bluuebunny/crossref_metadata_2025 huggingface dataset:
78CHRONOLOGICAL EVENTS OF IN VITRO MATURATION IN CAMEL (CAMELUS
DROMEDARIES) OOCYTES
Authors: Wani N.A. , Wernery U. , Nowshari M.A.
Published: February 2004

Experiments were conducted to investigate the chronological events and optimum time for in vitro oocyte maturation in the dromedary camel. Follicles measuring 3–12mm were isolated from ovaries obtained from an abattoir and the oocytes harvested by teasing apart these follicles under a stereo microscope. Pooled oocytes were randomly distributed to 4-well dishes (20–25per well) (Nunc, Denmark) containing 400μL of the maturation medium (TCM-199 supplemented with 0.6mgmL−1 calcium lactate, 0.1mgmL−1 L-glutamine, 0.8mgmL−1 sodium bicarbonate, 1.4mgmL−1 HEPES, 0.25mgmL−1 pyruvate, 50μgmL−1 gentamicine, 10μgmL−1 FSH, 10μgmL−1 LH, 1μgmL−1 estradiol and 10% heat-inactivated estrous camel serum) and incubated at 38.5°C under 5% CO2 for 4 to 48h. After every 4h (starting from 0 to 48h), oocytes were denuded by treating them with hyaluronidase (1mgmL−1) followed by repeated pipetting. Denuded oocytes were mounted on glass slides and fixed in 3:1 ethanol:acetic acid for 24h. Oocytes were stained with 1% aceto-orcein and examined under a phase contrast microscope at 400times. for each experimental group, 3 to 7 replications were made. Based on the visualization of the chromatin, oocytes were categorized as at germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase (Ana), metaphase-II (M-II) stage and those with no visible chromatin as NVC. At the start of maturation, 75.4% (43/57) of oocytes were at GV stage; however, none of the oocytes revealed a GV at 28 h of maturation (0/97). At 8h of maturation 49.3% (34/69) of oocytes were at DK stage, and after 16h of maturation 50% (49/98) of oocytes were at M-I stage. At 24h of maturation the maximum number of oocytes were in Ana (24.7%, 21/85) stage. At 44h the maximum number of oocytes had reached M-II stage (52%, 103/198) whereas, 10.6% (21/198) of the oocytes were at Ana stage. After 48h the proportion of oocytes with NCV increased to 52.9% (45/85) and the proportion of M-II stage oocytes decreased to 37.6% (32/85). It may be concluded that 40–44h of in vitro maturation yields the highest proportion of matured (M-II stage) oocytes suitable for further use in assisted reproductive technologies in camel.
8RESYNCHRONIZATION OF OVULATION AND TIMED INSEMINATION IN LACTATING DAIRY COWS USING THE OVSYNCH AND HEATSYNCH PROTOCOLS INITIATED 7 DAYS BEFORE PREGNANCY DIAGNOSIS ON DAY 30 BY ULTRASONOGRAPHY
Authors: Bartolome J.A. , Sozzi A. , McHale J. , Swift K. , Kelbert D. , Archbald L.F. , Thatcher W.W.
Published: February 2004

Rapid re-synchronization of ovulation and insemination in cows found nonpregnant to a previous service is important to maintain high reproductive efficiency. The objective was to compare pregnancy rate (PR) and pregnancy losses (PL) in cows subjected to Ovsynch or Heatsynch protocols 7 days before pregnancy diagnosis by ultrasonography (U/S) on Day 30. Initiation of both protocols included administration of GnRH on Day 23, which was considered the optimal time according to the distribution of interestrus intervals in cows previously inseminated. The study was conducted in a large dairy herd located in north central Florida from March to May, 2003. Of 593 cows treated with GnRH on Day 23 (±1) after insemination and found nonpregnant at U/S on Day 30 (±1; experimental Day 0), 75% (445) had a CL (diestrus) and were sequentially assigned to the two experimental groups. Cows in the Ovsynch Group (n=225) received 25mg (i.m.) PGF2α (Lutalyse, Pharmacia, Kalamazoo, MI, USA) on Day 0, 100μg (i.m.) GnRH (Cystorelin, Merial Limited, Iselin, NJ, USA) on Day 2, and were timed inseminated 16h later. Cows in Heatsynch Group (n=220) received 25mg (i.m.) PGF2α on Day 0, and 1mg (i.m.) estradiol cypionate (ECP, Pharmacia, Kalamazoo, MI, USA) on Day 1, and were timed-inseminated 36h later. Pregnancy at Day 30 was determined by U/S and at Day 55 by rectal examination of the genital tract. Pregnancy rate and PL were evaluated using multiple logistic regression. Pregnancy rate at Day 30 for Ovsynch (64/225, 28.4%) and Heatsynch (63/220, 28.6%) and PR at Day 55 for Ovsynch (58/225, 25.8%) and Heatsynch (54/220, 24.5%) were not different. In addition, PL between Days 30 and 55 for Ovsynch (6/64, 9.4%) and Heatsynch (9/63, 14.3%) were not different. There were no effects of parity, inseminator and days in milk on PR. However, PR at day 30 was higher in lots with cooling systems (46/141, 32.6%) and lots of first calf heifers (41/135, 30.4%) compared to lots of multiparous cows late in lactation without cooling systems (32/148, 21.6%). In conclusion, Heatsynch and Ovsynch initiated 7 days before pregnancy diagnosis on Day 30 had comparable PR of 25% at Day 55 in lactating dairy cows during the period of March–May.
83ANALYSIS OF RAPID COOLING V. SLOW COOLING COMBINED WITH ICE
CRYSTAL SEEDING FOR CRYOPRESERVATION OF PRIMATE EMBRYONIC STEM CELLS
Authors: Baran S. , Ware C.
Published: February 2004

