Temperature dependence of the rotation and hydrolysis activities of F1-ATPase

F₁-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F₁-ATPase over the temperature range 4-50°C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 × 10⁶ M⁻¹ s⁻¹ at 4°C to 4.3 × 10⁷ M⁻¹ s⁻¹ at 40°C, whereas the torque estimated at 2 mM ATP remained around 40 pN·nm over 4-50°C. The rotation was stepwise at 4°C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65°C. F₁-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30°C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile.     

Development of a novel methodology for cryopreservation of melanoma cells applied to CSF470 therapeutic vaccine

CSF470 vaccine is a mixture of four lethally irradiated melanoma cell lines, administered with BCG and GM-CSF, which is currently being tested in a Phase II/III Clinical trial in stage II/III melanoma patients. To prepare vaccine doses, irradiated melanoma cell lines are frozen using dimethyl sulfoxide (Me₂SO) and stored in liquid nitrogen (liqN₂). Prior to inoculation, doses must be thawed, washed to remove Me₂SO and suspended for clinical administration. Avoiding the use of Me₂SO and storage in liqN₂ would allow future freeze-drying of CSF470 vaccine to facilitate pharmaceutical production and distribution. We worked on the development of an alternative cryopreservation methodology while keeping the vaccine’s biological and immunogenic properties. We tested different freezing media containing trehalose suitable to remain as excipients in a freeze-dried product, to cryopreserve melanoma cells either before or after gamma irradiation. Melanoma cells incorporated trehalose after 5 h incubation at 37 °C by fluid-phase endocytosis, reaching an intracellular concentration that varied between 70–140 mM depending on the cell line. Optimal freezing conditions were 0.2 M trehalose and 30 mg/ml human serum albumin, at −84 °C. Vaccine doses could be frozen in trehalose at −84 °C for at least four months keeping their cellular integrity, antigen expression and apoptosis/necrosis profile after gamma-irradiation as compared to Me₂SO control. Non-irradiated melanoma cell lines also showed comparable proliferative capacity after both cryopreservation procedures. Trehalose-freezing medium allowed us to cryopreserve melanoma cells, either alive or after gamma irradiation, at −84 °C avoiding the use of Me₂SO and liqN₂ storage. These cryopreservation conditions could be suitable for future freeze-drying of CSF470 vaccine.     

Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury,but respond favorably to dimethyl sulfoxide

We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham’s F10 medium (isotonic control) or to this medium plus NaCl (350–1000 mOsm), sucrose (369 and 479 mOsm), 1 M glycerol (1086 mOsm) or dimethyl sulfoxide (Me₂SO, 1151 mOsm) for 10 min. Each sample then was diluted back into Ham’s medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me₂SO had no influence (P > 0.05), NaCl and sucrose solutions affected sperm motility (P < 0.05), but not membrane integrity. Motility of sperm exposed to <600 mOsm NaCl or sucrose was less (P < 0.05) than fresh ejaculate, but comparable (P > 0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to <5%. In a separate study, ejaculates were diluted in Test Yolk Buffer containing 1 M glycerol or Me₂SO and cooled from 5 °C to −120 °C at −57.8 °C, −124.2 °C or −67.0 °C/min, frozen in LN₂, thawed in a water bath for 30 s at 37 °C or 10 s at 50 °C, and then assessed for motility, plasma- and acrosomal membrane integrity. Cryopreservation markedly (P < 0.05) reduced sperm motility by 70% compared to fresh samples. Higher (P < 0.05) post-thaw motility (20.0 ± 1.9% versus 13.5 ± 2.1%) and membrane integrity (51.2 ± 1.7% versus 41.5 ± 2.2%) were observed in samples cryopreserved in Me₂SO than in glycerol. Cooling rates influenced survival of sperm cryopreserved in glycerol with −57.8 °C/min being advantageous (P < 0.05). The findings demonstrate that although maned wolf spermatozoa are similar to domestic dog sperm in their sensitivity to osmotic-induced motility damage, the plasma membranes tolerate dehydration, and the cells respond favorably to Me₂SO as a cryoprotectant.     

