Sensitive and rapid HPLC quantification of tenofovir from hyaluronic acid-based nanomedicine

The purpose of this study was to develop and validate a rapid, sensitive, and specific reversed-phase high-performance liquid chromatography method for the quantitative determination of native tenofovir (TNF) for various applications. Different analytical performance parameters such as linearity, precision, accuracy, limit of quantification (LOQ), limit of detection (LOD), and robustness were determined according to International Conference on Harmonization (ICH) guidelines. A Bridge™ C18 column (150 × 4.6 mm, 5 μm) was used as stationary phase. The retention time of TNF was 1.54 ± 0.03 min (n = 6). The assay was linear over the concentration range of 0.1–10 μg/mL. The proposed method was sensitive with LOD and LOQ values equal to 50 and 100 ng/mL, respectively. The method was accurate with percent mean recovery from 95.41% to 102.90% and precise as percent RSD (relative standard deviation) values for intra-day, and inter-day precision were less than 2%. This method was utilized for the estimation of molar absorptivity of TNF at 259 nm (ε ₂₅₉ = 12,518 L/mol/cm), calculated from linear regression analysis. The method was applied for determination of percentage of encapsulation efficiency ( 22.93 ± 0.04%), drug loading (12.25 ± 1.03%), in vitro drug release profile in the presence of enzyme (43% release in the first 3 h) and purification analysis of hyaluronic acid-based nanomedicine.     

Hydrogen formation by an arsenate-reducing Pseudomonas putida, isolated from arsenic-contaminated groundwater in West Bengal, India

Anaerobic growth of a newly isolated Pseudomonas putida strain WB from an arsenic-contaminated soil in West Bengal, India on glucose, l-lactate, and acetate required the presence of arsenate, which was reduced to arsenite. During aerobic growth in the presence of arsenite arsenate was formed. Anaerobic growth of P. putida WB on glucose was made possible presumably by the non-energy-conserving arsenate reductase ArsC with energy derived only from substrate level phosphorylation. Two moles of acetate were generated intermediarily and the reducing equivalents of glycolysis and pyruvate decarboxylation served for arsenate reduction or were released as H₂. Anaerobic growth on acetate and lactate was apparently made possible by arsenate reductase ArrA coupled to respiratory electron chain energy conservation. In the presence of arsenate, both substrates were totally oxidized to CO₂ and H₂ with part of the H₂ serving for respiratory arsenate reduction to deliver energy for growth. The growth yield for anaerobic glucose degradation to acetate was Y _Glucose = 20 g/mol, leading to an energy coefficient of Y _ATP = 10 g/mol adenosine-5'-triphosphate (ATP), if the Emden–Meyerhof–Parnas pathway with generation of 2 mol ATP/mol glucose was used. During growth on lactate and acetate no substrate chain phosphorylation was possible. The energy gain by reduction of arsenate was Y _Arsenate = 6.9 g/mol, which would be little less than one ATP/mol of arsenate.     

Comparison of metabolite levels in callus of <Emphasis Type="Italic">Tecoma stans</Emphasis> (L.) Juss. ex Kunth. cultured in photoperiod and darkness

Tecoma stans is a tropical plant from the Americas. Antioxidant activity and both phenolic compound and flavonoid total content were determined for callus tissue of T. stans cultured in either a set photoperiod or in darkness. Callus lines from three explant types (hypocotyls, stem, and leaf) were established on B5 culture medium supplemented with 0.5 μM 2,4-D and 5.0 μM kinetin. While leaf-derived callus grew slower under a 16-h photoperiod (specific growth rate, μ = 0.179 d⁻¹, t _D = 3.9 d) than in darkness (μ = 0.236 d⁻¹, t _D = 2.9 d), it accumulated the highest amount (p < 0.05) of both phenolics (86.6 ± 0.01 mg gallic acid equivalents/g) and flavonoids (339.6 ± 0.06 mg catechin equivalents/g). Similarly, antioxidant activity was significantly higher (p < 0.05) when callus was cultured in period light than when grown in extended darkness. Antioxidant activity measured with a 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS)-based assay was 350.5 ± 15.8 mmol Trolox/g extract for callus cultured under a defined photoperiod compared to 129.1 ± 7.5 mmol Trolox/g extract from callus cultured in darkness. Content of phenolic compounds and flavonoids was in agreement with a better antioxidant power (EC₅₀ = 450 μg extract/mg 1,1-diphenyl-2-picrylhydrazyl) and antiradical efficiency. Results of the present study show that calli of T. stans are a source of compounds with antioxidant activity that is favored by culture under a set photoperiod.     

