Oestradiol and the preovulatory surges of luteinising hormone and follicle stimulating hormone in ewes during the breeding season and transition into anoestrus

This study was designed to see if giving exogenous oestradiol, during the follicular phase of the oestrous cycle of intact ewes, during the breeding season or transition into anoestrus, would alter the occurrence, timing or magnitude of the preovulatory surge of secretion of luteinising hormone (LH) or follicle stimulating hormone (FSH). During the breeding season and the time of transition, separate groups of ewes were infused (intravenously) with either saline (30 ml h⁻¹; n = 6) or oestradiol in saline (n = 6) for 30 h. Infusion started 12 h after removal of progestin-containing intravaginal sponges that had been in place for 12 days. The initial dose of oestradiol was 0.02 μg h⁻¹; this was doubled every 4 h for 20 h, followed by every 5 h up to 30 h, to reach a maximum of 1.5 μg h⁻¹. Following progestin removal during the breeding season, peak serum concentrations of oestradiol in control ewes were 10.31 ± 1.04 pg ml⁻¹, at 49.60 ± 3.40 h after progestin removal. There was no obvious peak during transition, but at a time after progestin removal equivalent to the time of the oestradiol peak in ewes at mid breeding season, oestradiol concentrations were 6.70 ± 1.14 pg ml⁻¹ in ewes in transition (P < 0.05). In oestradiol treated ewes, peak serum oestradiol concentrations (24.8 ± 2.1 pg ml⁻¹) and time to peak (41.00 ± 0.05 h) did not differ between seasons (P > 0.05). During the breeding season, all six control ewes and four of six ewes given oestradiol showed oestrus with LH and FSH surges. The two ewes not showing oestrus did not respond to oestrus synchronisation and had persistently high serum concentrations of progesterone. During transition, three of six control ewes showed oestrus but only two had LH and FSH surges; all oestradiol treated ewes showed oestrus and gonadotrophin surges (P < 0.05). The timing and magnitude of LH and FSH surges did not vary with treatment or season. In blood samples collected every 12 min for 6 h, from 12 h after the start of oestradiol infusion, mean serum concentrations of LH and LH pulse frequency were lower in control ewes during transition than during mid breeding season (P < 0.05). Oestradiol treatment resulted in lower mean serum concentrations of LH in season and lower LH pulse frequency in transition (P < 0.05). We concluded that enhancing the height of the preovulatory peak in serum concentrations of oestradiol during the breeding season did not alter the timing or the magnitude of the preovulatory surge of LH and FSH secretion and that at transition into anoestrus, oestradiol can induce oestrus and the surge release of LH and FSH as effectively as during the breeding season.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Copper(II) complexes with (2-hydroxybenzyl-2-pyridylmethyl)amine–Hbpa: syntheses, characterization and crystal structures of the ligand and [Cu(II)(Hbpa)2](ClO4)2·2H2O

Copper(II) complexes were synthesized and characterized by means of elemental analysis, IR and visible spectroscopies, EPR and electrochemistry, as well as X-ray structure crystallography. The group consists of discrete mononuclear units with the general formula [Cu(II)(Hbpa)₂](A)₂·nH₂O, where Hbpa=(2-hydroxybenzyl-2-pyridylmethyl)amine and A=ClO₄ ⁻, n=2 (1), CH₃COO⁻, n=3 (2), NO₃ ⁻, n=2 (3) and SO₄ ²⁻, n=3 (4). The structures of the ligand Hbpa and complex 1 have been determined by X-ray crystallography. Complexes 1–4 have had their UV–Vis spectra measured in both MeCN and DMF. It was observed that the compounds interact with basic solvents, such that molecules coordinate to the metal in axial positions in which phenol oxygen atoms are coordinated in the protonated forms. The values were all less than 1000 M⁻¹ cm⁻¹. EPR measurements on powdered samples of 1–3 gave g/A values between 105 and 135 cm⁻¹, typical for square planar coordination environments. Complex 4·3H₂O exhibits a behaviour typical for tetrahedral coordination. The electrochemical behaviour for complexes 1 and 2 was studied showing irreversible redox waves for both compounds.     

