Delayed treatment with GnRH agonist, hCG and progesterone and reduced embryonic mortality in buffaloes

The present study examined the effect of delayed treatment with tropic hormones and progesterone (P₄) on embryonic mortality in buffaloes. Buffaloes with a conceptus on Day 25 after AI were assigned to the following treatments: Control (n = 41), i.m. physiological saline; GnRH agonist (n = 36), i.m. 12 μg buserelin acetate; hCG (n = 33), i.m. 1500 IU hCG; P₄ (n = 38), i.m. 341 mg P₄ every 4 days on three occasions. Control buffaloes had an embryonic mortality of 41.4% (17/41) between Days 25 and 45, and this was reduced (P < 0.01) by treatment with GnRH agonist (11.1%, 4/36), hCG (9.0%, 3/33) and P₄ (13.1%, 5/38). On Day 45, buffaloes treated with hCG and which ovulated had greater (P < 0.05) concentrations of P₄ in whey (453 ± 41 pg/ml) than buffaloes in the same treatment that did not ovulate (297 ± 32 pg/ml). A similar but non-significant trend was observed for buffaloes treated with GnRH agonist. It was concluded from the findings that the treatment of buffaloes on Day 25 after AI with tropic hormones or P₄ is beneficial to processes associated with embryonic implantation.     

The effect of GnRH,eCG and progestin type on estrous synchronization following laparoscopic AI in ewes

The aim of the experiment was to evaluate the effects of GnRH and/or eCG and progestin type (implant versus CIDR) on the induction of estrus and pregnancy rate following laparoscopic AI (LAI) with frozen semen. In the first trial, ewes (n = 129) were treated with norgestomet implants for 14 days. At implant removal ewes received eCG (400 IU) and/or GnRH (25 μg) 36 h after removal, resulting in control, eCG, GnRH, and eCG/GnRH groups (n = 30–34/group). In trial 2, ewes (n = 36) were treated with intravaginal fluorogestone acetate sponges (FGA) or CIDR for 12 days. After withdrawal, half of the ewes from each progestin group received eCG (400 IU), resulting in sponge, sponge/eCG, CIDR and CIDR/eCG groups (n = 8–10/group). In both trials, estrous activity was assessed using a vasectomized ram from the time of progestin removal to laparoscopic AI with frozen semen 58–60 h (trial 1) or 54–56 h (trial 2) following cessation of treatment. In trial 1, GnRH decreased (P < 0.05) the percentage of ewes in estrus (GnRH, 75.8% versus control, 93.8% versus eCG/GnRH, 94.1%), however pregnancy rates were similar in all groups (control, 53.1%; eCG, 70.0%; GnRH, 51.5%; eCG/GnRH, 55.9%, respectively). In trial 2, neither the type of progestin nor eCG treatment effected the percentage of ewes in estrus (sponge, 75.0%; sponge/eCG, 100.0%; CIDR, 100.0%; CIDR/eCG, 90.0%). However, pregnancy rates following LAI were higher (P < 0.05) when ewes were treated with eCG (progestin + eCG, 73.7% versus progestin alone, 41.2%). Results demonstrate that the source of progestin does not influence the expression of estrus or the proportion of ewes pregnant following LAI. When progestin treatment protocols are used in combination with eCG, pregnancy rates can be increased. A dose of GnRH near the end of progestin treatment may decrease the estrous response, by inducing ovulation before normal expression of estrus.     

Synchronization of estrus with short- and long-term progestagen treatments and the use of GnRH prior to short-term progestagen treatment in ewes

