An easy-to-use practical method to measure coincidence in the flow cytometer--the case of platelet-granulocyte complex determination

Cell complexes composed of two different cells labeled with different fluorophores can be detected as double positive events in the flow cytometer. Double positivity can originate not only from real complexes but from non-interacting coinciding cells as well. Coincidence has a high impact on the determination of the amount of platelet–granulocyte complexes since platelet concentration is in the orders of magnitude higher than that of the granulocytes. Mixtures of non-interacting fluorescent beads as well as EDTA anticoagulated or citrated blood samples were analyzed in the flow cytometer in the presence and absence of fluorescent beads at various dilutions. Experimental data were evaluated by mathematical means. The bead or platelet concentration dependence of double positivity was converted into linear functions using Poisson distribution. This linearised form contains information on the detection volume as well as on the presence/absence of dilution independent complexes. The presence of appropriate fluorescent beads in the blood sample makes possible to estimate the fraction of double positivity originating from coincidence if data collection is triggered by the granulocytes or by the fluorescent beads, alternatively. Mixing fluorescent beads into a blood sample is a simple experimental method to distinguish double positivity originating from real cell–cell complexes from the coincidence of cells in a flow cytometer, thus providing a tool for the determination of the real amount of cell–cell complexes.     

Caprine luteinizing hormone isoforms during the follicular phase and anestrus

The relative proportion of the circulating luteinizing hormone isoforms in goats during follicular phase (pre-ovulatory peak; F) and anestrus (A) was investigated. Estrus was synchronized in six goats with a prostaglandin analogue. After estrus was detected, blood samples were taken at 1 h intervals for 24 h. Four anestrous goats received 100 μg i.v. of GnRH and blood samples were collected every 15 min for 5 h. Samples with the greatest LH concentration in follicular phase and after GnRH administration (anestrus) were analyzed by chromatofocusing and eluted with a pH gradient from 10.5 to 3.5. For quantification purposes eluted LH was grouped into basic (pH ≥ 7.5), neutral (pH 7.4–6.5) and acidic isoforms (pH ≤ 6.4) as well as by pH unit. In both physiological conditions (PC), basic and acidic isoforms were greater than the neutral. With this grouping criteria, there was an interaction between PC and pH group, with the proportion of neutral isoforms being greater (p < 0.05) in A (12.0 ± 0.8%) as compared with F (5 ± 2%). Analysis by pH unit showed a very basic group of eluted isoforms (pH ≥ 10), which amounted to a percentage of 6.0 ± 0.4% of the total observed during A, and 3 ± 1% during F (p < 0.05). Predominant isoforms in A eluted in the pH range 9.99–9.0 (42 ± 3%) as compared to 7 ± 3% (p < 0.01) in that pH range in F. In contrast, the predominant isoforms in F eluted in the pH range 8.99–8.0, representing 55 ± 8%, while in A the proportion was 11 ± 2% (p < 0.01). Isoforms eluted at the pH range 7.9–7 represented a significantly greater proportion during A (5.0 ± 0.6%) as compared with F (3 ± 1%). This is the first report on goat LH circulating isoforms. During A the LH isoforms secreted by the pituitary are more basic than during F.     

Isolation and characterization of heparin and gelatin binding buffalo seminal plasma proteins and their effect on cauda epididymal spermatozoa

