravenously injected sCT in 8 hr. It can be concluded that the intranasal absorption of mono-PEG-sCTs was inversely related to the molecular weights of the PEG attached. Of the PEGylated sCTs examined, mono-PEG2K-sCT showed the most pronounced hypocalcemic effect. Therefore the intranasal application would probably be an alternative route of administration for mono-PEG-sCTs in achieving sustained calcium-lowering effects.

Arginine deiminase: a potential inhibitor of angiogenesis and tumour growth.

Hydrolysis of plasma arginine to citrulline by arginine deiminase (ADI) was recently shown to suppress lipopolysaccharide-induced nitric oxide (NO) synthesis. Since arginine is the precursor of NO, and the latter modulates angiogenesis, we explored whether ADI treatment significantly affected tube-like (capillary) formation of human umbilical vein endothelial cells. Inhibition occurred in a dose-dependent manner, both in the chorioallantoic membrane and the murine Matrigel plug assay. Inhibition of angiogenesis by ADI was reversed when a surplus of exogenous arginine was provided, indicating that its antiangiogenic effect is primarily due to arginine depletion, although other pathways of interference are not entirely excluded. Arginine deiminase is also shown to be as a potent inhibitor of tumour growth in vitro as in vivo, being effective at nanogram quantities per millilitre in CHO and HeLa cells. Thus, it could be highly beneficial in cancer therapy because of its two-pronged attack as both an antiproliferative and an antiangiogenic agent.

Isolation and characterization of a bacterium that produces hydrocarbons extracellularly which are equivalent to light oil.

A halotorelant bacterial strain that produces a significant amount of lipids from short-chain fatty acids was isolated from the sludge of a sewage disposal plant. This strain displayed a significant extracellular accumulation of lipids. The yield of lipids including hydrocarbons was highest (120% of cell dry weight) at the end of the linear growth phase. Fractionation of the lipids using thin-layer chromatography and subsequent gas chromatography showed that hydrocarbons were also obtained following an increase in total lipids. Their yield was the highest (50% of cell dry weight) in the linear growth phase. Additional analysis using infrared absorption spectrum and gas chromatography-mass spectrometry showed that the hydrocarbon fraction was composed of alkanes, such as C15H32, C18H38, C21H44, C22H46 and C24H50. Homology analysis of the 16s rDNA sequence as well as studies of the morphological and physiological characteristics indicated that the bacterium is a strain of Vibrio furnissii.

Identification of the modifying sites of mono-PEGylated salmon calcitonins by capillary electrophoresis and MALDI-TOF mass spectrometry.

A capillary electrophoretic method (CE) was developed for the determination of the PEG-modification sites of three positional isomers of mono-PEG modified salmon calcitonins (mono-PEG-sCTs). Resistance to proteolytic degradation on the PEG modification sites resulted in different patterns of CE electropherograms for the tryptic digested mono-PEG-sCTs isomers, and the PEG modification sites were assigned accordingly. The PEG-modification sites were also confirmed directly by determining the molecular masses of the tryptic digested PEG-modified fragments of respective mono-PEG-sCT by the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry.

Escherichia coli DNA polymerase III tau- and gamma-subunit conserved residues required for activity in vivo and in vitro.

The Escherichia coli DNA polymerase III tau and gamma subunits are single-strand DNA-dependent ATPases (the latter requires the delta and delta' subunits for significant ATPase activity) involved in loading processivity clamp beta. They are homologous to clamp-loading proteins of many organisms from phages to humans. Alignment of 27 prokaryotic tau/gamma homologs and 1 eukaryotic tau/gamma homolog has refined the sequences of nine previously defined identity and functional motifs. Mutational analysis has defined highly conserved residues required for activity in vivo and in vitro. Specifically, mutations introduced into highly conserved residues within three of those motifs, the P loop, the DExx region, and the SRC region, inactivated complementing activity in vivo and clamp loading in vitro and reduced ATPase catalytic efficiency in vitro. Mutation of a highly conserved residue within a fourth motif, VIc, inactivated clamp-loading activity and reduced ATPase activity in vitro, but the mutant gene, on a multicopy plasmid, retained complementing activity in vivo and the mutant gene also supported apparently normal replication and growth as a haploid, chromosomal allele.

