Artificial culture of Labyrinthuloides haliotidis (Protozoa: Labyrinthomorpha), a pathogenic parasite of abalone

1987 ◽  
Vol 65 (8) ◽  
pp. 2013-2020 ◽  
Author(s):  
Susan M. Bower
Keyword(s):  
Pinus Contorta ◽  
Sea Water ◽  
Calf Serum ◽  
Minimum Essential Medium ◽  
Artificial Culture ◽  
Zoospore Production ◽  
Pine Pollen ◽  
Pathogenic Parasite ◽  
Student’S T ◽  
Student’S T Test

Labyrinthuloides haliotidis was isolated from infected abalone (Haliotis kamtschatkana) and successfully cultured in minimum essential medium with 10% fetal calf serum at 10 °C for at least 1 year. On transfer to sea water, some subcultures produced numerous motile biflagellate zoospores while zoospore production of other subcultures was poor. On return to minimum essential medium, zoospores transformed into rapidly dividing vegetative forms. Labyrinthuloides haliotidis was not fastidious in its nutrient requirements and vegetative forms grew well in several different liquid media, on agar containing 10% bovine serum, and on pine pollen (Pinus contorta) in sea water. The mean diameter of the round vegetative forms often varied significantly (Student's t-test, P < 0.05) but the overall range in diameter (3.1 to 16.2 μm) observed in the various media was similar. Best growth occurred at 10 °C and in media made up with 30‰ sea water. No growth occurred at 28 °C or above, or in thioglycollate culture medium at 10 °C. Although L. haliotidis grew on pine pollen in sea water, zoosporoblasts and zoospores were not produced. The disappearance of precipitated proteins in agar medium around colonies of L. haliotidis and the destruction of host tissue around the parasite in infected abalone suggest that extracellular digestion occurs with this organism.

162 IMPROVED EMBRYO SURVIVAL AND QUALITY WITH EMCARE II

2006 ◽  
Vol 18 (2) ◽  
pp. 189
Author(s):  
A. Harvey ◽  
M. Lane ◽  
J. Thompson
Keyword(s):  
Calf Serum ◽  
Cell Number ◽  
Tunel Staining ◽  
Embryo Survival ◽  
Improved Outcomes ◽  
Student’S T ◽  
The Impact ◽  
Student’S T Test

Collection of embryos exposes them to a number of stresses, including light, air, and changes in temperature. Improvement of holding media to reduce the impact of handling stresses on the embryo during in vivo collection and transfer is therefore beneficial to ensure maintenance of viability following transfer. The aim of this study was to compare the effect of holding IVP-derived blastocysts at 25°C in Emcare I (ECMI, Emcare, Dallas, TX, USA) with those held in Emcare II (ECMII), a proprietry formulation designed to reduce in vitro-induced stress. In vitro-produced bovine embryos were generated using standard protocols. Blastocysts were randomly allocated to either ECMI or ECMII (ICPBio, Aukland, New Zealand) on Day 7 and were held at 25°C for a period of 24 h, after which they were cultured in Cook Bovine Blast (Cook Australia, Brisbane, Australia) supplemented with 10% fetal calf serum for 48 h. At 24 and 48 h, embryos were scored for hatching, and a cohort removed for TUNEL staining at each time point. Differences were analyzed by Student's t-test. At both 24- and 48-h culture, hatching rates tended to be higher for embryos held in ECMII than in ECMI (Table 1). The level of apoptosis at 48 h was reduced in blastocysts held in ECMII (P = 0.06). Moreover, the total cell number of hatched blastocysts at 48 h was significantly increased (1.5-fold) in those held in ECMII (P = 0.01). Results suggest that the formulation of ECMII improves the ability of IVP bovine blastocysts to re-expand and hatch following an imposed stress (25°C for 24 h). Furthermore, ECMII improves overall embryo quality through a reduction in the percentage of cells undergoing apoptosis as well as through increased cell numbers, evident 48 h following cessation of the stress. We suggest that Emcare II reduces the impact of (or increases the embryo's tolerance to and recovery from) an imposed stress, which, although severe in the present study, may provide improved outcomes following embryo transfer in field situations. Table 1. Hatching and apoptosis of blastocysts held at 25°C for 24 h in Emcare I or Emcare II This work was supported with funding by ICPBio (NZ).