Seventy semen ejaculates were obtained from 14 Murrah buffalo bulls and were subjected to plasma separation immediately after collection by centrifugation at 2000 rpm for 20 min and stored in liquid nitrogen until analysis. In the seminal plasma the total protein concentration were estimated and the heparin and gelatin binding (HB and GB) proteins were isolated using heparin and gelatin affinity column chromatography. The molecular weight of individual isolated HB and GB protein was determined by SDS–PAGE analysis. Buffalo bull spermatozoa was collected from cauda epididymis under aseptic conditions and was used for the in vitro fertility tests (i.e. bovine cervical mucus penetration test (BCMPT) and hypo-osmotic swelling test (HOST)). The heparin and gelatin binding buffalo seminal plasma proteins were used in six concentrations i.e. 10, 20, 30, 40, 50 and 60 μg/ml to test their effect on in vitro fertility assessment of cauda epididymal spermatozoa. The overall mean values of total protein, HB and GB proteins were recorded as 29 ± 2.7, 2.61 and 0.2 mg/ml, respectively. Eighteen total protein bands were observed in the range of 12–127 kDa. Eight major HB proteins were isolated in the range of 13–71 kDa. Seven major GB proteins were isolated in the range of 13–61 kDa in the buffalo seminal plasma. The mean penetration distance (mm) travelled by the buffalo cauda spermatozoa was maximum in HB proteins (26.9 ± 0.6) followed by GB proteins (25.4 ± 0.6) and control (21.2 ± 1.4). The difference in BCMPT values between protein treated and control group was significant (P < 0.05). Almost similar trend in the effect of protein on values of HOST percentage in both HB and GB proteins treated semen samples were recorded (66.4 ± 0.65 and 66.1 ± 0.6, respectively). The difference in HOST values between proteins treated and control group (50.4 ± 2.0) was significant (P < 0.05). The present results indicate that among the isolated proteins, 4 proteins were commonly seen in both the heparin and gelatin–sepharose affinity column chromatography, and the addition of buffalo seminal plasma proteins improved the in vitro sperm functions (40 μg/ml gave best results) of buffalo cauda spermatozoa.     

A method to reduce the invasiveness of fetal catheterization in the cow

Surgical intervention in general anesthesia (GA) of the cow in late gestation is a stressful condition for both mother and fetus, potentially leading to premature delivery or fetal death. The present study hypothesized that fetal catheterization at days 246–253 (90% of gestation) is done with less physical and metabolic stress for the mother and fetus, when the surgery is performed on a standing cow and local anesthesia (LA) rather than on a recumbent cow in general anesthesia. Fetal and uterine maternal intra-vascular catheters were implanted during general anesthesia (GA, n=24) or local analgesia (LA, n=7). Blood gases and metabolite levels in the fetal calves and their mothers were measured during surgery and for 5 days post-operatively. During surgery, venous blood pH was higher (7.44±0.01 versus 7.39±0.01, P<0.05) and hemoglobin and oxygen contents lower in LA cows compared with GA cows (9.3±0.3 mg/dl versus 11.8±0.2 mg/dl, P<0.001 and 10.1±0.3 ml/dl versus 12.6±0.6 ml/dl, P<0.05). The differences between the two groups of fetuses reflected those of their dams in that LA fetuses showed lower arterial oxygen pressure (18.3±1.4 mmHg versus 24.8±1.4 mmHg, P<0.05) and hemoglobin (7.81±0.30 mg/dl versus 9.22±0.21 mg/dl P<0.01) and furthermore, they also showed higher blood glucose (2.4±0.2 mM versus 1.4±0.1 mM, P<0.01). During the 5 days post-surgery, 10 GA fetuses (42%) and 1 LA fetus (14%) died in utero. Bacterial contamination was implicated in six of the GA deaths and in the one LA death. In the dams with surviving calves, differences in hemoglobin (9.49±0.21 mg/dl versus 11.17±0.23 mg/dl P<0.001) and O₂ct (10.9±0.3 ml/dl versus 12.5±0.5 ml/dl, P<0.05) were still present, and in addition, blood glucose was higher in LA versus GA cows (4.3±0.2 mM versus 3.8±0.1 mM, P<0.05). The choice of surgical method did not affect post-surgery blood chemistry in the surviving fetuses, except that the higher blood glucose in the LA fetuses at surgery tended to be maintained also post-operatively (2.0±0.2 mM versus 1.5±0.1 mM, P=0.07). The observed differences in blood chemistry parameters between the two methods of surgery and possibly in the fetal death may be explained by differences in catheterization method and the associated differences in physical and metabolic stress during and after surgery. Thus, surgery upon a standing cow in local anesthesia should be considered as an alternative to surgery in universal anesthesia for fetal catheterization in the cow in late gestation.     