Complexation of Al(III) with reduced glutathione in acidic aqueous solutions

The complexation of reduced glutathione (GSH) in its free and Al(III)-bound species in acidic aqueous solutions was characterized by means of multi-analytical techniques: pH-potentiometry, multinuclear (¹H, ¹³C and ²⁷Al) and two-dimensional nuclear Overhauser enhancement NMR spectroscopy (¹H, ¹H-NOESY), electrospray mass spectroscopy (ESI-MS), and ab initio electronic structure calculations. The following results were found. In the 25 °C 0.1 M KCl and 37 °C 0.15 M NaCl ionic medium systems, Al³⁺ coordinates with the important biomolecule GSH through carboxylate groups to form various mononuclear 1:1 (AlHL, AlH₂L and AlH₋₁L), 1:2 (AlL₂) complexes, and dinuclear (Al₂H₅L₂) species, where H₄L⁺ denotes totally protonated GSH. Besides the monodentate complexes through carboxylate groups, the amino groups and the peptide bond imino and carbonyl groups may also be involved in binding with Al³⁺ in the bidentate and tridentate complexes. The present data reinforce that the glycine carboxylate group of GSH has a higher microscopic complex formation constant than γ-glutamyl carboxylate. Compared with simple amino acids, the tripeptide GSH displays a greater affinity for the Al³⁺ ion and thus may interfere with aluminum’s biological role more significantly.     

Bio-kinetic analysis on treatment of textile dye wastewater using anaerobic batch reactor

An anaerobic digestion technique was applied to textile dye wastewater aiming at the colour and COD removal. Pet bottles of 5 L capacity were used as reactor which contains methanogenic sludge of half a liter capacity which was used for the treatment of combined synthetic textile dye and starch wastewater at different mixing ratios of 20:80, 30:70, 40:60, 50:50 and 60:40 with initial COD concentrations as 3520, 3440, 3360, 3264 and 3144 mg L⁻¹, respectively. The reactor was maintained at room temperature (30 ± 3 °C) with initial pH of 7. The maximum COD and colour removal were 81.0% and 87.3% at an optimum mixing ratio of 30:70 of textile dye and starch wastewaters. Both Monod’s and Haldane’s models were adopted in this study. The kinetic constants of cell growth under Haldane’s model were satisfactory when compared to Monod’s model. The kinetic constants obtained by Haldane’s model were found to be in the range of μ_max = 0.037-0.146 h⁻¹, K_s = 651.04-1372.88 mg L⁻¹ and K_i = 5681.81-18727.59 mg L⁻¹.     

Enhanced resistance to peroxide stress in Escherichia coli grown outside their niche temperatures

Extended exposure of Escherichia coli to temperatures above and below their growth optimum led to significant changes in oxidant production and antioxidant defense. At 20 °C an increase in the intracellular H₂O₂ concentration and oxidized glutathione (GSSG) level was observed against a background of low levels of reduced glutathione (GSH) and decreased catalase and glutathione reductase (GOR) activities. The intracellular H₂O₂ and GSSG concentrations had minimal values at 30 and 37 °C, but rose again at 42 °C, suggesting that oxidative processes were intensified at high temperatures. An increase in temperature from 20 to 42 °C led to an elevation in the oxygen respiration rate and superoxide production; a 5-fold increase in the intracellular GSH concentration and in the GSH:GSSG ratio occurred simultaneously. Catalase HPI and GOR activities were elevated 4.4- and 1.5-fold, respectively. Prolonged exposure to sublethal temperatures facilitated an adaptation to subsequent oxidative stress produced by the addition of H₂O₂.     