Primate embryonic stem (ES) cells have the ability to self-renew indefinitely while maintaining the ability to differentiate. This unique property allows scientists to study the factors necessary for stem cell self-renewal and differentiation in vitro that reflect in vivo processes. Work with primate ES cells is handicapped by the poor survival (1–5%) of rhesus and human ES cells following standard tissue culture methods of rapid cryopreservation. The purpose of this study was to compare and contrast two cryopreservation techniques, slow cooling combined with ice crystal seeding commonly used for mammalian embryos v. rapid cooling commonly used for tissue culture, to find a method for efficient primate ES cell cryopreservation. A combination of trials was run to compare dimethyl sulfoxide (DMSO) v. ethylene glycol as a cryoprotectant, a cooling rate
of 0.3°C per minute following ice crystal seeding at −7°C v. placement at −80°C with no seeding, and rapid thaw with step-wise cryoprotectant removal v. one-step sucrose cryoprotectant removal. Cell survival was assessed through a combination of cell surface markers, alkaline phosphatase staining and morphology to look for undifferentiated cells and quantitate survival. All cryopreservations were performed with the same cell density. The survival of the cells with slow embryo-style cooling in DMSO with a step-wise cryoprotectant removal was 64.0% v. 12.8% with rapid cooling.
85ASSISTED HATCHING IMPROVES POST-WARMING IN VITRO VIABILITY OF VITRIFIED PORCINE BLASTOCYSTS
Authors: Beebe L.F.S. , Cameron R.D.A. , Blackshaw A.W. , Keates H. , Nottle M.B.
Published: February 2004

The objective of this study was to improve the vitrification protocol that was used to obtain the first reported litter of pigs resulting from the transfer of vitrified zona pellucida-intact blastocysts (Beebe et al. 2000 Theriogenology 53: 249 abstr). We had previously noticed that vitrified and warmed early blastocysts rarely hatched after 48h in vitro culture, starting to degenerate after 24h. These experiments examined whether assisted hatching, initially done by removing the zona, then by zona thinning, would improve post-warming in vitro viability. Early blastocysts were surgically recovered from mature Large White x Landrace sows five days after mating. Embryos were washed 3 times in modified phosphate-buffered saline (mPBS; Quinn et al. 1982 J. Reprod. Fert. 66:161–168), and then washed twice and cultured in 40-μL droplets of NCSU23+10% fetal bovine serum under mineral oil at 39°C until ready for vitrification. Embryos were cultured for 25min with 7.5μgmL−1 cytochalasin B, placed into a 0.5-mL eppendorf tube in 30μL of the culture medium, centrifuged for 13min at 13000g and then recovered back to the culture medium. All media involved in the vitrification protocol included cytochalasin B. Centrifuged embryos were equilibrated in 2M ethylene glycol in mPBS at 25°C for 5min, washed briefly in 8M ethylene glycol+7% polyvinylpyrrolidone in mPBS for approximately 20sec, loaded into open pulled straws and plunged into liquid nitrogen. Warming and rehydration was performed by immersing the end of the straw containing the embryos into 1.2mL 1M sucrose in mPBS at 39°C, allowing the thawing media to enter the straw and recovering the embryos from the straw with a pipette, followed by 2min in 1M ethylene glycol in mPBS, and 2min in 0.5M ethylene glycol, both at 25°C. The zona-intact group (ZI) was placed in mPBS at 39°C for at least 5min, then cultured in vitro for 24h. The zona-free group (ZF) was washed briefly in mPBS, and the zona was removed with 0.5% pronase in PBS for 30sec, then washed extensively in mPBS, all at 25°C, and then washed and cultured as described for 24h. After 24h embryos that had reformed the blastocoel and expanded were considered viable. Viable embryos were fixed with 100% ethanol and the nuclei stained with 10ngmL−1 bisbenzimide, visualized and counted using fluorescent microscopy. There were 3 experiments. Removing the zona did not affect survival rates (ZI 81% n=21; ZF 91% n=23) but improved the cell count by 56% (cell number±SEM; ZI 39.1±2.8 n=17; ZF 60.8±4.3 n=20; P<0.05 by ANOVA). A subsequent series of experiments found that zona-thinned embryos (0.25% pronase for 10sec) had the same survival and cell count as zona-free embryos. These experiments show that vitrified porcine blastocysts benefit from assisted hatching, whether the zona pellucida is removed or just thinned.
90EFFECTIVE VIABILITY THRESHOLD FOR PRESERVED HONEY BEE SEMEN
Authors: Collins A.M.
Published: February 2004