Effect of Selenium,Zinc, and Copper Supplementation on Blood Metabolic Profile in Male Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) Calves

Twenty male buffalo calves (15 months, 200.2 ± 9.75) were divided into four groups of five animals in each and fed diets without (T1) or supplemented with 0.3 ppm selenium (Se) + 40 ppm zinc (Zn) (T2), 0.3 ppm Se + 40 ppm Zn + 10 ppm copper (Cu) (T3), and 40 ppm Zn + 10 ppm Cu (T4) for 120 days, during which blood samples were collected on days 0, 40, 80, and 120. Concentrations of glucose, total protein, albumin, globulin, urea, uric acid, and creatinine were similar in all the four groups. The level of different serum enzymes viz. lactate dehydrogenase, alkaline phosphatase, glutamate pyruvate transaminase, and glutamate oxaloacetate transaminase, and hormones viz. T₃, T₄, testosterone and insulin were similar (P > 0.05) among the four groups but the ratio of T₄/T₃ was reduced (P < 0.05) in the groups (T2 and T3) where selenium was supplemented at 120th day of supplementation. It was deduced that supplementation of 0.3 ppm Se and/or 10.0 ppm of Cu with 40 ppm Zn had no effect on blood metabolic profile in buffalo calves, except the ratio of T₄ and T₃ hormone which indicates that selenium plays an important role in converting T₄ hormone to T₃ which is more active form of thyroid hormone.     

NMR Reinvestigation of the Caffeine–Chlorogenate Complex in Aqueous Solution and in Coffee Brews

Caffeine complexation by chlorogenic acid (3-caffeoylquinic acid, CAS Number [327-97-9]) in aqueous solution as well as caffeine–chlorogenate complex in freshly prepared coffee brews have been investigated by high-resolution ¹H-NMR. Caffeine and chlorogenic acid self-associations have also been studied and self-association constants have been determined resorting to both classical isodesmic model and a recently introduced method of data analysis able to provide also the critical aggregation concentration (cac). Furthermore, caffeine–chlorogenate association constant was measured. For the caffeine, the average value of the self-association constant determined by isodesmic model (K _i = 7.6 ± 0.5 M⁻¹) is in good agreement with the average value (K _a = 10 ± 1.8 M⁻¹) determined with the method which permits the determination of the cac (8.43 ± 0.05 mM). Chlorogenic acid shows a slight decreased tendency to aggregation with a lower average value of association constants (K _i = 2.8 ± 0.6 M⁻¹; K _a = 3.4 ± 0.6 M⁻¹) and a critical concentration equal to 24 ± 1 mM. The value of the association constant of the caffeine–chlorogenate complex (30 ± 4 M⁻¹) is compatible with previous studies and within the typical range of reported association constants for other caffeine–polyphenol complexes. Structural features of the complex have also been investigated, and the complex conformation has been rediscussed. Caffeine chemical shifts comparison (monomeric, complexed, coffee brews) clearly indicates a significant amount of caffeine is complexed in beverage real system, being chlorogenate ions the main complexing agents.     

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B₁

Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B₁ and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin B₁. We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as 2-keto hydrolyzed fumonisin B₁. Pyruvate was found to be the preferred co-substrate and amino group receptor (K _M = 490 μM at 10 μM hydrolyzed fumonisin B₁) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10 μM. The enzyme showed activity in the range of pH 6 to 10 with an optimum at pH 8.5, and in the range of 6°C to 50°C with an optimum at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation at pH 4.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1 h, and the residual activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters K _M = 1.1 μM and k _cat = 104/min were determined with 5 mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification of hydrolyzed fumonisins seems possible, if the enzyme properties are considered.     

Magnesium Sulfate in Emergency Department Patients with Hypertension

To compare the effect of IV magnesium with other antihypertensives in emergency department (ED) patients with hypertension. ED patients with a systolic BP > 135 mmHg or diastolic BP > 85 were approached for entry into the study. Those granting consent were randomly placed into one of three treatment groups: (1) 1.5 gm IV MgSO₄ (n = 42), (2) a parenteral or oral antihypertensive agent (n = 41), (3) both IV MgSO₄ and an antihypertensive agent (n = 44). Systolic and diastolic blood pressures were measured at entry into the study and at 15, 30, 45, and 60 min after magnesium or other antihypertensive medications were given. The main outcome measure was blood pressure at 60 min, and results were compared using one-way analysis of variance with the post hoc Tukey HSD test. Compared to systolic and diastolic blood pressures at time 0, both were lower at 15, 30, 45, and 60 min in all groups (p < 0.05). No significant difference in systolic or diastolic BP at any time point was observed when response to treatment was compared between the three groups. Intravenous MgSO₄ is as effective as antihypertensives at lowering BP in emergency department patients.     