Nuclear maturation and development of IVM/IVF canine embryos in synthetic oviductal fluid or in co-culture with buffalo rat liver cells

In vitro embryo production in the domestic bitch can provide valuable insights for conservation of endangered canids. In the present study, canine oocytes underwent in vitro maturation (IVM) in simple or complex media, with production of in vitro matured and fertilized (IVM/IVF) canine embryos. Cumulus–oocyte complexes (COCs) were harvested from ovaries by slicing and subjected to IVM in four media (SOF, TCM 199, Ham-F10, and DMEM/F12). After culture for 48 h, oocytes were stained and examined for nuclear maturation. There were no significant differences in the mean (±S.D.) percentage of nuclear maturation (metaphase II) of oocytes cultured in SOF (18.6 ± 7.6%), TCM 199 (18.3 ± 4.5%), Ham-F10 (13.9 ± 8.2%), or DMEM/F12 (11.9 ± 4.2%). For assessment of embryo development, oocytes were matured for 48 h in synthetic oviductal fluid (SOF), fertilized with frozen-thawed sperm, and presumptive zygotes were cultured for 7 d, either in SOF or as co-cultures with BRL cells in TCM 199. Percentages of IVM/IVF oocytes that developed to the 2-cell, 3–4-cell, and 5–7-cell stages were higher (P < 0.05) following culture in SOF versus BRL cell co-cultures (33.6 ± 1.2% vs 13.7 ± 1.2%, 24.7 ± 0.5% vs 8.7 ± 1.1%, and 15.1 ± 2.2% vs 4.3 ± 1.3%, respectively). However, none of the embryos developed beyond the 8–16-cell stage. In conclusion, simple or complex media successfully induced resumption of meiosis and nuclear maturation of canine oocytes. Furthermore, SOF supported in vitro development of IVM/IVF canine embryos to the 8–16-cell stage.     

Mobility of lipid in complexes of amylose–fatty acids by deuterium and C solid state NMR

Palmitic and lauric acid complexes with amylose were studied by solid state methods: ¹³C CP/MAS NMR, deuterium NMR, X-ray powder diffraction and differential scanning calorimetry (DSC). The crystalline amylose complexes were found to be in a V-type sixfold single chain helix. The melting points of the complexes were over 100°C, at least 40–50°C higher than the melting points of the free fatty acids. CP/MAS ¹³C NMR spectra revealed two resonance peaks at 33.6 and 32.4 ppm for the palmitic acid, which were assigned as free and complexed fatty acid, respectively. A single resonance peak at 32.4 ppm was found for the lauric acid and assigned to the complex. The chemical shift of 32.4 ppm for the complexed fatty acids suggests a combined trans and gauche conformation for the fatty acid chain in the complex. T₁ relaxation measurements on the two palmitic acid resonances show different behavior: a very slow relaxation for the 33.6 ppm and a much faster relaxation (1.2 s) for the 32.4 ppm resonances. The latter was similar to the relaxation of the single resonance of the lauric acid (1.1 s). Temperature dependent deuterium spectra of the amylose–lauric acid and amylose–palmitic acid complexes suggest a complete complexation for the amylose–lauric acid, whereas the amylose–palmitic acid complex is partially disassociated by the thermal treatment. Based on the overall data, a partially disordered model is proposed: an imperfect helix with the fatty acid partly inside and partly out, depending on crystallization conditions and the necessity of placing the carboxyl head outside the V-helix.     

Twice single doses of 100,000 IU of vitamin D in winter is adequate and safe for prevention of vitamin D deficiency in healthy children from Ushuaia, Tierra Del Fuego, Argentina