The honey bee, Apis mellifera, is one of very few insects for which artificial insemination is possible, and preservation of semen has been attempted. Honey bee queens normally mate with 7–20 males early in life, store the semen in the spermatheca and release approximately 30 sperm to fertilize each egg. Fertilized eggs are females (queens or workers) and unfertilized eggs are males (drones). The queen controls release of spermatozoa, laying only worker eggs across large areas of comb, and drone eggs in small separate groups. As stored spermatozoa are depleted, a queen will begin to lay mixed groups of workers and drones. Semen cryopreserved following existing protocols (Harbo JR 1983 Annals Entomol. Soc. Amer. 76, 890–891) has less than 20–25% live spermatozoa (based on percentage of workers reared). This viability level is insufficient to successfully fertilize all of the 1000–1500 eggs per day from an active queen, and the colony slowly dwindles and dies. Using dual fluorescent staining (Collins AM and Donoglye AM 1999 Theriogenology 51, 1513–1523). I have determined that semen with 46% live spermatozoa (50% fresh and 50% freeze-killed semen v/v) or more, produces consistent laying patterns with all fertilized eggs (workers) from inseminated queens (Collins AM 2000 Apidologie 31, 421–429). The current study was done to determine how long queens inseminated with mixed fresh:frozen semen would continue to produce normal brood (collective term for all eggs, larvae and pupae), as compared to those inseminated with all fresh semen, or naturally mated in flight. In spring, sister queens were reared and inseminated with [1] all fresh semen, [2] half fresh and half freeze-killed semen, or [3] ¼ fresh and ¾ freeze-killed semen, or [4] were allowed to mate naturally. The queens were evaluated monthly in individual colonies for percentage of worker offspring v. drone offspring and area of comb with eggs, larvae and pupae, until the queen failed. As expected, the inseminated queens produced less brood than did the naturally mated [4] queens. All of the queens with only fresh semen [1] produced normal brood with 100% workers, and lived well into the winter. Eighty-eight percent of the queens inseminated with half freeze-killed semen [2] performed as well, although some of those failed within a few months. The remaining 22% began laying with mixed worker and drone brood. In treatment [3], 60% of the queens produced variable but high levels of drones in reduced areas of egg laying. Those queens in this group that did lay well (40%) also survived into the winter. These results mean that preserved semen that has 50% or better viable sperm has a good probability of producing inseminated queens that will lay normally for a complete beekeeping season. This is sufficient time for breeders to rear daughter queens from such matings, and incorporate desirable genotypes into a breeding program.
92THE ADDITION OF SEMINAL PLASMA FROM INDIVIDUAL BOARS TO FREEZING
EXTENDER CAN IMPROVE THE POST-THAW SPERM SURVIVAL
Authors: Cremades T. , Carvajal G. , Hernandez M. , Vazquez J.M. , Martinez E.A. , Roca J.
Published: February 2004

Contradictory results have been reported about the effect of seminal plasma (SP) on the freezability of mammalian spermatozoa. In pigs, current methods for sperm cryopreservation involve removing seminal plasma. Therefore, no conclusive evidence of the potential effect of SP on the freezability of boar spermatozoa has been reported. In this study, we evaluate the effect of the addition of low concentrations of SP from individual boars to the freezing extender on post-thaw sperm survival. Sperm cryopreservation procedure included: dilution of sperm-rich fraction in Beltsville Thaw Solution extender (BTS), cooling to 17°C for 16h, centrifugation at 2400g for 3min, dilution in lactose/egg-yolk/glycerol/Equex Stem (freezing extender) to a final concentration of 1×109 spermmL−1, dispensing into 0.5-mL straws, and freezing in a programmable cell freezer at 20°Cmin−1. Thawing was carried out in a waterbath at 37°C for 20s. Post-thaw sperm survival was assessed by progressive sperm motility (PSM) using a CASA system (SCA); plasma membrane integrity (PMI) and acrosome membrane integrity (AMI) were assessed by flow cytometric procedures (SYBR-14/PI and FITC-PNA/PI, respectively) at 30 and 150min post-thawing in BTS-diluted thaw spermatozoa held in a waterbath at 37°C. Four individual seminal plasma donors (SP1 to SP4) were selected in a preliminary study in which 48 ejaculates from 12 boars (4 ejaculates/boar) were cryopreserved. Then the boars were classified into 3 groups (good, moderate and bad freezers) based on their post-thaw sperm survival. SP1 and SP2 were good freezers (>60% PSM and PMI), SP3 was a moderate freezer (40–60% PSM and PMI) and SP4 was a bad freezer (<40% PSM and PMI). In the main experiment, pooled sperm-rich fractions collected from 9 mature hybrid boars were divided into five aliquots and each was diluted with freezing extender supplemented with 0% (control) or 10% of SP (1–4). Data from eight replicates were analyzed as a split plot design using a PROMIXED model. The addition of SP to freezing extender had a significant effect (P<0.05) on post-thaw sperm survival compared to control. Moreover, there were significant differences (P<0.05) between SP donors. PSM, PMI and AMI were significantly (P<0.05) higher in SP1 (56.71±4.30; 57.16±4.01 and 57.22±4.01, respectively) and SP2 (59.48±4.30; 60.17±4.01 and 60.05±4.01, respectively) compared to control (50.39±4.30; 49.98±4.01 and 49.54±4.01, respectively). There were no differences (P>0.05) between SP3, SP4 and control. These results indicate that the addition of SP from particular boars (good freezers) to freezing extender may improve post-thaw sperm survival. Individual differences in the SP composition should explain the above results. Supported by INIA (RZ01-019) and MCYT (AGL2001-0471).
95FIRST REPORT ON CLEAVAGE DEVELOPMENT FOLLOWING CRYOPRESERVATION OF
ADULT AND PREPUBERTAL RHESUS MONKEY (MACACA MULATTA) OOCYTES
Authors: Dinnyes A. , Wei S. , Li Y. , Zheng P. , Ji W.
Published: February 2004