Ratio Spectra Derivative and Zero-Crossing Difference Spectrophotometric Determination of Olmesartan Medoxomil and Hydrochlorothiazide in Combined Pharmaceutical Dosage Form

Two simple, economical, rapid, precise, and accurate methods for simultaneous determination of olmesartan medoxomil and hydrochlorothiazide in combined tablet dosage form have been developed. The first method is based on ratio spectra derivative spectrophotometry, and the second method is zero-crossing difference spectrophotometry. The amplitudes in the first derivative of the corresponding ratio spectra at 231.0 and 271.0 nm were selected to determine olmesartan medoxomil and hydrochlorothiazide, respectively. Measurements of absorbance were carried out at zero-crossing wavelengths 257.8 and 240.2 nm for olmesartan medoxomil and hydrochlorothiazide by zero-crossing difference spectrophotometric method. Beer’s law is obeyed in the concentration range of 08–24 μg/mL for olmesartan medoxomil (OLM) and 05–15 μg/mL for hydrochlorothiazide (HCT) by ratio spectra derivative and 05–30 μg/mL for OLM and HCT by zero-crossing difference spectrophotometric method. The results of the assay were found to be 100.46 ± 0.95 for OLM and 100.4 ± 0.27 for HCT by ratio spectra derivative and 99.06 ± 1.14 for OLM and 100.05 ± 0.90 for HCT by zero-crossing difference spectrophotometric method. These methods passes F test and t test. Both methods were validated statistically and by performing recovery study.     

Purification and characterisation of a novel starch synthase selective for uridine 5′-diphosphate glucose from the red alga Gracilaria tenuistipitata

Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules in the cytosol. Starch synthase activity in crude extracts of Gracilaria tenuistipitata Chang et Xia was almost 9-fold higher with UDP[U-¹⁴C]glucose than with ADP[U-¹⁴C]glucose. The activity with UDP[U-¹⁴C]glucose was sensitive to proteolytic and oxidative inhibition during extraction whilst the activity with ADP[U-¹⁴C]glucose appeared unaffected. This indicates the presence of separate starch synthases with different substrate specificities in G. tenuistipitata. The UDPglucose: starch synthase was purified and characterised. The enzyme appears to be a homotetramer with a native M_r of 580 kDa and displays kinetic properties similar to other α-glucan synthases such as stimulation by citrate, product (UDP) inhibition and broad primer specificity. We propose that this enzyme is involved in cytosolic starch synthesis in red algae and thus is the first starch synthase described that utilises UDPglucose in vivo. The biochemical implications of the different compartmentalisation of starch synthesis in red algae and green algae/plants are also discussed. Received: 29 January 1999 / Accepted: 11 March 1999     

Highly Sensitive Resonance Scattering Detection of DNA Hybridization Using Aptamer-Modified Gold Nanopaticle as Catalyst

Gold nanoparticle particles in size of 10 nm were used to label the thiol-modified single-stranded DNA aptamer (SH-ssDNA) to obtain an aptamer-modified gold nanoparticle probe (AussDNA) for target DNA (tDNA). In pH 7.4 NaH₂PO₄–Na₂HPO₄ buffer solution, the hybridization reaction between AussDNA and tDNA took place to form larger aptamer-modified gold nanoparticle cluster complex. The excess aptamer-modified gold nanoparticle probe in the supernatant solutions was obtained by centrifuging and can be used as nanocatalyst for the 0.276 mmol/L CuSO₄-65.4 mmol/L potassium-sodium tartrate-0.37 mmol/L glucose system at 70 °C. The cubic Cu₂O particles generated by the nanocatalytic reducing exhibit a strong resonance scattering (RS) peak at 620 nm. In the selected conditions, the RS intensity at 620 nm decreased with addition of tDNA, and the decreased intensity ΔI _{620 nm} is proportional to tDNA concentration (C _tDNA) from 0.12 to 72 pM, with regress equation of ΔI _620 nm = 1.29C _tDNA + 4.05, correlation coefficient of 0.9917, and detection limit of 0.084 pM tDNA.     