In order to improve vitamin D status of children from Ushuaia (55°S), at the South of Argentina, double supplementation with 100.000 IU of vitamin D was administered at the beginning of winter (March 2004), and 3 months later during winter (June 2004). In 2004, serum 25-hydroxyvitamin D (25OHD) was measured before the first supplementation, a month after, and 3 months after receiving the second supplementation (March, April and September). We studied 18 healthy children from Ushuaia, age (mean ± S.D.) 7.3 ± 4.4 years old (range 1.2–14.6), seven girls and 11 boys. Before treatment, serum 25OHD was 29.3 ± 5.9 ng/ml. It increased significantly 1 month after the first supplementation (April): 35.3 ± 4.4 ng/ml (p < 0.001), and decreased significantly 3 months after the second supplementation: 22.4 ± 4.6 ng/ml (September (p < 0.001). No child was neither deficient (<10 ng/ml) nor insufficient (10–15 ng/ml) of vitamin D. On April, a month after the first supplementation, no children had vitamin D intoxication levels (>50 ng/ml). These results disclosed that to prevent vitamin D deficiency for children at zones of risk at the south of our country, double supplementation of 100,000 IU of vitamin D during autumn and winter, would be adequate and safe.     

Enhancing long-term thermal stability in mesophilic glutamate dehydrogenase from Clostridium symbiosum by eliminating cysteine residues

Glutamate dehydrogenase from Clostridium symbiosum has two cysteine residues, C144 and C320. The single mutant C320S and a double mutant with both cysteines replaced by serine have been compared with one another in terms of long-term stability and other properties. Specific activities and kinetic parameters were relatively little affected, but stability was improved—e.g. at 25 °C sterile, sealed samples of wild-type enzyme, C320S and the double mutant at 0.1 mg/ml in 0.1 M phosphate buffer, pH 7 lost 50%, 42% and 32% of activity over 60 days. For the first two proteins this loss was partly reversible with dithiothreitol. When wild-type enzyme was deliberately contaminated with 1 μM Cu²⁺ it became less stable and formed aggregates, whereas the double mutant was not affected. The double mutation thus removes a source of instability through –SH oxidation that would be accentuated by any heavy metal contamination of solutions.     

The effect of intravenous PACAP38 on cerebral hemodynamics in healthy volunteers

PACAP38 is an endogenous peptide located in trigeminal perivascular nerve fibers in the brain. It reduces neuronal loss and infarct size in animal stroke models and has been proposed a candidate substance for human clinical studies of stroke. The effect on systemic hemodynamics and regional cerebral blood flow (rCBF) is not well understood. We here present the first study of the effect of PACAP38 on cerebral hemodynamics in humans.

PACAP (10 pmol kg^− 1 min^− 1) or placebo (0.9% saline) was infused for 20 min into 12 healthy young volunteers in a cross over, double blind study. rCBF was measured with SPECT and ¹³³Xe inhalation and mean blood flow velocity in the middle cerebral artery was measured with transcranial Doppler ultrasonography. End tidal partial pressure of CO₂ (P_etCO₂) and vital parameters were recorded throughout the 2 hour study period.

PACAP38 decreased rCBF in all regions of interest (ROIs) by  3–10%, though not uniformly significant. P_etCO₂ decreased significantly during PACAP38 infusion compared to placebo (P = 0.032), peak decrease was 8.9 ± 3.8%. After correction for P_etCO₂, rCBF remained unchanged in most ROIs. Heart rate increased 61.9 ± 22.4% (P < 0.0001 vs. placebo).

These findings suggest that PACAP38 has no major direct effect on rCBF in healthy volunteers. The marked increase in heart rate and the reduction in rCBF caused by decreased P_etCO₂ are important dose-limiting factors to consider in future clinical studies.     

The effect of follicle-stimulating hormone on follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor-I in the goat ovary