Seed germination of Lasia spinosa as a function of temperature,light, desiccation,and storage

Lasia spinosa seeds were not dormant at maturity in early spring. The most favorable temperatures for germination were between 25 and 30 °C, and final percentage and rate of germination decreased with an increase or decrease in temperature. When L. spinosa seeds were transferred to 25 °C, after 60 days at 10 °C (where none of the seeds germinated), final germination increased from 0% to 78%. Seeds germinated to high percentage both in light and in dark, although dark germination took more than twice as long as in the light. During desiccation of seeds at 15 °C and 45% relatively humidity, moisture loss decreased exponentially from 2.02 to 0.13 g H₂O g⁻¹ dry wt within 16 days, and only a few seeds (12%) survived 0.13 g H₂O g⁻¹ dry wt moisture content. Seeds stored at 0.58 g H₂O g⁻¹ dry wt moisture content at four constant temperatures (4, 10, 15, and −18 °C) for up to 6 months exhibited a well-defined trend of decreasing viability with decreasing temperature. Thus, we concluded that freshly harvested L. spinosa seeds are non-dormant and recalcitrant. Also, the seeds with 0.58 g H₂O g⁻¹ dry wt moisture content could be effectively stored for a few months between 10 and 15 °C although the most appropriate temperature for wet storage appears to be 10 °C, as it is close to the minimum temperature for germination and so there will be less pre-sprouting compared to 15 °C.     

Heart valve cryopreservation: protocol for addition of dimethyl sulphoxide and amelioration of putative amphotericin B toxicity

Aim

To investigate the need for stepwise addition of dimethyl sulphoxide to heart valves and amelioration of putative amphotericin B toxicity.

Methods

There were four groups: an untreated control (Group 1) and three experimental groups. For the latter, porcine heart valves were exposed to the antibiotic/antimycotic mixture used for disinfecting heart valves in the Bristol Heart Valve Bank, for 24 h at 22 °C. Dimethyl sulphoxide (Me₂SO, 10% v/v) was added either in two steps (5% then 10%) (Group 2) or in a single step. For single-step addition, valves were either first placed in Hanks’ balanced salt solution for 10 min before transfer to the cryoprotectant solution (Group 3) or immersed directly in the 10% cryoprotectant solution (Group 4). The valve leaflets were dissected from the valves and frozen in 10% Me₂SO in multi-well tissue culture plates at 1 °C/min to −80 °C. After storage overnight, the valve leaflets were warmed at approximately 11 °C/min and the cryoprotectant was removed by single-step dilution in excess Hartmann’s solution. Each leaflet was then divided into four pieces, which were placed in separate wells of a culture plate. Outgrowth of cells from the explants was monitored daily and graded according to the extent of cell growth.

Results

After freezing and thawing, only 77% of the explants from valves placed directly into 10% Me₂SO (Group 4) showed outgrowth of cells after freezing compared with 89% with two-step addition of Me₂SO (Group 2) and 95% with one-step addition after the extra rinse in Hanks’ solution (Group 3) (χ², p = 0.001). 92% of unfrozen control explants showed outgrowth of cells (Group 1). Only 37% of Group 4 explants reached confluence compared with 63 and 56%, respectively, of Groups 2 and 3 explants (χ², p = 0.007). The rates of cell growth in Group 2 (two-step addition of Me₂SO) and Group 3 (one-step addition of Me₂SO with additional Hanks’ solution rinse) were similar and faster than the Group 4 (one-step addition of Me₂SO without the additional Hanks’ rinse).

Conclusion

Single-step addition of Me₂SO before freezing gave similar results to two-step addition provided an additional rinse in Hanks’ solution was introduced after exposure to the antibiotic/antimycotic mixture. This suggests that antibiotic/antimycotic carryover may have been harmful during freezing and that the additional rinse in Hanks before one-step addition of Me₂SO, and the 5% Me₂SO step in the two-step protocol, merely served to reduce this carryover.     