74 EFFECT OF SIZE AND CULTURE PERIOD OF FROZEN–THAWED BOVINE TROPHOBLASTIC VESICLES ON INTERFERON-τ SECRETION

2014 ◽  
Vol 26 (1) ◽  
pp. 151
Author(s):  
Y. Hashiyada ◽  
H. Takahashi ◽  
D. Yamaguchi ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Significant Positive Correlation ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Calf Serum ◽  
Culture Period ◽  
Fetal Bovine ◽  
Student’S T ◽  
Student’S T Test

Frozen–thawed bovine trophoblastic vesicles (bTV) derived in vivo could secrete interferon-τ (IFN-τ) at the same level as fresh bTV on Days 4 to 6 after thawing. However, amounts of IFN-τ decreased following continuous in vitro culture (Hashiyada et al. 2012 38th IETS). Co-transfer of frozen–thawed bTV improved pregnancy rate of embryos due to the effects of IFN-τ secreted by bTV (Hashiyada et al. 2008 41th SSR). However, the relation between bTV size and IFN-τ secretion level during culture has not been well documented. The objective of present study was to characterise the concentration of IFN-τ related bTV volume and culture period after thawing of cryopreserved bTV. The bTV were prepared from Day 16 elongating blastocysts recovered nonsurgically. The dissected trophoblastic fragment, 1 to 1.5 mm in width, was cultured using TCM-199 supplemented with 20% (vol/vol) fetal bovine serum and 0.1 mM β-mercaptoethanol. Formed vesicles after 24 h of culture were cryopreserved using D-PBS supplemented with 20% calf serum and 1.8 M ethylene glycol. After thawing, bTV were cultured individually with 100 μL/well/day until Day 2 (i.e. the day of thawing was defined as Day 0), and thereafter changed to 200 μL/well/day to termination at Day 10. Collection of culture media and measurement of bTV diameter were performed before cryopreservation and after thawing for every day. Interferon-τ in collected media was measured by radioimmunoassay. The estimated bTV volume was calculated based on the diameter. Data were analysed by Student's t-test. Nine fresh bTV before cryopreservation were used to assess the IFN-τ secretion for 24 h in relation to bTV volume. A significant positive correlation was observed between secreted IFN-τ (mean ± s.e.M, 19.9 ± 3.1 ng mL–1) and bTV volume (1.49 ± 0.6 mm3, r = 0.91; P < 0.01). Initial IFN-τ secretion from bTV cultured for 24 h after thawing was significantly decreased compared with that before cryopreservation (29.1 ± 2.1 ng mL–1 and 58.4 ± 4.8 ng mL–1; P < 0.01, n = 27). In continuous culture of bTV (n = 8), IFN-τ secretion increased gradually from Day 2 (23.1 ± 9.0 ng mL–1) to Day 4 (32.2 ± 8.4 ng mL–1), and then maintained this level until Day 7 (33.4 ± 14.9 ng mL–1). However, this amount of IFN-τ tended to decrease on Day 8 (24.8 ± 5.0 ng mL–1), 9 (16.5 ± 4.4 ng mL–1), and 10 (12.0 ± 1.7 ng mL–1). Interferon-τ secretion from bTV on Day 9 and 10 was lower than that on Day 3, 4, 5, 6, and 8, respectively (P < 0.05). Volume of bTV increased also from Day 2 (0.2 ± 0.1 mm3) to Day 5, 6 (0.8 ± 0.3 mm3) and 7 (0.7 ± 0.2 mm3). However, bTV volumes shrank drastically on Day 8 (0.3 ± 0.1 mm3), 9, and 10 (0.2 ± 0.1 mm3). In comparison with bTV during culture, volumes on Day 4, 5, and 7 were greater than those on Day 2 and 3, and volumes on Day 6 and 7 were greater than on Day 8, 9, and 10 (P < 0.05). These results indicate that the dynamics of IFN-τ secretion reflected the expansion or reduction of bTV in continued culture after thawing. Interferon-τ secretion might be related to bTV volume. Moreover, we reconfirmed that cryopreserved bTV highly express IFN-τ during 4 to 7 days after thawing.