Pharmacokinetic disposition of polyethylene glycol-modified salmon calcitonins in rats.

This study first reports the pharmacokinetic disposition of polyethylene glycol (PEG)-modified salmon calcitonin (sCT) based on the number of attached PEG molecules. PEG-modified sCT was prepared by covalent linkage with succinimidyl carbonate monomethoxy polyethylene glycol. Mono- and di-PEG-sCTs were separated by size exclusion and reverse phase HPLC, and radioiodinated by the chloramine-T method with Na125I. 125I-mono-PEG sCT, 125I-di-PEG-sCT and unmodified 125I-sCT were administered to rats by i.v. injection. Serial blood samples, urine and various tissue samples were taken for the determination of radioactivity. Di-PEG-sCT exhibited significantly reduced systemic clearance (2.3 vs. 11.1 ml/min/kg) and steady-state volume of distribution (229.9 vs. 603.1 ml/kg), while mono-PEG-sCT showed a prolonged elimination half-life (189.1 min vs. 59.8 min) compared with unmodified sCT. The extent of urinary excretion of the PEG-modified sCTs was higher than for the unmodified sCT, but all these chemicals were excreted in urine in small quantities (< or = 0.6%). There was a tendency toward reduced accumulation of PEGylated sCTs in tissues, with its reduction being inversely proportional to the molecular size. Accumulation of the total radioactivity of the unmodified and PEG-modified sCTs was highest in the liver, followed by kidneys, lungs, spleen, heart and thyroid. When expressed per tissue gram weight, however, the highest radioactivity was found in the kidneys. PEGylated sCTs may have greater therapeutic potential via reduced systemic clearance and prolonged elimination half-life over unmodified sCT.

Stability and cytotoxicity of Fab-ricin A immunotoxins prepared with water soluble long chain heterobifunctional crosslinking agents.

The effects of the hindered and non-hindered water soluble long-chain disulfide bonds on the stability and cytotoxicity of the ricin A chain (RTA) immunotoxin were examined. The RTA immunotoxins were prepared with the Fab fragments of anti-common acute lymphoblastic leukemia antigen (CALLA) monoclonal antibody (Fab-RTA) using sulfosuccinimidyl-6-[(-methyl-(-(2-pyridyldithio)toluamido]hexanoate (S-LC-SMPT) and sulfosuccinimidyl-6-[3-(2-pyridyldithio)-propionamido]hexanoate (S-LC-SPDP). The prepared Fab-RTA immunotoxins were evaluated for their conjugation yield, immunoreactivity, thermal and disulfide bond stability and cytotoxicity. The conjugation yield of the Fab-RTA immunotoxin from the water soluble long chain crosslinking agents, S-LC-SMPT and S-LC-SPDP, were comparable. Both Fab-RTA immunotoxins exhibited a similar immunoreactivity and thermal stability in aqueous solution. However, S-LC-SMPT -mediated Fab-RTA, sterically hindered, showed an enhanced disulfide bond stability in vitro over S-LC-SPDP mediated one. In the cytotoxicity against antigenic cell Daudi, the S-LC-SMPT -mediated RTA immunotoxin maintained a comparable cytotoxicity, compared with S-LC-SPDP mediated Fab-RTA immunotoxin.

Isolation, characterization, and stability of positional isomers of mono-PEGylated salmon calcitonins.