Preliminary studies on cryopreservation of snakehead (Channa striata) embryos

This paper reports the findings of the ongoing studies on cryopreservation of the snakehead, Channa striata embryos. The specific objective of this study was to collect data on the sensitivity of C. striata embryo hatching rate to low temperatures at two different developmental stages in the presence of four different cryoprotectants. Embryos at morula and heartbeat stages were selected and incubated in 1 M dimethyl sulfoxide (Me₂SO), 1 M ethylene glycol (EG), 1 M methanol (MeOH) and 0.1 M sucrose solutions at different temperatures for a period of time. Embryos were kept at 24 °C (control), 15 °C, 4 °C and −2 °C for 5 min, 1 h and 3 h. Following these treatments, the embryos were then transferred into a 24 °C water bath until hatch to evaluate the hatching rate. The results showed that there was a significant decrease of hatching rate in both developmental stages following exposure to 4 °C and −2 °C at 1 h and 3 h exposure in each treatment. Heartbeat stage was more tolerant against chilling at −2 °C for 3 h exposure in Me₂SO followed by MeOH, sucrose and EG. Further studies will be conducted to find the best method to preserve embryos for long term storage.     

Pre-freeze mortality in three species of aphids from sub-Antarctic Marion Island

Understanding the mechanisms by which aphids survive low temperature is fundamental in forecasting the risk of pest outbreaks. Aphids are chill susceptible and die at a temperature close to that at which a small exothermal event is produced. This event, which can be identified using differential scanning calorimetry (DSC), normally occurs at a higher temperature than the supercooling point (SCP) and has been termed a pre-freeze event (PFE). However, it is not known what causes the PFE or whether it signifies the death of the aphid. These questions are addressed here by using a sensitive DSC to quantify the PFE and SCP and to relate these thermal events to the lower lethal temperature (LT₅₀) of sub-Antarctic aphids acclimated to low temperatures. PFEs were observed in each of the 3 species of aphids examined. They occurred over a narrower temperature range and at a higher temperature range than the SCP (−8.2 to −13.8 and −5.6 to −29.8 °C, respectively). Increased acclimation temperature resulted in increased SCPs in Myzus ascalonicus but not in Rhopalosiphum padi. The LT₅₀ reduced by approximately 1 °C from −9.3 to −10.5 °C with reduced acclimation temperature (10–0 °C). The LT₅₀ was close to the temperature at which the PFE occurred but statistically significantly higher than either the PFE or the SCP. In the majority of cases the PFE exotherm occurred well before the main exotherm produced by the bulk of the insect’s body water freezing (SCP). However, in a few cases it occurred at the same temperature or before the super-cooling point making the term, pre-freeze event (PFE), rather misleading. The possible origins of the PFE are discussed.     

Selective oxidation of sulfide catalysed by Cu complex of a triazamacrocyclic ligand with carbamoyl pendant arms

Copper(II) complex with a new ligand 1,4,7-tris(carbamoylethyl)-1,4,7-triazacyclononane (L) has been synthesized and characterized by elemental analysis, FT-IR, ES-MS, UV-Vis and cyclic voltammetry. Determined by X-ray analysis, the crystal structure shows the metal center is six-coordinated. The compound can catalyze the oxygenation of ethyl phenyl sulfide (EPS) utilizing H₂O₂ under ambient conditions. EPS was converted to the corresponding sulfoxide and sulfone step by step which was confirmed by ¹H NMR spectra. The existence of sulfoxide and sulfone was identified by GC-MS. The gradually disappearance of EPS’s ultraviolet absorption at 290 nm was significantly correlated with the rates of sulfide consumption. The initial reaction rate during the first 3 h is consistent with the first-order law in substrate concentration. The averaged pseudo-first-order rate constant is calculated to be (2.25 ± 0.42) × 10⁻³ min⁻¹ at 25 °C and (4.44 ± 0.17) × 10⁻³ min⁻¹ at 30 °C. The oxidation product is almost sulfoxide by choosing the molar concentrations of Cu complex (2% of substrate) and H₂O₂ (seven times as much as substrate).     