The objective of the present study was to determine the efficacy of the synchronization of estrus using short- and long-term progestagen treatments in ewes at the onset of the breeding season, and to evaluate the effect of the exogenous GnRH administration immediately prior to short-term progestagen treatment on the reproductive performance. A total of 240 Tahirova cross-bred ewes, aged 18–24 months, and 40 rams, aged 2–4 years-old, were used in the trial. Ewes were divided equally into 3 groups (n = 80 per group). Intravaginal progestagen sponges containing FGA (30 mg) were inserted in the ewes for 7 d in the FGA1 (short-term) and GnRH treatment groups, and for 12 d in the FGA2 group (long-term). The ewes in the GnRH group received 10.5 μg busereline acetate i.m. at the time of sponge insertion. Tiaprost tromethamol (PGF_2α; 0.294 mg) and eCG (400 IU) were injected i.m. on the 6th day of progestagen treatment in the GnRH and FGA1 groups, and on the 11th day in the FGA2 group following sponge insertion. All ewes were hand-mated once at the detection of estrus. The estrous response, fertility rate, multiple birth rate and litter size recorded was 88.7, 87.3, 51.6% and 1.6 in the FGA1 group, 92.5, 71.6, 50.9% and 1.5 in the FGA2 group, and 96.2, 89.6, 71.0% and 1.8 in the GnRH group, respectively. No significant difference in estrous response between the groups was recorded, but the fertility rate in the FGA1 and GnRH groups was significantly (P < 0.05) higher than in the FGA2 group. The occurrence of multiple births and litter sizes were significantly (P < 0.05) higher in the GnRH group, compared to both the FGA1 and FGA2 groups, with the number of single lambs being significantly (P < 0.05) higher in the FGA1 (48.4%) and FGA2 (49.0%) groups than in the GnRH (29.0%) group. However, the differences recorded between any of the groups in terms of the number of twin and triplet lambs were insignificant. In conclusion, it can be said that estrous synchronization using the 12-d-FGA-eCG-PGF_2α regimen could be replaced with the 7-d-FGA-eCG-PGF_2α regime in sheep at the onset of the breeding season. However, the combination of GnRH with the latter regimen (7-d-GnRH-FGA-eCG-PGF_2α) increased the multiple birth rate and litter size in the ewes.     

Effects of feeding nutritionally balanced rations on animal productivity,feed conversion efficiency,feed nitrogen use efficiency,rumen microbial protein supply,parasitic load,immunity and enteric methane emissions of milking animals under field conditions

Milking animals produce milk commensurate with their genetic potential only when they are fed a nutritionally balanced ration in an amount that provides nutrients to express their genetic potential. As animals kept by smallholder farmers are rarely fed a balanced ration, a programme to feed balanced rations to animals of such farmers was launched in India. Based on their milk yield, the animals were categorized as: low (<8 kg/d), medium (8–12 kg/d) and high (>12 kg/d) yielders. Milk yield, milk fat and net daily income to milk producers were recorded before and after feeding a balanced ration. Nutritional status of animals showed that, for 71% of animals’, crude protein (CP) and metabolizable energy intakes were higher and, for 65% of animals’, calcium and phosphorus intakes were lower than requirements. Ration balancing improved milk yield by 2–14% and its milk fat proportion by 0.2–15%. Feed conversion efficiency, as kg of fat corrected milk (FCM)/kg of dry matter intake of buffaloes (n = 1131) before and after feeding balanced rations was 0.6 and 0.7, respectively, and in cows (n = 540) the values were 0.6 and 0.8. Dietary N secreted into milk increased from 0.16 to 0.25 and 0.16 to 0.19 in low and medium yielding cows and buffaloes, respectively. Rumen microbial CP synthesis also increased (P<0.05) by 36 and 38% in cows and buffaloes, respectively. On feeding balanced rations, levels (mg/ml) of plasma immunoglobulins IgG, IgM and IgA increased from 14.48 to 22.11, 2.69 to 3.29 and 0.48 to 0.67, and the parasitic load was reduced from 168 to 81 eggs/g of faeces. Enteric CH₄ emissions (g/kg milk yield) was reduced by 15–20% (P<0.05) in these lactating animals. Results demonstrate that feeding nutritionally balanced rations increased milk production and reduced enteric CH₄ emissions and N excretion from lactating cows and buffaloes. While implementation of a ration balancing programme under small holding systems is challenging, large scale use of this programme in tropical countries can help improve productivity of milking animals with available feed resources in an environmentally sustainable manner.     