The crystal and molecular structures of lithium trimethylenediaminetetraacetatoferrate(III) trihydrate Li[Fe(trdta)]·3H2O and sodium ethylenediamine-N,N′-diacetato-N,N′-di-3-propionatoferrate(II) pentahydrate Na[Fe(eddda)]·5H2O

The crystal structures of Li[Fe(trtda)]·3H₂O and Na[Fe(eddda)]·5H₂O (trtda = trimethylenediaminetetraacetate and eddda = ethylenediamine-N,N′-diacetate-N,N′-di-3-propionate) have been determined by single crystal X-ray diffraction techniques. The former crystal was monoclinic with the space group P2₁/n,a = 17.775(3),b = 10.261(1),c = 8.883(2)Å, β = 95.86(4)° and Z = 4. The latter was also monoclinic with the space group P2₁/n,a = 6.894(2),b = 20.710(6),c = 13.966(3)Å, β = 101.44(2)° and Z = 4. Both complex anions were found to adopt an octahedral six-coordinated structure with all of six ligand atoms of trdta⁴⁻ or eddda⁴⁻ coordinated to the Fe(III) ion, unlike the corresponding edta⁴⁻ complex which is usually seven-coordinate with the seventh coordination site occupied by H₂O. Of the three geometrical isomers possible for the eddda complex, the trans(O₅) isomer was actually found in the latter crystal. Factors determining the structural types of metal–edta complexes are discussed in detail.     

A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg_DCW⁻¹ (dry cell weight). The kinetic parameters, K_m and V_max, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min⁻¹ mg_DCW⁻¹, respectively. The measured apparent activation energy, 25.54 kJ mol⁻¹, indicated a partial control by mass transport, more likely through the cell wall.

UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg  ml⁻¹) indicated: lower diffusional control (E_a=37.73 kJ mol⁻¹); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg  ml⁻¹ of resting cells, with a maximum volumetric productivity of 0.5 g l⁻¹ h⁻¹.     

Structures and magnetism of rubidium and cesium salts of dimeric oxovanadium(IV) tartrate(4−) complexes

Complexes of type A₄[VO(tart)]₂·nH₂O, where A = Rb or Cs and tart =d,l-tartrate(4−) (n = 2) or d,d-tartrate(4−) (n = 2 for Rb and n = 3 for Cs), were prepared from an aqueous mixture of V₂O₅, AOH and H₄tart. These complexes were studied by single-crystal X-ray diffraction methods: Rb₄[VO(d,l-tart)]₂·2H₂O, space group P1 with a = 8.156(1),b = 8.246(1),c = 8.719(1)Å, = 66.09(1)°, β = 65.07(1)°, γ = 82.40(1)°,Z = 2, 1917 observed reflections, and final R_w = 0.035; Cs₄[VO(d,l-tart)]₂·2H₂O, space group P₂₁/c with a = 9.350(1),b = 13.728(2),c = 8.479(1)Å, β = 106.77(1)°,Z = 4, 2235 observed reflections, and final R_w = 0.054; Rb₄[VO(d,d-tart)]₂·2H₂O, space group P₄₁₂₂ with a = 8.072(1),c = 32.006(3)Å,Z = 8, 1014 observed reflections and final R_w = 0.038; Cs₄[VO(d,d-tart)]₂·3H₂O, space group P₁₂₂ with a = 8.184(1),c = 33.680(5)Å,Z = 8, 1310 observed reflections, and final R_w = 0.063. Bulk magnetic susceptibility data (1.5–300 K) for these compounds and A₄[VOl,l-tart)]₂·nH₂O (A = Rb, Cs) were obtained on polycrystalline samples. These data were analyzed in terms of a Van Vleck exchange coupled S = 1/2 model which was modified to include an interdimer exchange parameters Θ. Analysis of the low-temperature (1.5–20 K) susceptibility data gave 2J = +1.30 cm⁻¹ and Θ = −1.86 K for Rb₄[VO(d,l-tart)]₂·2H₂O, 2J = +1.16 cm⁻¹ and Θ = −1.69 K for Cs₄[VO(d,l-tart)]₂·2H₂O, 2J = +1.90 cm⁻¹ and Θ = −0.82 K for Rb₄[VO(d,d-tart)]₂·2H₂O, 2J = +2.04 cm⁻¹ and Θ = −0.80 K for Rb₄[VO(l,l-tart)]₂·2H₂O, 2J = +1.52 cm⁻¹ and Θ = −0.25 K for Cs₄[VO(d,d-tart)]₂·3H₂O, and 2J = +1.64 cm⁻¹ and Θ = −0.31 K for Cs₄[VO(l,l-tart)]₂·3H₂O. These results suggest the magnitudes of intradimer (ferromagnetic and interdimer (antiferromagnetic) exchange interactions are similar in these complexes, as observed for the analogous Na salts.     