The effect of FSH on goat follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor (IGF)-I were studied through both in vivo and in vitro experiments. The FSH treatment was begun on Day 9 after estrus and consisted of injections twice a day for 3 days in decreasing doses (7.5–7.5–5.0–5.0–2.5–2.5 mg). Does in both treatment and control groups were slaughtered for ovaries on Day 12. Granulosa cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Expression of IGF-I and IGF-II mRNA was determined by RT–PCR, while concentrations of progesterone (P4), estradiol (E2), IGF-I and IGF-II were measured by radioimmunoassay (RIA). Following parameters increased significantly (P<0.05) after the FSH treatment: follicle number (5.0±1.5 versus 9.0±2.0 per ovary), the level of E2 (0.1±0.1 ng/ml versus 0.7±0.2 ng/ml), the E2/P4 ratio (0.7±0.4 versus 4.7±3.0) and the concentrations of IGF-I (0.5±0.2 ng/ml versus 119.4±15.1 ng/ml) and IGF-II (0.12±0.03 ng/ml versus 40.9±18.7 ng/ml) in follicular fluid of the medium sized (3–5 mm) follicles and in the ovarian cortex the relative quantity of IGF-I mRNA (0.37±0.17 versus 0.90±0.12 Max OD). In contrast, the ratio of apoptotic granulosa cells in these follicles was reduced significantly (0.53±0.1 versus 0.10±0.01, P<0.05). In large (>5 mm) follicles, however, only the follicle number (2.3±0.7 versus 7.0±1.5 per ovary) and the level of IGF-I (38.4±11.0 ng/ml versus 87.3±13.9 ng/ml) increased significantly (P<0.05), whereas other values did not change. In vitro culture of granulosa cells showed that FSH significantly (P<0.05) enhanced IGF-I production (12.7±2.1 ng/ml versus 26.±21.9 ng/ml) by these cells, and both FSH and IGF-I reduced the ratios of apoptotic cells (from 0.7±0.07 to 0.3±0.1 and 0.2±0.04, respectively) and the effect was additive when both were used together. H89, the PKA pathway inhibitor, blocked the effect of FSH on granulosa cell apoptosis and IGF-I production in vitro. These results indicated that FSH mainly enhanced the development of medium sized follicles in the goat by suppressing the apoptosis of granulosa cells via increasing production of IGF-I and steroids, possibly through the PKA pathway.     

A novel spectrophotometric method for determination of kinetic constants of aldehyde oxidase using multivariate calibration method

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1–30.0 μM and the absorption spectra of the solutions were recorded in the range of 210–280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis–Menten constants from a Lineweaver–Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04 ± 0.01 and 0.03 ± 0.01 μM (mean ± SD, n = 5), respectively. Using this method, the K_m value for the oxidation of phenanthridine was calculated as 1.72 ± 0.09 μM (mean ± SD, n = 3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.     

Effects of radiograph projection parameter uncertainty on TKA kinematics from model-image registration

Model-image registration techniques have been used extensively for the measurement of joint kinematics in vivo. These techniques typically utilize an explicit measurement of X-ray projection parameters (principal distance, principal point), which is easily done for prospective studies. However, there is vast opportunity to derive useful information from previously collected clinical radiographic films where the projection parameters are unknown. The purpose of this study was to determine variation in measured knee arthroplasty kinematics when the X-ray projection parameters were unknown, but bounded. Based on the clinical radiographic protocol, a nominal principal point was chosen and eight additional points ±2 and ±5 cm in the horizontal and vertical directions were defined. Tibiofemoral kinematics were determined for all nine projection parameter sets for a series of 10 lateral radiographs. In addition, the principal distance was varied ±15 cm and tibiofemoral kinematics were determined for these two projection sets. Measured joint kinematics varied less than 0.6° and 0.4 mm for ±2 cm variations in principal point location, and 0.7° and 0.6 mm for ±5 cm variations in principal point location. Measured joint kinematics varied less than 0.6° and 0.7 mm for ±15 cm variations in principal distance. Variation in X-ray principal point and principal distance over clinically bounded ranges has a small effect on knee arthroplasty kinematics computed from model-image registration with high-quality clinical radiographs.     

Critical thermal maximum and body water loss in first instar larvae of three Cetoniidae species (Coleoptera)

Critical thermal maximum (CT_max) and body water losses were measured in first instar larvae of Gnorimus nobilis, Osmoderma eremita (Trichiinae) and Cetonischema aeruginosa (Cetoniinae) when air temperature was increased gradually (0.5 °C/min) from 20 °C to the critical thermal maximum (CT_max), in dry air (near 0% R.H.).

The CT_max was significantly lower in O. eremita (45.6±0.7 °C) than in G. nobilis (48.5±0.6) and C. aeruginosa (51.4±0.9 °C).