PURPOSE: To separate and characterize the different positional isomers of mono-PEGylated salmon calcitonins (mono-PEG-sCTs) and to evaluate the effects of the PEGylation site on the stability of different mono-PEG-sCTs in rat kidney homogenate. METHODS: Mono-PEG-sCTs were prepared using succinimidyl carbonate monomethoxy polyethylene glycol (5,000 Da) and separated by gel-filtration HPLC followed by reversed-phase HPLC. To characterize PEGylated sCTs, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and reversed-phase HPLC of the trypsin digested samples were performed. Mono-PEG-sCTs and sCT in rat kidney homogenates were measured by column-switching reversed-phase HPLC with on-line detection of the radioiodinated samples using a flow-through radioisotope detector. RESULTS: Three different mono-PEGylated sCTs were separated by reversed-phase gradient HPLC. From the MALDI-TOF MS analysis, the average molecular weight of mono-PEG-sCTs was confirmed as around 8650 Da. The presence of PEG moiety in the mono-PEG-sCTs was also manifested by the fact that the distance between two adjacent mass spectum lines was 44 Da which corresponds to PEG monomer unit. Tryptic digestion analysis demonstrated that these mono-PEG-sCTs are 3 positional isomers of N-terminus, Lys18- and Lys11-residue modified mono-PEGylated sCTs. The degradation half-life of these 3 positional isomers in rat kidney homogenates significantly increased in order of the N-terminus (125.5 min), Lys11- (157.3 min), and Lys18 residue modified mono-PEGylated sCT (281.5 min) over the native sCT (4.8 min). CONCLUSION: Three positional isomers of mono-PEGylated sCTs were purified and characterized. Of these, the resistance to proteolytic degradation was highest for the Lys18-residue modified mono-PEG-sCT. These studies demonstrate that the in vivo stability of PEGylated sCTs is highly dependent on the site of PEG molecule attachment.

Preparation and characterization of polyethylene-glycol-modified salmon calcitonins.

The conjugation of salmon calcitonin (sCT) by covalent linkage of polyethylene glycol (PEG) was attempted to overcome several disadvantages of sCT as a therapeutic drug, namely its rapid clearance from blood circulation and enzymatic degradation. The polymer employed was succinimidyl carbonate monomethoxypolyethylene glycol (12 kDa). Superose HR size-exclusion chromatography was applied to separate the PEGylated sCTs (mono-PEG-sCT and di-PEG-sCT) from the unmodified sCT. The PEGylation of sCT was verified by an electrophoresis gel stained with iodine and by MALDI-TOF mass spectrometry. The molecular weights of mono-PEG-sCT and di-PEG-sCT were determined to be 16,094 and 29,077 Da, respectively. PEGylated sCTs showed a substantially improved stability in rat liver homogenates as compared to the intact sCT, indicating that PEG molecules protected sCT from various degrading enzymes. These PEGylated sCTs exhibited similar biological activity to the intact sCT by adenosine cyclic 3',5'-phosphate (cAMP) assay. In clearance studies in the rat, PEGylated sCTs had significantly longer circulating half-lives than the intact sCT (11.2 min for mono-PEG-sCT and 54.0 min for di-PEG-sCT versus 4.7 min for intact sCT).

MR findings of secondary hemochromatosis: transfusional vs erythropoietic.

OBJECTIVE: The aim of this study was to demonstrate the MR characteristics of secondary hemochromatosis (transfusional versus erythropoietic). MATERIALS AND METHODS: Magnetic resonance images of five patients with transfusional (n = 3) or erythropoietic (n = 2) hemochromatosis were reviewed. RESULTS: The liver of all patients had low signal intensity in all pulse sequences. The spleen had low signal intensity in all patients with transfusional iron overload, but normal signal intensity in erythropoietic hemochromatosis, which had similar MR findings to idiopathic hemochromatosis. However, the pancreas had variable signal intensity. CONCLUSION: On MRI the signal intensity of the spleen may allow distinction between transfusional and erythropoietic hemochromatosis.

Regulation of tyrosine biosynthesis by phenylalanine in anthramycin-producing Streptomyces refuineus.

The regulation of tyrosine production in the anthramycin-producing organism Streptomyces refuineus var. thermotolerans has been studied with wild-type and tyrosine auxotrophic organisms. Growth of the auxotroph on minimal medium plus phenylalanine suggested that phenylalanine may increase the supply of tyrosine. In incubation with whole cells, tyrosine levels increased in response to added phenylalanine. However, no radiolabeled tyrosine was detected after incubation with 14C-phenylalanine. Thus, no phenylalanine hydroxylase is present. Phenylalanine was found to feedback inhibit prephenate dehydratase, resulting in an increase in NAD-dependent prephenate dehydrogenase activity, thus channeling prephenic acid toward tyrosine.