Rate of electron transfer between plastocyanin, cytochrome ƒ, related proteins and artificial redox reagents in solution

The rate of electron transfer between reduced cytochrome ƒ and plastocyanin (both purified from parsley) has been measured as k = 3.6 · 10⁷ M⁻¹ · s⁻¹, at 298 °K and pH 7.0, with activation parameters ΔH^‡ = 44 kJ · mole⁻¹ and ΔS^‡ = +46 J · mole⁻¹ · °K⁻¹. Replacement of cytochrome ƒ with red algal cytochrome c-553, Pseudomonas cytochrome c-551 and mammalian cytochrome c gave rates at least 30 times slower: k = 5 · 10⁵, 7.5 · 10⁵ and 1.0 · 10⁶ M⁻¹ · s⁻¹, respectively.

Similar measurements made with azurin instead of plastocyanin gave k = 6 · 10⁶ and approx. 2 · 10⁷ M⁻¹ · s⁻¹ for reaction of reduced azurin with cytochrome ƒ and algal cytochrome respectively.

Rate constants of 115 and 80 M⁻¹ · s⁻¹ were found for reduction of plastocyanin by ascorbate and hydroquinone at 298 °K and pH 7.0. The rate constants for the oxidation of plastocyanin, cytochrome ƒ, Pseudomonas cytochrome c-551 and red algal cytochrome c-553 by ferricyanide were found to be between 3 · 10⁴ and 8 · 10⁴ M⁻¹ · s⁻¹.

The results are discussed in relation to photosynthetic electron transport.     

Effect of growth hormone and γ-aminobutyric acid on Brachionus plicatilis (Rotifera) reproduction at low food or high ammonia levels

Growth hormone (GH, 0.0025 and 0.025 I.U. ml⁻¹) and γ-aminobutyric acid (GABA, 50 μg ml⁻¹) enhance rotifer population growth in batch cultures. In order to further understand the mechanism of their actions, we conducted experiments culturing isolated females at low food and high free ammonia levels. At an optimum food level of 7×10⁶ Nannochloropsis oculata cells ml⁻¹ or at low free ammonia level of 2.4 μg ml⁻¹, the F₁ offspring of rotifers treated with GH at 0.0025 I.U. ml⁻¹ had significantly higher population growth rate (r) and net reproduction rate (Ro), and shorter generation time than untreated rotifers. At a lower food level of 7×10⁵ cells ml⁻¹ or at high free ammonia level of 3.1 μg ml⁻¹, rotifers treated with GABA at 50 μg ml⁻¹ had significantly higher r and Ro, and shorter generation time. These results indicate that GABA is effective in enhancing rotifer reproduction when rotifers are cultured under stress whereas GH enhances rotifer reproduction when culture conditions are optimal. Significant effects were also observed in F¹ and F² generations which were not treated with hormones. These data may be useful for treating rotifer mass cultures to mitigate the effects of stress caused by high population densities.     