The induction of oestrus in ewes during the non-breeding season using pre-used CIDRs and oestradiol-17β treatment

The fertility obtained in sheep after the use of intravaginal progesterone devices is related to the content of progesterone of the device. The hypothesis of this study was that the reproductive response of anoestrous ewes to the ram-effect could be improved by the administration of oestradiol-17β in conjunction with CIDRs treatment—using previously used CIDRs in a 5-day progestagen priming. Therefore, the objective was to determine if oestradiol-17β treatment increases fertility of anoestrous ewes primed with used CIDRs and stimulated by the ram-effect. The hypothesis was tested with CIDRs that had been previously used for 12 or 18 days. The trial was performed during the non-breeding season using 158 Corriedale ewes. Ewes had been isolated from rams since Day −35 (Day 0 = introduction of the rams). A CIDR (0.3 g progesterone, InterAg, Hamilton, New Zealand) was inserted on Day −5 in all ewes with CIDR that had been previously used for 12 days (n = 62) or 18 days (n = 96). Also on Day −5, 29 and 53 ewes that had received CIDRs of 12 or 18 days, respectively, received an intra-muscular treatment of 50 μg of oestradiol-17β (E groups). The ewes that did not receive the oestradiol-17β treatment remained as the control group (C group). Overall the treatment groups were thus: C12 (n = 33), C18 (n = 43), E12 (n = 29), and E18 (n = 53). On Day 0 all CIDRs were withdrawn, and ewes were placed with 18 rams and 20 ewes hormonally induced to exhibit oestrus. Sexual receptivity of ewes treated with CIDRs was estimated from marks on the rumps of the ewes daily from Day 0 to Day 5, and the pregnancy status diagnosed with transrectal ultrasonography on Day 40. The percentage of ewes exhibiting oestrus and pregnancy rates were lower in ewes synchronized with previously used CIDRs for 18 days, compared to those used for 12 days. The responses of ewes in oestrus were 39.4, 14.0, 65.5, and 32.1% for the C12, C18, E12, and E18 groups respectively, with pregnancy rates of 30.3, 14.0, 34.5, and 17.0%. Administration of oestradiol-17β increased the frequency of oestrous response in ewes that were treated with CIDRs previously used for 12 days (P < 0.05), but not in those treated with CIDRs used for 18 days. It could be concluded that the administration of oestradiol-17β only improved the percentage of ewes responding to oestrus when CIDRs previously used for 12 days were used for 5 days before the introduction of rams. No positive effect on fertility was observed irrespective of the period during which CIDR had been previously used.     

Design,synthesis and evaluation of dual pharmacology β2-adrenoceptor agonists and PDE4 inhibitors

A novel series of formoterol–phthalazinone hybrids were synthesised and evaluated as dual pharmacology β₂-adrenoceptor agonists and PDE4 inhibitors. Most of the hybrids displayed high β₂-adrenoceptor agonist and moderate PDE4 inhibitory activities. The most potent compound, (R,R)-11c, exhibited agonist (EC₅₀ = 1.05 nM, pEC₅₀ = 9.0) and potent PDE4B2 inhibitory activities (IC₅₀ = 0.092 μM).     

Optimal whole-body vibration settings for muscle strength and power enhancement in human knee extensors

This study compared the effects of 6-week whole-body vibration (WBV) training programs with different frequency and peak-to-peak displacement settings on knee extensor muscle strength and power. The underlying mechanisms of the expected gains were also investigated. Thirty-two physically active male subjects were randomly assigned to a high-frequency/high peak-to-peak displacement group (HH; n = 12), a low-frequency/low peak-to-peak displacement group (LL; n = 10) or a sham training group (SHAM; n = 10). Maximal voluntary isometric, concentric and eccentric torque of the knee extensors, maximal voluntary isometric torque of the knee flexors, jump performance, voluntary muscle activation, and contractile properties of the knee extensors were assessed before and after the training period. Significant improvement in knee extensor eccentric voluntary torque (P < 0.01), knee flexor isometric voluntary torque (P < 0.05), and jump performance (P < 0.05) was observed only for HH group. Regardless of the group, knee extensor muscle contractile properties (P < 0.05) were enhanced. No modification was observed for voluntary muscle activation or electrical activity of agonist and antagonist muscles. We concluded that high-frequency/high peak-to-peak displacement was the most effective vibration setting to enhance knee extensor muscle strength and jump performance during a 6-week WBV training program and that these improvements were not mediated by central neural adaptations.     