Scanning force microscopy studies of X-ray-induced double-strand breaks in plasmid DNA

We present an analysis of X-ray-induced damage in ΦX174 plasmid DNA, applying doses between D = 250 and 1,500 Gy. To analyse this damage in detail, the distribution of plasmid fragments after irradiation have been determined by scanning force microscopy. The results show that even for the lowest dose of D = 250 Gy, a significant amount of double-strand breaks are observed. For increasing dose, the percentage of small fragments increases and is accompanied by a shortening of the average fragment length from < L> = 1,400 nm for a dose of D = 250 Gy to < L> = 1,080 nm after irradiation with D = 1,500 Gy. The most crucial parameter, the average number of double-strand breaks per broken plasmid (<DSB_b> ) has been determined for the first time for the applied doses. The results show that the average number of DSBs per broken plasmid <DSB_b> increases almost linearly from a value of <DSB_b> = 1.3 after irradiation with D = 250 Gy to <DSB_b> = 1.7 after exposure to D = 1,500 Gy. The presented results show that the amount of DSBs induced by X-ray radiation in plasmid DNA can be calculated with high accuracy by means of scanning force microscopy, providing relevant information regarding the interaction of X-rays with DNA molecules.

Experimental and theoretical investigations on the structure-properties interrelationship of the gadolinium-vanadate-germanate glasses

Glasses in the system xGd₂O₃·(100-x)[GeO₂·V₂O₅] with 0 ≤ × ≤ 20 mol% have been prepared from the melt quenching method. In this paper, we investigated changes in germanium coordination number in gadolinium-vanadate-germanate glasses through molar volume analysis, measurements of densities, investigations of FTIR and UV-VIS spectroscopy, calculations of density functional theory (DFT). Analyzing the structural changes resulted from the IR spectra we found that the gadolinium ions have a pronounced affinity toward [VO₄] structural units which contain non-bridging oxygens necessary for the charge compensation. The introduction of the excess of oxygen yields the formation of [VO₅] structural units. This attains maximum value at 5 mol% Gd₂O₃, in agreement with the density measurements. Further, the addition of the surplus of oxygen implies the transformation of [VO₅] to [VO₄] structural units and the formation of VO₄⁻³ orthovanadate structural units. The UV-VIS spectra show a broad UV absorption band located in the 300–500 nm region. These bands are assumed to originate from the combination of vanadium ions possibly present in the three states of valence. The presence of Ge-Ge wrong bonds attains its maximum values in the samples with x = 5 and 15 mol% Gd₂O₃ (bands centered in the 250–300 nm range). DFT calculations show the massive vibrations of the [VO_n] structural units coupled with each other via [GeO₆] and [GeO₄] structural units. This leads to the splitting of the bridge modes and a multiplication of the number of these bands.     

Cavamax W7 composite ethosomal gel of clotrimazole for improved topical delivery: development and comparison with ethosomal gel

The present research work was aimed to formulate clotrimazole encapsulated Cavamax W7 composite ethosomes by injection method for improved delivery across epidermis. 3² factorial design was used to design nine formulations (F1-F9) and compared with ethosomal formulations (F10-F12). F9 with vesicle size of 202.8 ± 4.8 nm, highest zeta potential (−83.6 ± 0.96 mV) and %EE of 98.42 ± 0.15 was selected as optimized composite ethosome and F12 as reference ethosomal formulation. As revealed by transmission electron microscopy F9 vesicles were more condensed, uniformly spherical in shape than F12 vesicles. Vesicular stability studies indicated F9 to be more stable as compared to F12. Both F9 and F12 were incorporated in carbopol 934 gel base to get G1–G8 gel formulations and evaluated for in vitro skin permeability. Cavamax W7 composite ethosomal optimized gel (G5) showed higher in vitro percent cumulative drug permeation (88.53 ± 2.10%) in 8 h and steady state flux (J _ss) of 3.39 ± 1.45 μg/cm²/min against the J _ss of 1.57 ± 0.23 μg/cm²/min for ethosomal gel (G1) and 1.13 ± 0.06 μg/cm²/min for marketed formulation. The J _ss flux of G5 was independent of amount of drug applied/unit area of skin. In vivo confocal laser scanning microscopic study of G5 depicted uniform and deeper penetration of rhodamine B (marker) in epidermis from Cavamax W7 composite ethosomal gel in comparison to G1. Finally, G5 demonstrated better (p < 0.05) antifungal activity against Candida albicans and Aspergillus niger than G1 thus, signifying that Cavamax W7 composite ethosomes present a superior stable and efficacious vesicular system than ethosomal formulation for topical delivery of clotrimazole.     