Colocalization of NO and VIP in neurons of the submucous plexus in the rat intestine

Since very few previous studies have carried out the quantitative analysis for the colocalization of nitric oxide (NO) and vasoactive intestinal peptide (VIP) in the submucous neurons in the rat digestive tract, we applied in vivo treatment of colchicine to enhance the immunoreactivity and examined the colocalization of NO synthase (nNOS) and VIP in neurons of the submucous plexus throughout the rat digestive tract. The density of nNOS-containing neurons in the submucous plexus in the stomach corpus (103±25 cells/cm², n=3) and that in the antrum (157±9 cells/cm², n=3) were significantly lower than those in small and large intestine. However no difference was detected in the cell density among duodenum (1967±188 cells/cm², n=3), jejunum (2640±140 cells/cm², n=3), ileum (2070±42 cells/cm², n=3), proximal colon (2243±138 cells/cm², n=3) and distal colon (2633±376 cells/cm², n=3). The proportion of nNOS-immunoreactive (IR), nNOS/VIP-IR and VIP-IR neurons to the total number of submucous neurons was examined. nNOS/VIP-IR neurons comprised 45–55% of total number of submucous neurons from the duodenum to the proximal colon, however those comprised 66.4±5.1% in the distal colon. The results showed that the dense distribution of nNOS-containing neurons was found in the submucous plexus throughout the small and large intestine, and large population of submucous neurons co-stored nNOS and VIP.     

Procedural improvements for in vitro production of viable uterine stage caprine embryos

Efforts to improve proportions of caprine immature oocytes developing into viable uterine-stage embryos in vitro involved study of 1924 oocytes in experiments designed to examine influences of fertilization media, sperm incubation temperatures, sperm treatment procedures, different protein supplementations, and different insemination intervals. Oocytecumulus complexes (OCCs) were matured during 27 h in TCM-199 supplemented with 20% FBS, 100 μg LH ml⁻¹, 0.5 μg FSH ml⁻¹, and 1 μg Estradiol-17-β ml⁻¹at 38.5 °C in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere. Freshly collected sperm were washed and incubated at either 22 °C or 38.5 °C for 5 h and then treated with either 0.1 μM calcium ionophore A23187 for 1 min, or with 7.35 mM calcium lactate in the presence of oocytes during the insemination interval, or with 100 μg heparin +2 mM caffeine ml⁻¹ for 15 min. The interval for insemination was experimentally varied i.e. 14 or 24 h. Results showed that: (a) when used as a fertilization medium mDM supported more blastocyst development than TALP (10.5% vs. 0%, P < 0.05); (b) incubation temperatures of 22 °C or 38.5 °C prepared goat spermatozoa equally for capacitation in mDM containing 20% FBS; (c) when oocytes were inseminated with sperm incubated in mDM with 20% FBS and capacitated with calcium lactate more embryos reached the blastocyst stage (P < 0.05) than after incubation in the same conditions but after sperm capacitation with heparin, and A23187 (31.8% vs. 24.2% and 10.2%, respectively; (d) a 24 h insemination interval was not superior to 14 h when sperm were incubated with either 20% FBS or 6 mg BSA ml⁻¹ and capacitated with calcium lactate (P > 0.05). Three morulae resulting from the best conditions in this work (FBS, calcium lactate, 14 h insemination) were transferred into the uterine horn ipsilateral to the corpus luteum of a recipient and two normal female kids were born after normal gestation. This is the first report in which it has been possible to consistently take caprine development to the blastocyst stage in vitro, and to obtain offspring following uterine transfer. Methodology reported here should facilitate implementation of new reproductive and genetic strategies in goat breeding.     