Cryopreservation of non-human primate oocytes would allow a more efficient management of biological resources for medical research and endangered species preservation. Despite previous attempts, no cleavage-stage development has been reported following cryopreservation of rhesus monkey (Macaca mulatta) or other non-human primate oocytes. The extreme chilling sensitivity of rhesus monkey oocytes (Songsasen N et al., 2002 Fertil. Steril. 77, 818–825) might be overcomed by vitrification methods. Our aim was to test the Solid Surface Vitrification (SSV, Dinnyes A et al., 2000 Biol. Reprod. 63, 513–518) method on metaphase II (MII)-stage rhesus monkey oocytes and to achieve successful fertilization of warmed oocytes. Oocytes were obtained from hormonally stimulated adult and unstimulated prepubertal females and matured in vitro for 36 to 48h as described by Zhang P et al. (2001 Biol. Reprod. 64, 1417–1421). The vitrification solution contained 35% ethylene glycol (EG), 5% polyvinyl pyrrolidone, and 0.4M trehalose in Tyrode-lactate (TL)-HEPES medium with 3mgmL−1 BSA added. Oocytes at MII-stage were equilibrated in 4% EG in TL-HEPES at 37°C for 10 to 12min and then exposed to the vitrification solution at 37°C for about 20s. Oocytes in 2-μL droplets of vitrification solution were dropped onto a metal surface at about −180°C where they were vitrified instantaneously. Warming was performed by moving the vitrified droplets into 0.3M trehalose at 37°C. Recovered oocytes were exposed to 0.15M and then 0.075M trehalose for 2min each and then rinsed three times in TL-HEPES. Warmed oocytes were fertilized in vitro and then cultured for 96h in 50-μL drops of mCMRL-1066 containing 20% FCS at 37°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2, as described in details by Zhang (see above). A total of 21 MII oocytes were collected from 2 adult and 55 MII oocytes from 14 prepubertal animals. No difference has been found between the rate of adult-origin (15/19; 79%) v. prepubertal-origin (37/52; 71%) oocytes surviving the vitrification process
without lysis (P>0.05, chi-square analyses). Following subsequent IVF, 1/15 (7%) adult-origin and 4/23 (17%) prepubertal-origin oocytes cleaved further, which was lower than that of the corresponding controls (1/2 (50%) and 1/3 (33%), respectively). The furthest development observed following cryopreservation was 16-cell stage, verified by counting of stained nuclei. This is the first report on cleavage-stage development of cryopreserved rhesus monkey oocytes, demonstrating the feasibility of vitrification and the potential for gene banking of non-human primate oocytes, even from prepubertal animals. Further experiments are needed to achieve higher rates of cryosurvival and progeny development. This research was supported by a Chinese-Hungarian Bilateral Technological and Scientific Collaboration Project (No. CHN14/02).
99MACROMOLECULES FOR VITRIFYING BOVINE OOCYTES
Authors: Horvath G. , Seidel G.E.
Published: February 2004

Vitrification of oocytes would make them available for research and clinical purposes wherever and whenever needed. However, development rates and quality of blastocysts arising from vitrified oocytes have been low. Zona hardening following exposure to vitrification solutions and cooling could contribute to low fertilization rates. Addition of fetal calf serum (FCS) to handling media and vitrification solutions can prevent zona hardening, but FCS may be detrimental to resulting embryos and spread viral diseases, and its composition varies among batches. Fetuin is the component responsible for the protective effect of FCS (Landim-Alvarenga FC et al., 2002 Anim. Reprod. Sci. 71, 181–191). Our objective was to determine whether fetuin is a suitable substitute for FCS during vitrification. Oocytes derived from slaughterhouse ovaries were matured in a chemically defined medium with hormones and with or without 1mgmL−1 fetuin at 39°C in 5% CO2 in air. At 22h after the start of maturation, oocytes were transferred to one of the following handling media: 2% BSA, 2% BSA+1mgmL−1 fetuin, or 20% FCS in TCM-199+HEPES (HTCM-199). In the same media plus 100IUmL−1 hyaluronidase, cumulus cells were partly removed by gentle pipetting. Oocytes with approximately 3 layers of cumulus were chosen for two-step vitrification. First, they were exposed to VS1 (10% ethylene glycol (EG), 10% DMSO, 6% PVP in HTCM-199) for 30s, then to VS2 (20% EG, 20% DMSO, 6% PVP, 0.48M galactose in HTCM-199) for 25s, loaded into cryoloops in groups of five, and plunged into liquid nitrogen. Rapidly warmed oocytes were moved stepwise in 0.5, 0.25, 0.125, and 0M galactose in HTCM-199+20% FCS, 3 min each. All procedures were conducted at 39°C. Warmed oocytes were placed in maturation medium for an additional hour, and then fertilized and cultured according to standard procedures (Olson SE and Seidel GE Jr 2000 J. Anim. Sci. 78, 152–157). About 2000 oocytes were used in 11 replicates with semen of 4 bulls. The experimental treatments and controls were: A: maturation BSA, handling BSA; B: maturation BSA, handling FCS; C: maturation BSA, handling BSA+fetuin; D: maturation BSA+fetuin, handling BSA+fetuin; E: non-vitrified control, maturation BSA; F: non-vitrified control, maturation BSA+fetuin (Table 1). Controls did not differ (P>0.1) from each other, nor were there differences among vitrification treatments. Controls resulted in greater cleavage and more 8-cell embryos and blastocysts than vitrified treatments, and also tended to have higher cell numbers. In summary, fetuin can replace FCS during handling without decreasing the success of vitrification.

Table 1
Development of vitrified oocytes and controls
Quaternary Organic Amines Inhibit Na,K Pump Current in a Voltage-dependent Manner
Authors: Peluffo R. Daniel , Hara Yukio , Berlin Joshua R.
Published: February 2004