An increase of 10 °C (30–40 °C) induced a 2-fold increase of the water loss in C. aeruginosa and O. eremita (Q₁₀=2.10±0.12 and 2.13±0.20, respectively). In the range from 40 to 45 °C to CT_max a strong increase of the water loss was observed in O. eremita and C. aeruginosa, respectively. Body water losses were significantly lower in C. aeruginosa than in O. eremita and G. nobilis over the range 20 °C—CT_max; no significant difference occurred between G. nobilis and O. eremita.     

Influence of incubation temperature and time on resistant starch type III formation from autoclaved and acid-hydrolysed cassava starch

Raw cassava starch, having 74.94 and 0.44 g/100 g resistant starch type II and III (RS II and RS III), respectively, was autoclaved at 121 °C in water, 1, 10 or 100 mmol/L lactic acid. The formation of RS III was evaluated in relation to variable incubation temperature (−20 to 100 °C), incubation time (6–48 h) and autoclaving time (15–90 min). Negligible to low quantities of RS III (0.59–2.42 g/100 g) were formed from autoclaved starch suspended in 100 mmol/L lactic acid, whereas intermediate to high quantities (2.68–9.97 g/100 g) were formed from autoclaved starch suspended in water, 1 or 10 mmol/L lactic acid, except for treatments with water or 10 mmol/L lactic acid incubated at 100 °C for 6 h (1.74 g/100 g). Autoclaving times corresponding to maximum RS III contents were 15 and 45 min for water and 10 mmol/L lactic acid, respectively. Whereas, the RS III fractions from cassava starch suspended in water had melt transitions between 158 and 175 °C with low endothermic enthalpies (0.2–1.6 J/g), the thermal transitions of the acid treated samples were indistinct.     

Effects of anti-oxidant additives on microscopic and oxidative parameters of Angora goat semen following the freeze–thawing process

The anti-oxidant system of reduced glutathione (GSH), glutathione peroxidase (GSH-PX), catalase (CAT), and superoxide dismutase (SOD) has been described as a defense functioning mechanism against lipid peroxidation (LPO) in semen, and is important in maintaining sperm motility and viability. This anti-oxidant capacity of sperm cells may be insufficient in preventing LPO during the freeze–thawing process. The aim of this study was thus to determine the influence of varying doses of anti-oxidant additives on standard semen parameters, lipid peroxidation and anti-oxidant activities after the freeze–thawing of goat semen. Ejaculate samples (artificial vagina) obtained from 4 mature Angora goats were evaluated and pooled at 37 °C. The semen samples diluted with a Tris-based extender, containing taurine (25, 50, 75 mM), trehalose (25, 50, 75 mM), and cysteine (5, 10, 15 mM), and an extender containing no anti-oxidant additives (control) were again evaluated. Diluted semen was cooled down to 5 °C and frozen in 0.25 ml French straws, prior to being stored in liquid nitrogen. Frozen straws were thawed in a water bath (37 °C) for 30 s for microscopic sperm evaluation. Upon evaluation of parameters for semen quality, the use of a Tris-based extender supplemented with anti-oxidant additives was found to cause no significant improvement in sperm mortality, when compared to the controls. Increasing doses of taurine and trehalose decreased (P < 0.05) the sperm motility following the freeze–thawing of the goat semen. In biochemical assays, the application of taurine (75 mM) produced the lowest level of malondialdehyde (MDA) (4.46 ± 0.31 nmol/ml), compared to the controls (P < 0.001). Lower GSH levels were higher in the groups in which cysteine was included at 10 and 15 mM (3.27 ± 0.11 and 3.45 ± 0.28 nmol/ml) – compared to the group which received 5 mM cysteine, as well as the controls (2.27 ± 0.08 and 2.50 ± 0.08 nmol/ml respectively, P < 0.001). Compared to the controls, taurine at a concentration of 25 and 75 mM, and increasing doses (50 and 75 mM) of trehalose, significantly increased the GSH-PX activity (P < 0.01). The maintenance of CAT activity was demonstrated to be higher with the addition of 10 and 15 mM cysteine, compared to the other groups (P < 0.001). Vitamin A (VitA) levels were significantly higher, compared to the controls (267.34 ± 9.68 mg/dl and 267.34 ± 9.68 mg/dl, respectively), when 25 mM taurine (329.61 ± 6.35 mg/dl) and 10 mM (318.64 ± 6.34 mg/dl) cysteine was added to the extender (P < 0.001). The results of this study provide a new approach to the cryopreservation of Angora goat semen and could contribute to the improvement of this technology in the goat industry.     