Impacts of chlorination and heat shocks on growth, pigments and photosynthesis of Phaeodactylum tricornutum (Bacillariophyceae)

The main impacts of cooling water from thermal (nuclear) power plants on aquatic organisms were caused by chlorination and temperature increase. In this study, we investigated the impacts of residual chlorine and short-term heat shocks on growth, pigment contents and photosynthesis of Phaeodactylum tricornutum. Growth of P. tricornutum was completely inhibited; Chlorophyll a and carotenoids contents deceased about 63.3% and 61.4% in 24 h treated with 0.2 mg L^− 1 chlorine. The negative effects of chlorination increased with enhanced concentration and prolonged exposure time. Relative electrode transfer rate (rETR) of P. tricornutum was significantly suppressed when treated with 0.2 mg L^− 1 residual chlorine for 24 h. Furthermore, the effective quantum yield (F_v'/F_m') decreased first but then recovered with prolonged exposure when residual chlorine ranged between 0.1 and 0.2 mg L^− 1. The cells were less sensitive to heat shocks compared with chlorination: the rETR and F_v'/F_m' was suppressed only when the temperature exceeded 35 °C for 1 h. When P. tricornutum was exposed to chlorination combined with heat shocks, the rETR was further inhibited at 35 °C. It indicated that both chlorination and heat shocks had negative impacts on the primary producers living in discharging coastal waters; furthermore, there were synergistic effects of heat shocks on chlorination toxicity.     

Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~ 250 μl volumes, at − 80 °C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at − 80 °C, unless a cryopreservative is present. Our “small volume” approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.     

Diurnally cycling temperature and ventilation affect young turkeys' performance and sensible heat loss

The goals were to elucidate the effects of ventilation rate (VR) coupled with exposure to constant 20 °C or to diurnal temperature cycling on young turkeys' performance and sensible heat loss (SHL). In three experiments male British United Turkeys (BUT), from 3 to 6 weeks of age, were exposed to constant 20 °C, or to 35/25 °C or 30/20 °C diurnal temperature cycling, all at 50% RH and with VR (expressed as air velocity (AV)) ranging from 0.8 to 3.0 m s⁻¹. The 2nd and 3rd of these experiments included a positive control at constant 30 °C and VR of 1.5 m s⁻¹. Weight gain, feed intake, and feed efficiency were measured or calculated, as appropriate; SHL was calculated from measured surface temperature, and plasma concentrations of triiodothyronine (T₃) was determined in the 1st experiment. Changes of VR at constant 20 °C did not affect performance, but total SHL increased significantly with increasing VR. Under the 35/25 °C regime a significantly higher BW was recorded, with a similar pattern of feed efficiency, when VR during the hot part of the cycle was 1.5 or 2.0 m s⁻¹ than when it was 0.8 m s⁻¹. In the 3rd experiment, BW in the 35/20 °C treatment was significantly lower than that of the controls. In all experiments, turkeys maintained body temperature (T_b) within the normothermic range, and SHL varied with VR. It can be concluded that although diurnal temperature cycling reflects the natural situation, exposing young turkeys to constant 30 °C combined with optimal ventilation might yield the best performance results.     

The kinetics of the complex formation between iron(III)-ethylenediaminetetraacetate and hydrogen peroxide in aqueous solution

The kinetics of the formation of the purple complex [Fe^III(EDTA)O₂]³⁻, between Fe^III-EDTA and hydrogen peroxide was studied as a function of pH (8.22-11.44) and temperature (10-40 °C) in aqueous solutions using a stopped-flow method. The reaction was first-order with respect to both reactants. The observed second-order rate constants decrease with an increase in pH and appear to be related to deprotonation of Fe^III-EDTA ([Fe(EDTA)H₂O]⁻ ⇔ Fe(EDTA)OH]²⁻ + H⁺). The rate law for the formation of the complex was found to be d[Fe^IIIEDTAO₂]³⁻/dt=[(k₄[H⁺]/([H⁺] + K₁)][Fe^III-EDTA][H₂O₂], where k₄=8.15±0.05×10⁴ M⁻¹ s⁻¹ and pK₁=7.3. The steps involved in the formation of [Fe(EDTA)O₂]³⁻ are briefly discussed.     