The effects of organic quaternary amines, tetraethylammonium (TEA) chloride and benzyltriethylammonium (BTEA) chloride, on Na,K pump current were examined in rat cardiac myocytes superfused in extracellular Na+-free solutions and whole-cell voltage-clamped with patch electrodes containing a high Na+-salt solution. Extracellular application of these quaternary amines competitively inhibited extracellular K+ (K+o) activation of Na,K pump current; however, the concentration for half maximal inhibition of Na,K pump current at 0 mV (K0Q) by BTEA, 4.0 ± 0.3 mM, was much lower than the K0Q for TEA, 26.6 ± 0.7 mM. Even so, the fraction of the membrane electric field dissipated during K+o activation of Na,K pump current (λK), 39 ± 1%, was similar to λK determined in the presence of TEA (37 ± 2%) and BTEA (35 ± 2%), an indication that the membrane potential (VM) dependence for K+o activation of the Na,K pump current was unaffected by TEA and BTEA. TEA was found to inhibit the Na,K pump current in a VM-independent manner, i.e., inhibition of current dissipated 4 ± 2% of the membrane electric field. In contrast, BTEA dissipated 40 ± 5% of the membrane electric field during inhibition of Na,K pump current. Thus, BTEA inhibition of the Na,K-ATPase is VM-dependent. The competitive nature of inhibition as well as the similar fractions of the membrane electric field dissipated during K+o-dependent activation and BTEA-dependent inhibition of Na,K pump current suggest that BTEA inhibits the Na,K-ATPase at or very near the enzyme's K+o binding site(s) located in the membrane electric field. Given previous findings that organic quaternary amines are not occluded by the Na,K-ATPase, these data clearly demonstrate that an ion channel–like structure provides access to K+o binding sites in the enzyme.
State-dependent Block of CNG Channels by Dequalinium
Authors: Rosenbaum Tamara , Gordon-Shaag Ariela , Islas León D. , Cooper Jeremy , Munari Mika , Gordon Sharona E.
Published: February 2004

Cyclic nucleotide–gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca2+. Not surprisingly, they are blocked by a number of Ca2+ channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca2+-activated K+ channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3–5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10–20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.
Osmotic Properties of the Sealed Tubular System of Toad and Rat Skeletal Muscle
Authors: Launikonis Bradley S. , Stephenson D. George
Published: February 2004

A method was developed that allows conversion of changes in maximum Ca2+-dependent fluorescence of a fixed amount of fluo-3 into volume changes of the fluo-3–containing solution. This method was then applied to investigate by confocal microscopy the osmotic properties of the sealed tubular (t-) system of toad and rat mechanically skinned fibers in which a certain amount of fluo-3 was trapped. When the osmolality of the myoplasmic environment was altered by simple dilution or addition of sucrose within the range 190–638 mosmol kg−1, the sealed t-system of toad fibers behaved almost like an ideal osmometer, changing its volume inverse proportionally to osmolality. However, increasing the osmolality above 638 to 2,550 mosmol kg−1 caused hardly any change in t-system volume. In myoplasmic solutions made hypotonic to 128 mosmol kg−1, a loss of Ca2+ from the sealed t-system of toad fibers occurred, presumably through either stretch-activated cationic channels or store-operated Ca2+ channels. In contrast to the behavior of the t-system in toad fibers, the volume of the sealed t-system of rat fibers changed little (by <20%) when the osmolality of the myoplasmic environment changed between 210 and 2,800 mosmol kg−1. Results were also validated with calcein. Clear differences between rat and toad fibers were also found with respect to the t-system permeability for glycerol. Thus, glycerol equilibrated across the rat t-system within seconds to minutes, but was not equilibrated across the t-system of toad fibers even after 20 min. These results have broad implications for understanding osmotic properties of the t-system and reversible vacuolation in muscle fibers. Furthermore, we observed for the first time in mammalian fibers an orderly lateral shift of the t-system networks whereby t-tubule networks to the left of the Z-line crossover to become t-tubule networks to the right of the Z-line in the adjacent sarcomere (and vice versa). This orderly rearrangement can provide a pathway for longitudinal continuity of the t-system along the fiber axis.
Protons Block BK Channels by Competitive Inhibition with K+ and Contribute to the Limits of Unitary Currents at High Voltages
Authors: Brelidze Tinatin I. , Magleby Karl L.
Published: February 2004

Proton block of unitary currents through BK channels was investigated with single-channel recording. Increasing intracellular proton concentration decreased unitary current amplitudes with an apparent pKa of 5.1 without discrete blocking events, indicating fast proton block. Unitary currents recorded at pHi 8.0 and 9.0 had the same amplitudes, indicating that 10−8 M H+ had little blocking effect. Increasing H+ by recording at pHi 7.0, 6.0, and 5.0 then reduced the unitary currents by 13%, 25%, and 53%, respectively, at +200 mV. Increasing K+i relieved the proton block in a manner consistent with competitive inhibition of K+i action by H+i. Proton block was voltage dependent, increasing with depolarization, indicating that block was coupled to the electric field of the membrane. Proton block was not described by the Woodhull equation for noncompetitive voltage-dependent block, but was described by an equation for cooperative competitive inhibition that included voltage-dependent block from the Woodhull equation. Proton block was still present after replacing the eight negative charges in the ring of charge at the entrance to the intracellular vestibule by uncharged amino acids. Thus, the ring of charge is not the site of proton block or of competitive inhibition of K+i action by H+i. With 150 mM symmetrical KCl, unitary current amplitudes increased with depolarization, reaching 66 pA at +350 mV (pHi 7.0). The increase in amplitude with voltage became sublinear for voltages >100 mV. The sublinearity was unaffected by removing from the intracellular solutions Ca2+ and Ba2+ ions, the Ca2+ buffers EGTA and HEDTA, the pH buffer TES, or by replacing Cl− with MeSO3−. Proton block accounted for ∼40% of the sublinearity at +200 mV and pH 7.0, indicating that factors in addition to proton block contribute to the sublinearity of the unitary currents through BK channels.
Human Topoisomerase IIα: Targeting to Subchromosomal Sites of Activity during Interphase and Mitosis
Authors: Agostinho Marta , Rino José , Braga José , Ferreira Fernando , Steffensen Soren , Ferreira João
Published: February 2004