Body condition and protein supplementation positively affect periovulatory ovarian activity by non LH-mediated pathways in goats

Effects of rumen undegradable intake protein (UIP) supplementation on ovarian activity and serum insulin, GH, and LH were evaluated in goats having low or high body condition (BC). Goats with either low BC (n = 16, 28.7 ± 0.8 kg BW, BC = 2.1 ± 0.3) or high BC (n = 16, 38.4 ± 0.8 kg, BC = 3.2 ± 0.3) received, during 40-days, one of the two protein supplementation levels: without UIP or with UIP (120 g goat⁻¹ d⁻¹). Oestrus was synchronized with two i.m. doses of PGF₂, and jugular blood samples were collected from 36 to 42 h after the second prostaglandin injection at 15 min intervals. Serum concentrations of insulin, LH, and GH were measured The number of preovulatory follicles and the number of corpora lutea (CL) were evaluated by transrectal ultrasonography at 1 and 4 days after the second prostaglandin dose, respectively. Does with higher BC had more CL than those in the lower condition group (2.8 ± 0.2 versus 1.8 ± 0.2, P < 0.05). Similarly, goats receiving UIP supplementation had more follicles (2.6 ± 0.2 versus 1.9 ± 0.2, P < 0.05) and tended to have more CL (2.6 ± 0.2 versus 2.0 ± 0.2, P = 0.05) than does not receiving UIP. Neither BCS nor UIP supplementation affected serum GH or LH concentrations, pulsatility, or area under the curve. High BC does produced more insulin (1.92 ± 0.17 versus 0.81 ± 0.17 ng/mL, P < 0.01 ng/mL) than lower BC goats; the same for UIP-supplemented (1.69 ± 0.18 versus 1.04 ± 0.18, P < 0.05). Results suggest that the increased ovarian activity observed in both UIP-supplemented and higher BC goats was not the result of changes in LH or GH, suggesting effects at a local level, through changes in insulin in a non-GnRH-gonadotrophin dependent manner.     

Development of a pharmacodynamic model of murine malaria and antimalarial treatment with dihydroartemisinin

Antimalarial treatment strategies based on in vitro studies are limited by the paucity of pharmacodynamic information for dosage regimen design. We postulated that a murine model could be used for pre-clinical stages of drug development, especially in dose–response studies and evaluation of combination therapies. Swiss mice infected with Plasmodium berghei parasites (2–5% starting parasitaemia) were given dihydroartemisinin (0–100 mg/kg single dose). Parasite density was regularly determined from thin blood films. A parasite population growth model comprising parasite multiplication, decline in erythrocyte count with increasing parasitaemia and parasite clearance after drug administration was developed. This model described the rise in parasitaemia following inoculation, the nadir following dihydroartemisinin administration, and the subsequent resurgence of parasitaemia (analogous to ‘recrudescence’). At doses of 10, 30 and 100 mg/kg dihydroartemisinin, there was a graded response with 2.5 ± 1, 5 ± 1 and 12 ± 4-fold decreases in parasitaemia, respectively. The nadir parasitaemia (at 21–27 h) was also dose-dependent. This study demonstrates that a murine malaria pharmacodynamic model is a valuable tool for understanding how single drugs and their dosing schedules alter the time course and level of infection.     