Investigating Fluorescence Quenching of ZnS Quantum Dots by Silver Nanoparticles

Water dispersible zinc sulfide quantum dots (ZnS QDs) with an average diameter of 2.9 nm were synthesized in an environment friendly method using chitosan as stabilizing agent. These nanocrystals displayed characteristic absorption and emission spectra having an absorbance edge at 300 nm and emission maxima (λ _emission) at 427 nm. Citrate-capped silver nanoparticles (Ag NPs) of ca. 37-nm diameter were prepared by modified Turkevich process. The fluorescence of ZnS QDs was significantly quenched in presence of Ag NPs in a concentration-dependent manner with K _sv value of 9 × 10⁹ M⁻¹. The quenching mechanism was analyzed using Stern–Volmer plot which indicated mixed nature of quenching. Static mechanism was evident from the formation of electrostatic complex between positively charged ZnS QDs and negatively charged Ag NPs as confirmed by absorbance study. Due to excellent overlap between ZnS QDs emission and surface plasmon resonance band of Ag NPs, the role of energy transfer process as an additional quenching mechanism was investigated by time-resolved fluorescence measurements. Time-correlated single-photon counting study demonstrated decrease in average lifetime of ZnS QDs fluorescence in presence of Ag NPs. The corresponding F?rster distance for the present QD–NP pair was calculated to be 18.4 nm.     

Chitosan and Glyceryl Monooleate Nanostructures Containing Gemcitabine: Potential Delivery System for Pancreatic Cancer Treatment

The objectives of this study are to enhance cellular accumulation of gemcitabine with chitosan/glyceryl monooleate (GMO) nanostructures, and to provide significant increase in cell death of human pancreatic cancer cells in vitro. The delivery system was prepared by a multiple emulsion solvent evaporation method. The nanostructure topography, size, and surface charge were determined by atomic force microscopy (AFM), and a zetameter. The cellular accumulation, cellular internalization and cytotoxicity of the nanostructures were evaluated by HPLC, confocal microscopy, or MTT assay in Mia PaCa-2 and BxPC-3 cells. The average particle diameter for 2% and 4% (w/w) drug loaded delivery system were 382.3 ± 28.6 nm, and 385.2 ± 16.1 nm, respectively with a surface charge of +21.94 ± 4.37 and +21.23 ± 1.46 mV. The MTT cytotoxicity dose-response studies revealed the placebo at/or below 1 mg/ml has no effect on MIA PaCa-2 or BxPC-3 cells. The delivery system demonstrated a significant decrease in the IC50 (3 to 4 log unit shift) in cell survival for gemcitabine nanostructures at 72 and 96 h post-treatment when compared with a solution of gemcitabine alone. The nanostructure reported here can be resuspended in an aqueous medium that demonstrate increased effective treatment compared with gemcitabine treatment alone in an in vitro model of human pancreatic cancer. The drug delivery system demonstrates capability to entrap both hydrophilic and hydrophobic compounds to potentially provide an effective treatment option in human pancreatic cancer.     

Rice germ oil as multifunctional excipient in preparation of self-microemulsifying drug delivery system (SMEDDS) of tacrolimus

Surmounting the constraints of limited solubilization efficiency and prime requisite of antioxidant for conventional lipid formulations, the research work explores an edge over formulation utilizing potential applicability of rice germ oil (RGO) as a multifunctional excipient. Self-microemulsifying drug delivery system (SMEDDS) of tacrolimus (TAC) was formulated with RGO, an indigenous source of gamma-oryzanol. Being the same biological source, RGO and rice bran oil (RBO) were compared and it was found that RGO have more solubilization potential for TAC (2.2-fold) as well as higher antioxidant activity (8.06-fold) than the RBO. TAC-SMEDDS was prepared using RGO/Capmul PG8 (2:3) as an oil phase, Cremophore EL as a surfactant, and Transcutol P as a cosurfactant. The approximate particle size of TAC-SMEDDS was found to be 38 nm by dynamic light scattering and 12 nm by small angle neutron scattering. The in vitro dissolution studies showed complete and rapid drug release in 30 min compared to a plain drug (<5%) and marketed capsule (<50%). AUC and C _max were found to be 45.05 ± 15.64 ng h/ml and 3.91 ± 1.2 ng/ml for TAC-SMEDDS, 12.59 ± 5.54 ng h/ml and 0.48 ± 0.12 ng/ml for plain TAC, and 30.23 ± 10.34 ng h/ml and 2.31 ± 0.68 ng/ml for marketed formulation, respectively. The improved pharmacokinetic profile of TAC-SMEDDS is correlating to the dissolution results. Thus, gamma-oryzanol-enriched RGO acts as a potential multifunctional excipient for lipid formulations.     