Mammalian topoisomerase IIα (topo IIα) plays a vital role in the removal of topological complexities left on DNA during S phase. Here, we developed a new assay to selectively identify sites of catalytic activity of topo IIα with subcellular resolution. We show that topo IIα activity concentrates at replicating heterochromatin in late S in a replication-dependent manner and at centric heterochromatin during G2 and M phases. Inhibitor studies indicate that this cell cycle-dependent concentration over heterochromatin is sensitive to chromatin structure. We further show that catalytically active topo IIα concentrates along the longitudinal axis of mitotic chromosomes. Finally, we found that catalytically inert forms of the enzyme localize predominantly to splicing speckles in a dynamic manner and that this pool is differentially sensitive to changes in the activities of topo IIα itself and RNA polymerase II. Together, our data implicate several previously unsuspected activities in the partitioning of the enzyme between sites of activity and putative depots.
Multicenter Randomized Comparison of the Efficacy and Safety of Xenon and Isoflurane in Patients Undergoing Elective Surgery
Authors: Rossaint Rolf , Reyle-Hahn Matthias , Schulte am Esch Jochen , Scholz Jens , Scherpereel Philippe , Vallet Benoit , Giunta Francesco , Del Turco Monica , Erdmann Wilhelm , Tenbrinck Rob , Hammerle Alfons F. , Nagele Peter
Published: February 2004

Background

All general anesthetics used are known to have a negative inotropic side effect. Since xenon does not have a negative inotropic effect, it could be an interesting future general anesthetic. The aim of this clinical multicenter trial was to test the hypothesis of whether recovery after xenon anesthesia is faster compared with an accepted, standardized anesthetic regimen and that it is as effective and safe.

Method

A total of 224 patients in six centers were included in the protocol. They were randomly assigned to receive either xenon (60 +/- 5%) in oxygen or isoflurane (end-tidal concentration, 0.5%) combined with nitrous oxide (60 +/- 5%). Sufentanil (10 mcirog) was intravenously injected if indicated by defined criteria. Hemodynamic, respiratory, and recovery parameters, the amount of sufentanil, and side effects were assessed.

Results

The recovery parameters demonstrated a statistically significant faster recovery from xenon anesthesia when compared with isoflurane-nitrous oxide. The additional amount of sufentanil did not differ between both anesthesia regimens. Hemodynamics and respiratory parameters remained stable throughout administration of both anesthesia regimens, with advantages for the xenon group. Side effects occurred to the same extent with xenon in oxygen and isoflurane-nitrous oxide.

Conclusion

This first randomized controlled multicenter trial on the use of xenon as an inhalational anesthetic confirms, in a large group of patients, that xenon in oxygen provides effective and safe anesthesia, with the advantage of a more rapid recovery when compared with anesthesia using isoflurane-nitrous oxide.
Effects of Recruitment Maneuver on Atelectasis in Anesthetized Children
Authors: Tusman Gerardo , Böhm Stephan H. , Tempra Alejandro , Melkun Fernando , García Eduardo , Turchetto Elsio , Mulder Paul G. H. , Lachmann Burkhard
Published: February 2004

Background

General anesthesia is known to promote atelectasis formation. High inspiratory pressures are required to reexpand healthy but collapsed alveoli. However, in the absence of positive end-expiratory pressure (PEEP), reexpanded alveoli collapse again. Using magnetic resonance imaging, the impact of an alveolar recruitment strategy on the amount and distribution of atelectasis was tested.

Methods

The authors prospectively randomized 24 children who met American Society of Anesthesiologists physical status I or II criteria, were aged 6 months-6 yr, and were undergoing cranial magnetic resonance imaging into three groups. After anesthesia induction, in the alveolar recruitment strategy (ARS) group, an alveolar recruitment maneuver was performed by manually ventilating the lungs with a peak airway pressure of 40 cm H2O and a PEEP of 15 cm H2O for 10 breaths. PEEP was then reduced to and kept at 5 cm H2O. The continuous positive airway pressure (CPAP) group received 5 cm H2O of continuous positive airway pressure without recruitment. The zero end-expiratory pressure (ZEEP) group received neither PEEP nor the recruitment maneuver. All patients breathed spontaneously during the procedure. After cranial magnetic resonance imaging, thoracic magnetic resonance imaging was performed.

Results

The atelectatic volume (median, first and third standard quartiles) detected in the ZEEP group was 1.25 (0.75-4.56) cm3 in the right lung and 4.25 (3.2-13.9) cm3 in the left lung. The CPAP group had 9.5 (3.1-23.7) cm3 of collapsed lung tissue in the right lung and 8.8 (5.3-28.5) cm3 in the left lung. Only one patient in the ARS group presented an atelectasis of less than 2 cm3. An uneven distribution of the atelectasis was observed within each lung and between the right and left lungs, with a clear predominance of the left basal paradiaphragmatic regions.

Conclusion

Frequency of atelectasis was much less following the alveolar recruitment strategy, compared with children who did not have the maneuver performed. The mere application of 5 cm H2O of CPAP without a prior recruitment did not show the same treatment effect and showed no difference compared to the control group without PEEP.
Developmental Changes of Laryngeal Dimensions in Unparalyzed, Sedated Children
Authors: Litman Ronald S. , Weissend Eric E. , Shibata Dean , Westesson Per-Lennart
Published: February 2004

Background

Knowledge of the influence of age on laryngeal dimensions is essential for all practitioners whose interest is the pediatric airway. Early cadaver studies documented that the larynx is conically shaped, with the apex of the cone caudally positioned at the nondistensible cricoid cartilage. These dimensions change during childhood, as the larynx assumes a more cylindrical shape. The authors analyzed laryngeal dimensions during development to determine if this relationship continues in unparalyzed children in whom laryngeal muscles are tonically active. The authors determined the relationships between the vocal cord, sub-vocal cord, and cricoid ring dimensions and the influence of age on these relationships.