Energy-minimized structures and MO levels of catalysts related to [RuO(hpsd)(bpy)] that competently hydroxylate benzene (hpsd(2-)=(2-hydroxyphenyl)salicyldiminato)

Two series of complexes with formal oxidation state assignments of {Ru^V(O²⁻)} have been examined by molecular mechanics and molecular orbital methods at the level of PM3 calculations in order to assess the origin of differences in the activity of these complexes in the conversion of benzene to phenol by oxygen transfer. The first series includes complexes of general formula [RuO(hpsd)(XY)]ⁿ⁺ with hpsd²⁻ (also known in the literature as amp²⁻)=(2-hydroxyphenyl)salicyldiminato; XY=bpy(2,2^′-bipyridine) and other py-X, wherein the second pyridyl group of bpy is changed to X=-CH₂N(CH₃)₂ (stronger σ-donor X), -CH₂P(CH₃)₂ (better π-acceptor X), -CO₂ ⁻ (weak π-donor X), -CH₂S⁻ (strong π-donor X), and -CH₂C(CH₃)₂ ⁻ (very strong σ-donor X).A second series of complexes, [RuO(TDL)(bpy)]ⁿ⁺ was also studied with TDL=(tridentate ligand) of the parent hpsd²⁻ (or amp²⁻); cpsd²⁻=(N-(2-carboxyphenyl)salicylaldiminato); cppc⁻=(N-2-carboxyphenylpyridine-2-carboxaldiminato); and hppc⁻=(2-hydroxyphenyl)2-pyridylcarboxaldiminato (or app⁻). Experimentally, the activity order based upon the percentage yields of oxygenated products for [RuO(TDL)(bpy)]ⁿ⁺ is as follows for TDL’s=hpsd²⁻ (91%) > cppc⁻ (87%) > cpsd²⁻ (84%) > hppc⁻ (80%). The rates approach toward saturation in reactivity as a function of the fractional positive charge on the apical O center: cppc⁻ (0.233) > hpsd²⁻ (0.166) > cpsd²⁻ (0.105) > hppc⁻ (0.041). The reactivity order follows chelate ring strain influences of the TDL, with 5,6-membered chelate rings; hpsd²⁻ and cppc⁻ > 6,6; cpsd²⁻ > 5,5; hppc⁻.It was determined that the general structures of these complexes are best described as pentagonal pyramidal (rather than pseudo-octahedral) with the RuO unit apical, the three donors of hpsd²⁻, cpsd²⁻, cppc⁻ or hppc⁻, and the two donors of XY ligands adopting a waffled arrangement around the Ru center as the remaining donors of the pentagonal set. The donor most trans to the apical RuO is approximately at 140°, rather than 180°. Ligands such as hpsd²⁻ (amp²⁻) are not retained in a single planar array, but rather with one of the aromatic donors turned upward to shield the approach of the RuO unit from one side. The ligand series [RuO(hpsd)(XY)]⁺ averages angles between adjacent atoms of the pentagonal set of 75.4° instead of a theoretical 72.0°; angles between the apical RuO and adjacent donors average 111° but with wide deviations (±30°) depending upon the donors of the TDL.Small changes in the donor atom positions, and in the capability of the “trans” donor’s σ-donor strength, and whether it is a π-acceptor or a π-donor, modulate the degree of mixing of ligand orbitals and the LUMO/SOMO energy gap which influences reactivity. The presence of a π-acceptor ligand provides the most destabilization of Ru-O π bonding, and this appears to be the best way to increase the activity of these catalysts toward oxidation of C₆H₆ to C₆H₅OH. Also, implicated in the activity of the catalysts is the need for two non-innocent phenolate donors that raise the energy of orbitals on the apical O atom. This increases the oxenoid character of the terminal O, and makes the insertion into a C-H bond more favored.     