Effect of water renewal on dominance hierarchy of juvenile Nile tilapia

Nile tilapia social position (Oreochromis niloticus) can be mediated by multiple channels, including chemical communication. Absence of chemical cues in the environment prevents hierarchical settlement among pairs, and enhances time spent in confrontations. The aim of this study was to test the effect of continuously renewed water flow on the establishment of hierarchical dominance in Nile tilapia juveniles. In this condition, a high frequency of attacks and disruption on hierarchical stability were expected because chemical cues for hierarchy maintenance could be washed out. After 3 days in isolation, the fish were paired by standard size but not by sex, and submitted to two conditions: continuously renewed water flow (RENEWED, n = 7) and non-renewed water flow (NONRENEWED, n = 8). The paired fish were placed in an aquarium (40 cm × 30 cm × 40 cm) for 3 h; four 10-min sessions were video-recorded: the first, immediately after the fish were paired and the others 1, 2, and 3 h after pairing. Hierarchy was identified by a dominance index (DI = given attacks/received + given attacks) for each fish. The hierarchical stability was achieved by analyzing the difference between dominant DI and subordinate DI (DI-D). Hierarchy was established in both groups after second session because the DI was significantly higher for one fish of the pair. The frequency of attacks of the dominant fish in RENEWED and NONRENEWED conditions was similar in all observation sessions. The attack frequency by subordinate fish was also similar during the first three sessions (2-h pairing). However, the frequency of attacks by subordinate fish in the RENEWED condition was higher than in the NONRENEWED situation at the fourth observation session (means ± S.E.: RENEWED = 2.83 ± 0.94 × 10 min⁻¹ and NONRENEWED = 0.25 ± 0.16 × 10 min⁻¹; Mann–Whitney, p = 0.04). At this point, a significant reduction of the DI-D was observed (means ± S.E.: RENEWED = 0.70 ± 0.11 and NONRENEWED = 1,00 ± 0.002; Mann–Whitney, p = 0.04). The changes in DI-D were related to more frequent attacks by the subordinated fish in renewed water flow. According to our results, the unsteady agonistic interaction under renewed water flow leads to social instability. Thus, continuous water renewing can wash out relevant chemical substances and therefore disturb the dominance recognition by subordinate fish.     

Serial measurements of whole blood nitrite in an intensive care setting

Nitrite plays an eminent role in cardiovascular physiology and pathology, mediating hypoxic vasodilation, reducing ischemia–reperfusion injury, and regulating cardiac energetics and function. The role of circulating nitrite in critically ill patients has not been examined so far. To investigate whether whole blood nitrite can be determined reproducibly in an intensive care setting, 30 patients from a cardiology intensive care unit were enrolled in this study, no matter what the underlying disease. Blood was drawn from an arterial catheter and whole blood nitrite was determined, using a tri-iodide/ozone-based chemiluminescence assay after incubation with a ferricyanide-containing stabilization solution. Whole blood nitrite levels ranged from 35 to 1193 nmol/L (mean ± SEM: 220 ± 20 nmol/L). Myocardial infarction was associated with lower whole blood nitrite levels (200 ± 53 nmol/L for elevated serum CK MB levels vs 432 ± 95 nmol/L in the normal CK MB range, p = 0.039). Neither impaired kidney function nor an inflammatory state was associated with higher or lower whole blood nitrite levels. In conclusion, whole blood nitrite can be measured easily and reproducibly in critically ill patients, regardless of renal function and inflammation. The origin of decreased nitrite levels in myocardial infarction is currently unclear and needs to be further elucidated.     

Influence of chelators and iron ions on the production and degradation of H2O2 by beta-amyloid-copper complexes

β-Amyloid peptide (Aβ) 1–42, involved in the pathogenesis of Alzheimer’s disease, binds copper ions to form Aβ · Cu_n complexes that are able to generate H₂O₂ in the presence of a reductant and O₂. The production of H₂O₂ can be stopped with chelators. More reactive than H₂O₂ itself, hydroxyl radicals HO (generated when a reduced redox active metal complex interacts with H₂O₂) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H₂O₂ associated to a pre-incubation step between ascorbate and Aβ · Cu_n allows to study the formation of H₂O₂ but also, at the same time, its transformation by the copper complexes. Aβ · Cu_n peptides produce but do not efficiently degrade H₂O₂. The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H₂O₂ production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from Aβ · Cu_n, whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with Aβ · Cu_n. The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.