Synthesis,characterization and magnetic properties of six new copper(II) complexes with aminoacids as bridging ligand,exhibiting ferromagnetic coupling

The synthesis, crystallographic analysis and magnetic studies of six new copper(II) complexes of formulae [Cu(μ-ala)(im)(H₂O)]_n(ClO₄)_n (1), [Cu(μ-ala)(pz)(μ-ClO₄)] (2), [Cu(μ-phe)(im)(H₂O)]_n(ClO₄)_n (3), [Cu(μ-gly)(H₂O)(ClO₄)]_n (4), [Cu(μ-gly)(pz)(ClO₄)]_n(5) and [Cu(μ-pro)(pz)(ClO₄)]_n (6) have been carried out (ala = alanine; phe = phenylalanine; gly = glycine; pro = proline; im = imidazole; pz = pyrazole). In all cases, the deprotonated aminoacid ligand acts as chelate through the N(amine) and one O(carboxylato), whereas the second O atom of the same carboxylato acts as a bridge to the neighbouring copper(II) ion. The coordination of copper(II) ions is square-pyramidal in all complexes but 2 (elongated O_h). All complexes (1–6) are uniform chains with syn–anti (equatorial–equatorial) coordination mode of the carboxylato bridging ligand, exhibiting intrachain ferromagnetic interactions.     

Effects of exposing pigs to moving and odors in a simulated slaughter chute

Pigs in the finishing stage are infrequently handled and can be difficult to handle when experiencing novel situations. This study sought to determine the effects of minimal training and a novel odor/taste reward on the ease of handling finishing pigs in a novel environment. Pigs were assigned to one of four treatments organized in a factorial arrangement: training and odor exposure at the barn or not (trained or non-trained, respectively) and provision or not of maple syrup in the simulated pre-stun area of a slaughter plant (reward or no reward, respectively). Trained pigs (n = 14 pens) were let out of their home pens and onto a trailer for 10 min/d for 10 d and could chew on maple syrup-soaked flags. Non-trained pigs (n = 14 pens) were not handled or exposed to maple syrup. After the 10 d, trained and non-trained pigs were transported, unloaded and then experienced a novel simulated pre-stun area. A maple syrup-soaked flag (reward) was dragged through the simulated pre-stun area and put in a simulated CO₂ stun box. Non-rewarded pigs were not exposed to maple syrup. Trained pigs unloaded the trailer and reached the resting pen faster (P = 0.014) than non-trained pigs. Trained pigs had fewer (P = 0.02) blood neutrophils and more (P = 0.03) lymphocytes than non-trained pigs. Rewarded pigs received fewer (P = 0.02) taps before reaching the simulated CO₂ stun box than non-rewarded pigs. Cortisol concentration increased (P = 0.004) when the total time to reach the simulated CO₂ stun box increased. Pigs that were allowed to exercise out of their home pen and were given access to an odor/taste reward moved faster and the neutrophil:lymphocyte ratio was decreased when exposed to a novel environment containing the same odor/taste reward.     

Synthesis and anti-acetylcholinesterase properties of novel β- and γ-substituted alkoxy organophosphonates

Activated organophosphate (OP) insecticides and chemical agents inhibit acetylcholinesterase (AChE) to form OP-AChE adducts. Whereas the structure of the OP correlates with the rate of inhibition, the structure of the OP-AChE adduct influences the rate at which post-inhibitory reactivation or aging phenomena occurs. In this report, we prepared a panel of β-substituted ethoxy and γ-substituted propoxy phosphonoesters of the type p-NO₂PhO-P(X)(R)[(O(CH₂)_nZ] (R = Me, Et; X = O, S; n = 2, 3; Z = halogen, OTs) and examined the inhibition of three AChEs by select structures in the panel. The β-fluoroethoxy methylphosphonate analog (R = Me, Z = F, n = 2) was the most potent anti-AChE compound comparable (k_i ～6 × 10⁶ M^?1 min^?1) to paraoxon against EEAChE. Analogs with Z = Br, I, or OTs were weak inhibitors of the AChEs, and methyl phosphonates (R = Me) were more potent than the corresponding ethyl phosphonates (R = Et). As expected, analogs with a thionate linkage (PS) were poor inhibitors of the AChEs.     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.     