Methods

Infants and children undergoing magnetic resonance imaging with propofol sedation had determinations of the transverse and anterior-posterior (AP) dimensions of the larynx at the most cephalad level of the larynx (vocal cords) and the most caudad level (cricoid). Most patients had an additional measurement (sub-vocal cord) at a level between the vocal cords and the cricoid ring. Relationships were obtained by plotting age against laryngeal dimensions and the ratio of laryngeal dimensions at different levels within the larynx.

Results

The authors measured transverse and AP laryngeal dimensions in 99 children, aged 2 months-13 yr. The relationship between the transverse and AP dimensions at all levels of the larynx did not change during development. Transverse and AP dimensions increased linearly with age at all levels of the larynx. In all children studied, the narrowest portion of the larynx was the transverse dimension at the level of the vocal cords. Transverse dimensions increased linearly in a caudad direction through the larynx ( P< 0.001), while AP dimensions did not change relative to laryngeal level. The shape of the cricoid ring did not change throughout childhood.

Conclusions

In sedated, unparalyzed children, the narrowest portions of the larynx are the glottic opening (vocal cord level) and the immediate sub-vocal cord level, and there is no change in the relationships of these dimensions relative to cricoid dimensions throughout childhood.
Difference in Risk Factors for Postoperative Nausea and Vomiting
Authors: Stadler Michaela , Bardiau Françoise , Seidel Laurence , Albert Adelin , Boogaerts Jean G.
Published: February 2004

Background

It is commonly stated that risk factors for postoperative nausea are the same as for vomiting. The authors designed a prospective study to identify and differentiate the risk factors for postoperative nausea and vomiting in various surgical populations in a clinical audit setting.

Methods

The study included 671 consecutive surgical inpatients, aged 15 yr or more, undergoing various procedures. The study focused on postoperative nausea visual analog scale scores every 4 h and vomiting episodes within 72 h. Both vomiting and retching were considered as emetic events. Patient-, anesthesia-, and surgery-related variables that were considered to have a possible effect on the proportion of patients experiencing postoperative nausea and/or vomiting were examined. The bivariate Dale model for binary correlated outcomes was used to identify selectively the potential risk factors of postoperative nausea and vomiting.

Results

Among the 671 patients in the study, 126 (19%) reported one or more episodes of nausea, and 66 patients (10%) suffered one or more emetic episodes during the studied period. There was a highly significant association between the two outcomes. Some risk factors were predictive of both nausea and vomiting (female gender, nonsmoking status, and general anesthesia). History of migraine and type of surgery were mainly responsible for nausea but not for vomiting. The predictive effect of risk factors was controlled for postoperative pain and analgesic drugs.

Conclusion

This study shows that differences exist in risk factors of postoperative nausea and vomiting. These could be explained by differences in the physiopathology of the two symptoms.
Parental Presence during Induction of Anesthesia
Authors: Kain Zeev N. , Caldwell-Andrews Alison A. , Mayes Linda C. , Wang Shu-Ming , Krivutza Dawn M. , LoDolce Megan E.
Published: February 2004

Background

The authors conducted a randomized controlled trial to determine whether parental presence during induction of anesthesia (PPIA) is associated with parental physiologic and behavioral manifestations of stress.

Methods

Children and their parents (N = 80) were randomly assigned to one of three groups: (1) PPIA; (2) PPIA plus 0.5 mg/kg oral midazolam; and (3) control (no PPIA or midazolam). The effect of the group assignment on parental heart rate (HR), parental blood pressure, and parental skin conductance level (SCL) were assessed. Both parental HR and parental SCL were monitored continually. Anxiety of the parent and child was also assessed.

Results

Parental HR increased from baseline until the induction of anesthesia (P = 0.001). A group-by-time effect ( P= 0.005) was also found. That is, throughout the induction period there were several time points at which parents in the two PPIA groups had a significantly higher HR than did parents in the control group (P < 0.05). Similarly, SCL was found to increase in all parents from baseline until induction of anesthesia (P = 0.001). Significant group differences in SCL changes over time were found as well (P = 0.009). State anxiety and blood pressure following induction of anesthesia did not differ significantly between groups ( P= nonsignificant). Examination of parental Holter data revealed no rhythm abnormalities and no electrocardiogram changes indicating ischemia.

Conclusions

The authors found that PPIA is associated with increased parental HR and SCL. However, no increased incidence of electrocardiogram abnormalities were found in parents present during induction of anesthesia.
Performance of Target-controlled Sufentanil Infusion in Obese Patients
Authors: Slepchenko Gregory , Simon Nicolas , Goubaux Bernard , Levron Jean-Claude , Le Moing Jean-Pierre , Raucoules-Aimé Marc
Published: February 2004

Background

Because obesity might affect pharmacokinetic parameters, the authors evaluated the accuracy of target-controlled sufentanil infusion in morbidly obese patients using a pharmacokinetic model usually applied to a normal-weight population.

Methods

Target-controlled propofol and sufentanil coinfusions were administered to 11 morbidly obese patients (body mass index: 45.0 +/- 6.5 kg/m2 ) undergoing laparoscopic gastroplasty. The target plasma propofol concentration was 3 micro g/ml. The effect-site sufentanil target concentration was initially 0.4 ng/ml but was modified during surgery as a function of blood pressure and heart rate. Plasma sufentanil concentrations were measured from the onset of infusion until 24 h after its termination. The predicted sufentanil target concentrations were calculated by STANPUMP software. Intrasubject data analyzed included calculation of performance error, median performance error, median absolute performance error, divergence, and wobble. Pharmacokinetic analysis was performed using a nonlinear mixed effect model.