Longevity and temperature response of pollen as affected by elevated growth temperature and carbon dioxide in peanut and grain sorghum

It is important to understand the effects of environmental conditions during plant growth on longevity and temperature response of pollen. Objectives of this study were to determine the influence of growth temperature and/or carbon dioxide (CO₂) concentration on pollen longevity and temperature response of peanut and grain sorghum pollen. Plants were grown at daytime maximum/nighttime minimum temperatures of 32/22, 36/26, 40/30 and 44/34 °C at ambient (350 μmol mol⁻¹) and at elevated (700 μmol mol⁻¹) CO₂ from emergence to maturity. At flowering, pollen longevity was estimated by measuring in vitro pollen germination at different time intervals after anther dehiscence. Temperature response of pollen was measured by germinating pollen on artificial growth medium at temperatures ranging from 12 to 48 °C in incubators at 4 °C intervals. Elevated growth temperature decreased pollen germination percentage in both crop species. Sorghum pollen had shorter longevity than peanut pollen. There was no influence of CO₂ on pollen longevity. Pollen longevity of sorghum at 36/26 °C was about 2 h shorter than at 32/22 °C. There was no effect of growth temperature or CO₂ on cardinal temperatures (T_min, T_opt, and T_max) of pollen in both crop species. The T_min, T_opt, and T_max identified at different growth temperatures and CO₂ levels were similar at 14.9, 30.1, and 45.6 °C, respectively for peanut pollen. The corresponding values for sorghum pollen were 17.2, 29.4, and 41.7 °C. In conclusion, pollen longevity and pollen germination percentage was decreased by growth at elevated temperature, and pollen developed at elevated temperature and/or elevated CO₂ did not have greater temperature tolerance.     

Crystal structures and spectroscopic properties of palladium complexes isolated from Pd-EDTA solutions

Two complexes were isolated from aqueous Pd(NO₃)₂-Na₂H₂EDTA solutions and studied by single crystal X-ray diffraction, IR/Raman spectroscopy, and photoelectron spectroscopy. The first complex, Pd(Hkpda)₂ (kpda = ketopiperazinediacetate dianion, C₈H₁₀N₂O₅), forms yellow parallelepipeds during slow evaporation of Pd(NO₃)₂-Na₂H₂EDTA solution at 25 °C, and is a result of EDTA oxidation. The second one, [Pd(μ-H₂EDTA)]₂ · 2NaNO₃ · 7.5H₂O, with two EDTA molecules acting as bridges between two palladium atoms, forms yellow bipyramides during fast evaporation at 60 °C. In both complexes, the palladium atoms adopt a planar trans-geometry by bonding to two nitrogen and two oxygen atoms of two ligand molecules.     

Synthesis of the cobalt analogues of Roussin’s red salt esters

Cobalt analogs of the Roussin’s red salt esters have been prepared by the reaction of organic thiol or selenol compounds with the readily prepared [Co(NO)₂Cl]₂. The compounds are unstable to light and decompose in ca. 1 week even in the dark at −78 °C. The IR spectra suggest that the compounds have C_2v symmetry with the possibility of a folded Co₂S₂ rhombus about the S-S axis.     

Temperature manifold for a stopped-flow machine to allow measurements from −10 to +40 °C

Conducting enzymatic stopped-flow experiments at temperatures far removed from ambient can be very problematic because extremes in temperature (<10 °C or >30 °C) can damage the machine or the enzyme. We have devised a simple manifold that can be attached to most commercial stopped-flow systems that is independently heated or cooled separate from the main stopped-flow system. Careful calibration of the flow circuit allows the sample to be heated or cooled to the measurement temperature (−8 to +40 °C) 1 to 2 s before mixing in the reaction chamber. This approach allows measurements at temperatures where the stopped flow or the protein is normally unstable. To validate the manifold, we investigated the well-defined ATP-induced dissociation of rabbit muscle myosin subfragment 1 (S1) from its complex with pyrene-labeled actin. This process has both temperature-dependent and -independent components. Use of ethylene glycol allowed us to measure the reaction below 0 °C and up to 42 °C, and as expected the second-order rate constant (K₁k₊₂) and the maximum rate of dissociation (k₊₂) both increased with temperature, whereas 1/K₁ is unaffected by the change in temperature.