STK15 rs2273535 polymorphism and cancer risk: A meta-analysis of 74,896 subjects

Background: It has been suggested that the serine/threonine kinase 15 (STK15) T91A rs2273535 polymorphism is associated with susceptibility to cancer. However, the results are conflicting. We performed this meta-analysis to derive a more precise estimation of the relationship. Methods: PubMed was searched to select studies. Case–control studies containing available genotype frequencies of the STK15 rs2273535 polymorphism were chosen, and the odds ratio (OR) with its 95% confidence interval (CI) was utilized to assess the strength of association. Results: 52 studies – including 34,057 cases and 40,839 controls – were identified. A significant effect of the STK15 rs2273535 polymorphism on cancer risk was found (AA vs. TT: OR = 1.13, 95%CI = 1.01–1.26, P_{heterogeneity} < 0.001; AA vs. TA/TT: OR = 1.12, 95%CI = 1.02–1.22, P_{heterogeneity} < 0.001; TA/AA vs. TT: OR = 1.06, 95%CI = 1.01–1.12, P_{heterogeneity} < 0.001). Stratified analysis by cancer type revealed that the STK rs2273535 polymorphism may contribute to the risk of breast cancer (AA vs. TT: OR = 1.21, 95%CI = 1.01–1.44, P_{heterogeneity} = 0.002), colorectal cancer (AA vs. TA/TT: OR = 1.24, 95%CI = 1.05–1.47, P_{heterogeneity} = 0.124), and esophageal cancer (AA vs. TA/TT: OR = 1.19, 95%CI = 1.02–1.39, P_{heterogeneity} = 0.148). Further subgroup analysis by ethnicity indicated that there was a statistically increased cancer risk in Asians (AA vs. TA/TT: OR = 1.20, 95%CI = 1.05–1.37, P_{heterogeneity} = 0.004). Conclusion: This meta-analysis suggests that the STK15 rs2273535 polymorphism is a candidate gene polymorphism for cancer susceptibility, especially in Asian populations.     

Development of a microfluidic device for determination of cell osmotic behavior and membrane transport properties

An understanding of cell osmotic behavior and membrane transport properties is indispensable for cryobiology research and development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system is developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithography and is comprised of microfluidic channels and a perfusion chamber for trapping cells. During experiments, rat basophilic leukemia (RBL-1 line) cells were injected into the inlet of the device, allowed to flow downstream, and were trapped within a perfusion chamber. The fluid continues to flow to the outlet due to suction produced by a Hamilton Syringe. Two sets of experiments have been performed: the cells were perfused by (1) hypertonic solutions with different concentrations of non-permeating solutes and (2) solutions containing a permeating cryoprotective agent (CPA), dimethylsulfoxide (Me₂SO), plus non-permeating solute (sodium chloride (NaCl)), respectively. From experiment (1), cell osmotically inactive volume (V_b) and the permeability coefficient of water (L_p) for RBL cells are determined to be 41% [n = 18, correlation coefficient (r²) of 0.903] of original/isotonic volume, and 0.32 ± 0.05 μm/min/atm (n = 8, r² > 0.963), respectively, for room temperature (22 °C). From experiment (2), the permeability coefficient of water (L_p) and of Me₂SO (P_s) for RBL cells are 0.38 ± 0.09 μm/min/atm and (0.49 ± 0.13) × 10⁻³ cm/min (n = 5, r² > 0.86), respectively. We conclude that this device enables us to: (1) readily monitor the changes of extracellular conditions by perfusing single or a group of cells with prepared media; (2) confine cells (or a cell) within a monolayer chamber, which prevents imaging ambiguity, such as cells overlapping or moving out of the focus plane; (3) study individual cell osmotic response and determine cell membrane transport properties; and (4) reduce labor requirements for its disposability and ensure low manufacturing costs.     