Clara cell protein (CC16) in serum and bronchoalveolar lavage fluid of subjects exposed to asbestos

The Clara cell protein (CC16) is a small and readily diffusible protein of 16kDa secreted by bronchiolar Clara cells in the distal airspaces. These epithelial cells are altered in several pulmonary pathological processes induced by various lung toxicants. In the search for a new biomarker of asbestos-induced lung impairment, we used a sensitive immunoassay to determine the levels of CC16 in bronchoalveolar fluid (BALF) and serum of subjects exposed to asbestos compared with a group of healthy controls. In the BALF of asbestos-exposed subjects there was an insignificant trend towards CC16 elevation compared with controls, with a (mean ±SD of 0.81 ±0.65mg l^-1 for asbestos-exposed subjects (n = 23) versus 0.39 ±0.19mg l^-1 for controls (n = 11) (p = 0.09). In serum, CC16 concentration was significantly increased among asbestos-exposed subjects, with values of 27.2 ±24.0 µg l^-1 for asbestos-exposed subjects (n = 34) versus 16.1 ±7.6 µg l^-1 for controls (n = 34) (p = 0.01). Regarding the effects of smoking, there were significant differences between generally lower CC16 levels in serum and BALF (p = 0.05 and 0.001, respectively) of smokers compared with the higher levels in non-smokers. Serum CC16 levels positively correlated with those in BALF, which is consistent with a diffusional transfer of CC16 from the bronchoalveolar space into the serum. No association, however, emerged between the levels of CC16 in serum or BALF and either the duration of asbestos exposure or the severity of the lung impairment as assessed by chest X-ray. These findings suggest that exposure to asbestos elicits early changes in the local and, importantly, also the systemic levels of CC16. This pneumoprotein therefore appears as a promising non-invasive biomarker of asbestos-induced lung injury and occupational disease in both smoking and non-smoking exposed subjects.     

The reaction between the superoxide anion radical and cytochrome c

1. 1. The superoxide anion radical (O₂⁻) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O₂⁻ and ferrocytochrome c.

2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 10⁶ M⁻¹ · s⁻¹ and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O₂⁻ and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O₂⁻ reacts with the form present below pH 7.45 with k = 1.4 · 10⁶ M⁻¹ · s⁻¹, the form above pH 7.45 with k = 3.0 · 10⁵ M⁻¹ · s⁻¹, and the form present above pH 9.2 with k = 0.

3. 3. The reaction has an activation energy of 20 kJ mol⁻¹ and an enthalpy of activation at 25 °C of 18 kJ mol⁻¹ both above and below pH 7.45. It is suggested that O₂⁻ may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.

4. 4. No reduction of ferricytochrome c by HO₂ radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO₂ radicals oxidize ferrocytochrome c with a rate constant of about 5 · 10⁵–5 · 10⁶ M⁻¹ · s⁻¹

.     

Thermodynamics of the disproportionation of adenosine 5'-diphosphate to adenosine 5'-triphosphate and adenosine 5'-monophosphate, II. Experimental data