O-Acetylation of plant cell wall polysaccharides: identification and partial characterization of a rhamnogalacturonan O-acetyl-transferase from potato suspension-cultured cells

 A microsomal preparation from suspension-cultured potato stem cells (Solanum tuberosum L. cv. AZY) was incubated with [¹⁴C]acetyl-CoA resulting in a precipitable radiolabeled product. Analysis of the product revealed that it consisted mostly of acetylated proteins and cell wall polysaccharides, including xyloglucan, homogalacturonan and rhamnogalacturonan I. Thus, acetyl-CoA is a donor-substrate for the O-acetylation of wall polysaccharides. A rhamnogalacturonan acetylesterase was used to develop an assay to measure and characterize rhamnogalacturonan O-acetyl transferase activity in the microsomal preparation. Using this assay, it was shown that the transferase activity was highest during the linear growth phase of the cells, had a pH-optimum at pH 7.0, a temperature optimum at 30 °C, an apparent K _m of 35 μM and an apparent V _max of 0.9 pkat per mg protein. Further analysis of the radiolabeled acetylated product revealed that it had a molecular mass >500 kDa. Received: 3 July 1999; Accepted: 27 September 1999     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Theoretical study of crown ethers with incorporated azobenzene moiety

A series of crown ethers containing the azobenzene moiety incorporated into crowns of various sizes [Cr(O₆)_, Cr(O₇) and Cr(O₈)] and their corresponding alkali metal cation (Li⁺, Na⁺, K⁺, Rb⁺) complexes have been studied theoretically. The density functional theory (DFT) method was employed to elucidate the stereochemical structural natures and thermodynamic properties of all of the target molecules at the B3LYP/6-31 G(d) and LANL2DZ level for the cation Rb⁺. The fully optimized geometries had real frequencies, thus indicating their minimum-energy status. In addition, the bond lengths between the metal cation and oxygen atoms, atomic torsion angles and thermodynamic energies for complexes were studied. Natural bond orbital (NBO) analysis was used to explore the origin of the internal forces and the intermolecular interactions for the metal complexes. The calculated results show that the most significant interaction is that between the lone pair electrons of electron-donating oxygens in the cis-forms of azobenzene crown ethers (cis-ACEs) and the LP* (1-center valence antibond lone pair) orbitals of the alkali-metal cations (Li⁺, Na⁺, K⁺ and Rb⁺). The electronic spectra for the cis-ACEs [cis-Cr(O₆), cis-Cr(O₇) and cis-Cr(O₈)] are obtained by the time-dependent density functional theory (TDDFT) at the B3LYP/6-31 G(d) level. The spectra of the cis-isomers show broad π → π* (S₀ → S₂) absorption bands at 310–340 nm but weaker n → π* (S₀ → S₁) bands at 480–490 nm. The calculated results are in good agreement with the experimental results.     

Cottonseed feeding delivers sufficient quantities of gossypol as a male deer contraceptive

To define the quantitative and qualitative effects of gossypol (GP) on deer (Cervus elaphus) semen, the animals were fed cottonseed (CS). Adult stags each received 350 g of CS for 109 days. Animals received 15 mg of gossypol per kilogram body weight per day. Quantitative and qualitative parameters of experimental ejaculates (n = 182) were compared to ejaculates (n = 571) of control animals (n = 5) collected during three previous natural reproductive seasons. Ejaculate fractions were evaluated by classical methods used in domestic animals. In this paper, we show that mature male deer fed CS exhibited morphological changes and decreased motility of spermatozoa and abnormalities in spermatogenesis. Radioimmunoassay measured concentrations of various steroid hormones (T-testosterone, A₄-androstenedione, and E₂-estradiol 17β) in separated ejaculate fractions of the CS group were compared to a control group of stags. Generally, mean steroid concentrations in CS-treated deer decreased during the entire sampling period in examined ejaculate fractions. These changes resulted in decreased semen quality with no detectable side effects in the animals. It seems that gossypol fed to the deer in the form of CS serves as an efficient male contraceptive.