Determinants of platelet activation in hypertensives with microalbuminuria

Microalbuminuria is a predictor of adverse outcome in hypertension. We evaluated in vivo platelet activation, by urinary 11-dehydrothromboxane (TX)B₂ and plasma P-selectin, in hypertensives with or without microalbuminuria, and its possible association with oxidative stress, by urinary 8-iso-prostaglandin (PG)F_2α and endothelial dysfunction. Sixty essential hypertensive patients, with (n = 30) or without (n = 30) microalbuminuria, and 30 controls were studied. Endothelial function was assessed by nitric oxide products, intercellular adhesion molecule (ICAM)-1, and asymmetric dimethylarginine (ADMA) levels. Urinary 11-dehydro-TXB₂ excretion was higher in microalbuminuric (median 805 pg/mg creatinine) compared to nonmicroalbuminuric patients or controls (414 and 291 pg/mg, respectively; P < 0.0001). Plasma P-selectin was significantly higher in patients with microalbuminuria (median 136 ng/ml) as compared to those without microalbuminuria or controls (85 and 65 ng/ml; P < 0.0001). Urinary 8-iso-PGF_2α excretion was also enhanced in microalbuminuric (median 279 pg/mg creatinine) compared to nonmicroalbuminuric patients or controls (157 and 146 pg/mg, respectively; P < 0.0001). A significant impairment in endothelial function was found in microalbuminuric patients, with decreased nitric oxide and increased ICAM-1 and ADMA levels. Multivariate regression analysis showed that urinary 8-iso-PGF_2α excretion (beta = 0.49; P < 0.0001) and microalbuminuria (beta = 0.36; P < 0.001) were independently related to 11-dehydro-TXB₂ in hypertensives. Vitamin E supplementation (900 mg daily for 1 month) in 10 hypertensives with microalbuminuria was associated with normalization in median 11-dehydro-TXB₂ and 8-iso-PGF_2α. We conclude that lipid peroxidation is a major determinant of persistent platelet activation in hypertensive patients with microalbuminuria.     

Structures,magnetic properties,and XPS of cyanide-bridged NdIII/SmIII/GdIII–CrIII complexes

Synthesis, structural characterization, and spectroscopic and magnetic properties of three new cyano-bridged 3d–4f bimetallic complexes, Ln^III(DMF)₄(H₂O)₃Cr^III (CN)₆ · nH₂O (Ln = Nd, Sm, Gd), have been described. The Nd–Cr complex crystallizes in the monoclinic P2₁/n space group with the following unit cell parameters: a = 20.063(7) Å, b = 8.967(4) Å, c = 18.023(6) Å, b = 96.12(3)°, V = 3224(2) Å³, and Z = 4. The neodymium (III) ion, which adopts anti-prism eight-coordination environment, is linked to the [Cr^III(CN)₆]³⁻ moiety through a bridging cyanide ligand with Nd–N = 2.550(4) Å and Nd–N–C = 164.4(4)°. The variable-temperature (0.5 T at 2–300 K) and variable-field (0–5 T at 2 and 5 K) magnetic measurements reveal that the weak interaction of Gd–Cr complexes differs from that of Nd–Cr and Sm–Cr ones mainly because of the lack of orbital angular momentum. The XPS and diffuse reflectance electronic spectra were also measured to discuss charge transfer transitions concerning π-backdonation from the viewpoint of magneto-optical functions.     

In vitro production of bovine embryos in medium supplemented with a serum replacer: Effects on blastocyst development,cryotolerance and survival to term

In this study, we evaluated a serum replacer (SR; Knockout SR^®, Invitrogen) in our in vitro culture systems. We hypothesized that SR would benefit bovine embryo development, since SR supported survival of embryonic stem cells (which originate from embryos). Experiment 1 compared oocyte maturation with SR versus fetal bovine serum (FBS). Following fertilization, blastocyst development was lower for oocytes matured with SR (21.5 versus 34.1, P < 0.05). Experiment 2 evaluated SR for culturing embryos. Following fertilization, embryos were cultured for 3 days in KSOM, and then assigned to treatments: (1) KSOM static culture (KNM); (2) fresh KSOM (KD3); (3) KSOM + SR or (4) KSOM + FBS and cultured to Day 7 (fertilization = Day 0). Blastocyst development in FBS or SR was higher than either KNM or KD3 (48.2, 47.2, 32.7, and 35.5, respectively, P < 0.05). Experiment 3 evaluated cryosurvival of embryos cultured in the same manner as Experiment 2. On Day 7, embryos were vitrified and upon warming, embryos cultured in SR had greater 24 h survival rates (70.6%) than all other treatments (P < 0.05). Finally, Experiment 4 evaluated effects of SR on pregnancy rate and development to term. Culture in SR was not detrimental to pregnancy or calving rates (50 and 50%, respectively), and SR calves had normal birth weights (mean = 38.8 kg ± 1.5). In conclusion, the use of SR for maturation of oocytes was not beneficial; however, SR enhanced embryo culture by improving development in vitro, cryotolerance and survival, effectively replacing serum in culture.     