High-pressure liquid-chromatography and microcalorimetry have been used to determine equilibrium constants and enthalpies of reaction for the disproportionation reaction of adenosine 5′-diphosphate (ADP) to adenosine 5′-triphosphate (ATP) andadenosine 5′-monophosphate (AMP). Adenylate kinase was used to catalyze this reaction. The measurements were carried out over the temperature range 286 to 311 K, at ionic strengths varying from 0.06 to 0.33 mol kg⁻¹, over the pH range 6.04 to 8.87, and over the pMg range 2.22 to 7.16, where pMg = -log a(Mg²⁺). The equilibrium model developed by Goldberg and Tewari (see the previous paper in this issue) was used for the analysis of the measurements. Thus, for the reference reaction: 2 ADp³⁻ (ao) AMp²⁻ (ao)+ ATp⁻ (ao), K° = 0.225 ± 0.010, ΔG° = 3.70 +- 0.11 kJ mol ⁻¹, ΔH° = −1.5 ± 1. 5 kJ mol ⁻¹, °S ° = −17 ± 5 J mol⁻¹ K⁻¹, and ACP_p°≈ = −46 J mo1l⁻¹ K⁻¹ at 298.15 K and 0.1 MPa. These results and the thermodynamic parameters for the auxiliary equilibria in solution have been used to model the thermodynamics of the disproportionation reaction over a wide range of temperature, pH, ionic strength, and magnesium ion morality. Under approximately physiological conditions (311.15 K, pH 6.94, [Mg²⁺] = 1.35 × 10⁻³ mol kg⁻¹, and I = 0.23 mol kg⁻¹) the apparent equilibrium constant (K_A′ = m(ΣAMP)m(ΣATP)/[ m(ΣADP)]²) for the overall disproportionation reaction is equal to 0.93 ± 0.02. Thermodynamic data on the disproportionation reaction and literature values for this apparent equilibrium constant in human red blood cells are used to calculate a morality of 1.94 × 10⁻⁴ mol kg⁻¹ for free magnesium ion in human red blood cells. The results are also discussed in relation to thermochemical cycles and compared with data on the hydrolysis of the guanosine phosphates.     

Sporulation of Metarhizium anisopliae in solid-state fermentation with forced aeration

Metarhizium anisopliae was grown by solid-state fermentation using a medium based on a mixture of rice bran and rice husk (1:1 on a dry weight basis) where the initial water content was fixed at a value of 47%. The experiments were performed in glass column bioreactors of three different sizes. Moist saturated air was continuously passed through the bottom of each column at a flow rate of 0.34 l h⁻¹ g⁻¹ initial dry matter, and cultivation was performed for two weeks at 27 ± 1°C. The effect of medium packing density was studied in the columns of small size. For initial apparent densities of 0.270 and 0.357 g ml⁻¹, no significant differences were observed either in total biomass production or spore yields; however, when the initial apparent density was increased up to a value of 0.496 g ml⁻¹, both growth and sporulation were strongly affected.

Thermal gradients in the medium and larger columns packed at an apparent density of 0.357 g ml⁻¹ were observed both in the radial and axial directions. Heat build-up effects were analyzed in the larger column. In this case, there were significant differences between different segments of the column both in the water content of the fermented matter and the sporulation pattern.     

Studies on composition and stability of a large membered bacterial consortium degrading phenol

A ten member microbial consortium (AS) consisting of eight phenol-degrading and two non-phenol-degrading strains of bacteria was developed and maintained in a fed-batch reactor by feeding 500 mg l⁻¹ phenol for four years at 28 ± 3 °C. The consortium could degrade 99% of 500 mg l⁻¹ phenol after 24 hours incubation with a biomass increase of 2.6 × 10⁷ to 4 × 10¹² CFU ml⁻¹. Characterization of the members revealed that it consisted of 4 principal genera, Bacillus, Pseudomonas, Rhodococcus, Streptomyces and an unidentified bacterium. Phenol degradation by the mixed culture and Bacillus subtilis, an isolate from the consortium was compared using a range of phenol concentrations (400 to 700 mg l⁻¹) and by mixing with either 160 mg l⁻¹ glucose or 50 mg l⁻¹ of 2,4-dichlorophenol in the medium. Simultaneous utilization of unrelated mixed substrates (glucose/2,4-dichlorophenol) by the consortium and Bacillus subtilis, indicated the diauxic growth pattern of the organisms. A unique characteristic of the members of the consortia was their ability to oxidize chloro aromatic compounds via meta pathway and methyl aromatic compounds via ortho cleavage pathway. The ability of a large membered microbial consortia to maintain its stability with respect to its composition and effectiveness in phenol degradation indicated its suitability for bioremediation applications.     