Results

Applied sufentanil target concentrations ranged from 0.3 to 0.65 ng/ml. The mean +/- SD plasma sufentanil concentration measured during spontaneous ventilation was 0.13 +/- 0.03 ng/ml. Median performance error (range) was -13% (-42 to 36%). Median absolute performance error was 26% (8-44%) during infusion and 17% (12-59%) for the 24 h after its completion. The pharmacokinetic sets used slightly overpredicted the concentrations, with a median divergence of -3.4% (-10.2 to 3.1%) during infusion. For body mass index greater than 40, the overestimation of plasma sufentanil concentrations was greater. A two-compartment model with proportional error for interindividual variability best fitted the data. The residual variability was modeled as an additive (0.016 ng/ml) or proportional error (23%). Clearance, central volume of distribution, intercompartmental clearance, and peripheral volume of distribution (coefficient of variation) were 1.27 l/min (23%), 37.1 l (20%), 0.87 l/min (44%), and 92.7 l (22%), respectively.

Conclusion

The pharmacokinetic parameter set derived from a normal-weight population accurately predicted plasma sufentanil concentrations in morbidly obese patients.
Halogenated Anesthetics Reduce Interleukin-1β-induced Cytokine Secretion by Rat Alveolar Type II Cells in Primary Culture
Authors: Giraud Olivier , Molliex Serge , Rolland Corinne , Leçon-Malas Véronique , Desmonts Jean-Marie , Aubier Michel , Dehoux Monique
Published: February 2004

Background

Alveolar epithelial type II (AT II ) cells participate in the intraalveolar cytokine network by secreting cytokines and are widely exposed to volatile anesthetics during general anesthesia. The aim of the current study was to evaluate the effects of halothane, enflurane, and isoflurane on rat AT II cell cytokine secretions in AT II primary cell cultures.

Methods

Alveolar epithelial type II primary cell cultures were obtained from adult rat lungs. AT II cells were stimulated by recombinant murine interleukin-1beta (rmIL-1beta) to mimic an inflammatory response, and immediately exposed for various duration to different concentration of halothane, enflurane, or isoflurane. Interleukin-6, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1) protein concentrations were then measured in cell culture supernatants. Recombinant mIL-1beta-stimulated AT II cells exposed to air served as control.

Results

Halothane, isoflurane, and enflurane (1 minimum alveolar concentration [MAC], 4 h) decreased rmIL-1beta-stimulated AT II cell secretions of interleukin-6, MIP-2, and MCP-1, but did not modify total protein secretion. Halothane exposure decreased rmIL-1beta-stimulated AT II cell secretions of interleukin-6, MIP-2, and MCP-1 in a dose- and time-dependent manner. Total protein concentrations remained unchanged except AT II 1.5 MAC of halothane, and no cytotoxic effect could be evidenced by lactate dehydrogenase release. These effects were transient as rmIL-1beta-stimulated AT II cell secretions of interleukin-6 and MIP-2 progressively reached control values between 4 and 24 h after the end of halothane exposure. However, MCP-1 inhibition persisted until 24 h. rmIL-1beta-induced MIP-2 and tumor necrosis factor-alpha mRNA expression were decreased by 36 and 24%, respectively, after halothane exposure.

Conclusions

The current study shows that exposure of rmIL-1beta-stimulated AT II cells to volatile anesthetics reversibly alters their cytokine secretion. Therefore, volatile anesthesia, by modulating pulmonary epithelial cell secretion of inflammatory cytokines, might affect the lung inflammatory response.
Systemic Inflammation Leads to Resistance to Atracurium without Increasing Membrane Expression of Acetylcholine Receptors
Authors: Fink Heidrun , Luppa Peter , Mayer Barbara , Rosenbrock Hilkea , Metzger Jochen , Martyn J. A. Jeevendra , Blobner Manfred
Published: February 2004

Background

Systemic inflammation may be associated with resistance to nondepolarizing neuromuscular blocking drugs, the mechanisms of which are, however, uncharacterized. The authors therefore investigated the pharmacodynamics of atracurium and its relation to the expression of nicotinic acetylcholine receptors and alpha1 -acid glycoprotein in a rat model of systemic inflammation.

Methods

To induce a systemic inflammation, male CD rats received 56 mg/kg corynebacterium parvum intravenously. On days 2, 4, 6, 8, 10, 12, 14, or 16 after infection, neuromuscular transmission was measured. The individual effective dose of atracurium was determined, followed by an atracurium infusion at a rate to establish a steady state neuromuscular block of 50%. Total and unbound plasma concentrations of atracurium for 50% paralysis were measured using high-performance liquid chromatography. Acetylcholine receptors were quantitated using 125I-alpha-bungarotoxin. alpha1 -Acid glycoprotein concentrations in the serum were measured using a competitive chemiluminescence immunoassay.

Results

The effective dose of atracurium was increased on days 4, 6, and 8. Total atracurium plasma concentrations at 50% neuromuscular paralysis were increased on days 4, 6, 8, and 10, with a peak at day 8 (8.0 +/- 1.3 micro g/ml) compared with control rats (4.23 +/- 0.82 micro g/ml). The alpha1 -acid glycoprotein concentrations were increased between days 2 and 10, with a peak on day 4 (6.52 +/- 1.45 mg/ml), and recovered to control values (0.61 +/- 0.33 mg/ml) on day 12. Unbound plasma concentrations of atracurium to achieve 50% depression, as well as the expression of acetylcholine receptors, did not differ between groups.

Conclusion

Resistance to atracurium during corynebacterium parvum-induced systemic inflammation is due to increased drug binding to alpha1 -acid glycoprotein and is unrelated to changes in acetylcholine receptor expression.