Differential dopamine receptor subtype regulation of adenylyl cyclases in lipid rafts in human embryonic kidney and renal proximal tubule cells

Dopamine D₁-like receptors (D₁R and D₅R) stimulate adenylyl cyclase (AC) activity, whereas the D₂-like receptors (D₂, D₃ and D₄) inhibit AC activity. D₁R, but not the D₅R, has been reported to regulate AC activity in lipid rafts (LRs). We tested the hypothesis that D₁R and D₅R differentially regulate AC activity in LRs using human embryonic kidney (HEK) 293 cells heterologously expressing human D₁ or D₅ receptor (HEK-hD₁R or HEK-hD₅R) and human renal proximal tubule (hRPT) cells that endogenously express D₁R and D₅R. Of the AC isoforms expressed in HEK and hRPT cells (AC3, AC5, AC6, AC7, and AC9), AC5/6 was distributed to a greater extent in LRs than non-LRs in HEK-hD₁R (84.5 ± 2.3% of total), HEK-hD₅R (68.9 ± 3.1% of total), and hRPT cells (66.6 ± 2.2% of total) (P < 0.05, n = 4/group). In HEK-hD₁R cells, the D₁-like receptor agonist fenoldopam (1μM/15 min) increased AC5/6 protein (+ 17.2 ± 3.9% of control) in LRs but decreased it in non-LRs (− 47.3 ± 5.3% of control) (P < 0.05, vs. control, n = 4/group). By contrast, in HEK-hD₅R cells, fenoldopam increased AC5/6 protein in non-LRs (+ 67.1±5.3% of control, P < 0.006, vs. control, n = 4) but had no effect in LRs. In hRPT cells, fenoldopam increased AC5/6 in LRs but had little effect in non-LRs. Disruption of LRs with methyl-β-cyclodextrin decreased basal AC activity in HEK-D₁R (− 94.5 ± 2.0% of control) and HEK-D₅R cells (− 87.1 ± 4.6% of control) but increased it in hRPT cells (6.8 ± 0.5-fold). AC6 activity was stimulated to a greater extent by D₁R than D₅R, in agreement with the greater colocalization of AC5/6 with D₁R than D₅R in LRs. We conclude that LRs are essential not only for the proper membrane distribution and maintenance of AC5/6 activity but also for the regulation of D₁R- and D₅R-mediated AC signaling.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

Modulation of intracellular chloride channels by ATP and Mg2+

We report the effects of ATP and Mg²⁺ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n = 112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5–2 mmol/l) modulated and/or inhibited the chloride channel activities (n = 38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n = 5/93) or reversible (n = 15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n = 6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5–2 mmol/l) without the presence of Mg²⁺ decreased the chloride channel current (n = 12/14), whereas Mg²⁺ significantly reversed the effect (n = 4/4). We suggest that ATP-intracellular chloride channel interactions and Mg²⁺ modulation of these interactions may regulate different physiological and pathological processes.     

Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as nitrogen source

Arthrospira (Spirulina) platensis (Nordstedt) Gomont was autotrophically cultivated for biomass production in repeated fed-batch process using urea as nitrogen source, with the aim of making large-scale production easier, increasing cell productivity and then reducing the production costs. It was investigated the influence of the ratio of renewed volume to total volume (R), the urea feeding time (t_f) and the number of successive repeated fed-batch cycles on the maximum cell concentration (X_m), cell productivity (P_x), nitrogen-to-cell conversion yield (Y_x/n), maximum specific growth rate (μ_m) and protein content of dry biomass. The experimental results demonstrated that R = 0.80 and t_f = 6 d were the best cultivation conditions, being able to simultaneously ensure, throughout the three fed-batch cycles, the highest average values of three of the five responses (X_m = 2101 ± 113 mg L^?1, P_x = 219 ± 13 mg L^?1 d^?1 and Y_x/n = 10.3 ± 0.8 g g^?1).