Antimalarial properties of soy-bean fat emulsions

Intralipid^® and Ivelip^® are commercial preparations of soy-bean lipid extracts used for intravenous supplementation of lipids in various clinical conditions. They were found to inhibit the growth of Plasmodium falciparum in culture with an IC₅₀ of 8.07 ± 2.13 and 13.32 ± 2.05 mg.ml⁻¹, respectively. Intralipid^® rapidly and efficiently inhibited nucleic acid synthesis in cultured P. falciparum, exhibiting full inhibitory activity in less than 2 h. Ivelip^® injected intraperitoneally, was found by the 4-day suppressive test to be active in vivo against P. vinckei petteri within the normal recommended regimen for dietary lipid supply (0.5–4 g.kg⁻¹), but it was impossible to obtain a radical cure even with very high doses (6.4 g.kg⁻¹). Ivelip^® was less effective against P. berghei and P. yoelii nigeriensis. As Ivelip^® showed no interference with the antimalarial activity of chloroquine, it could be considered for use in the treatment of severe human malaria in association with 4-aminoquinolines to expedite the clearance of parasites.     

Reduction of ferricytochrome c by hydrogen atoms: Evidence for intramolecular transfer of reducing equivalent

Direct evidence obtained by means of the technique of pulse radiolysis-kinetic spectrometry, with measurements in the time range 10⁻⁶ to 1 s, is presented that, consequent upon reaction of a single H-atom with a single molecule of ferricytochrome c, a reducing equivalent is transmitted via the protein structure to the ferriheme moiety. Such transmission accounts for at least 70% of the total reduction of the ferri to the ferro state of cytochrome c. The remainder of the total reduction takes place without stages resolvable on the time scale of these experiments. Reduction brought about by H atoms appears to follow a different course than reduction by hydrated electrons. In the latter case, intramolecular transmission of reducing equivalents could not be demonstrated (Lichtin, N. N., Shafferman, A. and Stein, G. (1973) Biochim. Biophys. Acta 314, 117–135).

Not every H-atom reacts with ferricytochrome c at a site which results in conversion of the Fe(III) state to the Fe(II) state. Approximately half of reacting H-atoms do not produce reduction.

The following second order rate constants have been determined in solutions of low ionic strength at 20±2 °C: k[H+ferricytochrome c] = (1.0±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0 and 6.7; k[H+ferrocytochrome c] = (1.3±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0; k[e⁻_aq + ferrocytochrome c] = (1.9±0.4) · 10¹⁰ M⁻¹ · s⁻¹ at pH 6.7.     

A computational formula for heat influx in animal experiments

1. 1.The bahavioural paradigm in which cold-exposed animals can work for pulses of infrared radiation has been extensively used in the literature, but a formula to calculate the amount of heat obtained has not been advanced.

2. 2.This paper describes a computational formula for heat influx in rats: E = 3.64 · 10⁻⁶ · n · d · I · M^0.6 where E is heat influx (kJ), n is number of rewards, d is reward duration (sec), I is irradiance (mW/cm²), and M is body mass (g).

Thiopurine methyltransferase activity: new high-performance liquid chromatographic assay conditions

This paper reports change to our previously published high-performance liquid chromatographic method for the measurement of 6-methylmercaptopurine (6-MMP) in red blood cell lysates. The extraction procedure and chromatographic conditions have been improved and the range of the calibration curves has been modified. The recoveries of 10 and 100 ng ml⁻¹ 6-MMP were 99.0±6.0% and 96.3±4.0% respectively and the limit of quantification was lowered to 5 ng ml⁻¹. This method, which does not require radioactive S-adenosyl- -methionine, is more sensitive, specific and reproducible and may prove useful for routine determination of thiopurine methyltranferase activity in red blood cells.     

Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml⁻¹ was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l⁻¹ h⁻¹, and in reactor, 104,000 U l⁻¹ h⁻¹ was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l⁻¹ h⁻¹, compared to shake-flask fermentations, 12,000 U l⁻¹ h⁻¹. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.