78 INTERFERON-τ SECRETION OF CRYOPRESERVED BOVINE TROPHOBLASTIC VESICLES DERIVED FROM IN VIVO

2012 ◽  
Vol 24 (1) ◽  
pp. 151
Author(s):  
Y. Hashiyada ◽  
H. Takahashi ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Continuous Culture ◽  
Culture Media ◽  
Calf Serum ◽  
Measure Concentration ◽  
Fetal Bovine ◽  
The Mean ◽  
Student’S T ◽  
Student’S T Test

The co-transfer of bovine trophoblastic vesicles (bTVs) prepared from in vivo recovered conceptuses is known to promote the successful implantation of embryos, which expected lower viability, through the effects of interferon-τ (IFN-τ) secreted by bTVs. We have reported that the pregnancy rate was improved for co-transferred embryos with frozen-thawed bTVs using the direct-transfer technique (Hashiyada et al. 2008, 41st SSR). However, the IFN-τ secretion level from cryopreserved bTVs is not well known. The objective of the present study was to measure concentration of IFN-τ released from frozen-thawed bTVs individually cultured in vitro. bTVs were prepared from elongating blastocysts 3 to 20 mm in length, following superstimulatory treatment and recovered on Day 16 post-AI, by dissection using a surgical blade. Each trophoblastic fragment, 1 to 1.5 mm in width, was cultured in a well of a 96-well plate using TCM-199 supplemented with 20% (v/v) fetal bovine serum and 0.1 mM β-mercaptoethanol. Formed vesicles after 24 or 48 h of culture were cryopreserved using D-PBS supplemented with 20% calf serum, 1.5 M ethylene glycol (EG) and 0.1 M sucrose or 1.8 M EG. After thawing, each bTVs was cultured for 2 days to compare IFN-τ secretion between the 2 cryoprotectants. Furthermore, transition of IFN-τ level was assessed in continuous culture until Day 10 (the day of thawing was defined as Day 0). The volume of culture medium was 100 μL well–1 day–1 until Day 2 and thereafter changed to 200 μL well–1 day–1 until termination. Exchange and collection of culture media were performed on Day 1, 2, 4, 6, 8 and 10. Collected culture media were stored at –30°C until use. IFN-τ was measured by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). Data were analysed by Student's t-test. Initial IFN-τ secretion from bTVs before cryopreservation did not differ between 24 and 48 h of culture period to form vesicles, 44.0 ± 2.9 (mean ± standard error of the mean, n = 64) and 52.8 ± 6.4 ng mL–1 (n = 27), respectively. IFN-τ secretion was no difference between the 1.5 M EG group and the 1.8 M EG group on Day 1 (41.2 ± 4.9 ng mL–1, n = 42 and 30.4 ± 2.2 ng mL–1, n = 31) and on Day 2 (38.0 ± 5.4 and 38.2 ± 4.5 ng mL–1), respectively. In the continuous culture group (n = 28), IFN-τ secretion tended to increase from Day 2 (25.2 ± 3.4 ng mL–1) to Day 4 (51.8 ± 12.3 ng mL–1) and 6 (55.4 ± 13.3 ng mL–1) (P < 0.05). However, this amount of IFN-τ on Day 6 significantly decreased on Day 8 (25.6 ± 2.7 ng mL–1; P < 0.05) and Day 10 (15.5 ± 2.2 ng mL–1; P < 0.01), gradually. These results indicate that cryopreserved bTVs could secrete IFN-τ at the same level as fresh bTVs on Day 4 to 6 after thawing and then these amounts of IFN-τ significantly decrease in vitro.

scholarly journals Angiotensin II modulates the activity of the Na+/K+ATPase in eel kidney

2000 ◽  
Vol 165 (1) ◽  
pp. 147-156 ◽  
Author(s):  
S Marsigliante ◽  
A Muscella ◽  
S Barker ◽  
C Storelli
Keyword(s):  
Angiotensin Ii ◽  
Atpase Activity ◽  
Calcium Release ◽  
Sea Water ◽  
T Test ◽  
Sodium Balance ◽  
Ang Ii ◽  
Tubule Cells ◽  
Student’S T ◽  
Student’S T Test

We have previously shown that angiotensin II (Ang II) has a role at the level of the eel gill chloride cell regulating sodium balance, and therefore osmoregulation; the purpose of the present study was to extend these findings to another important osmoregulatory organ, the kidney. By catalytic histochemistry Na(+)/K(+)ATPase activity was found in both sea water (SW)- and freshwater (FW)-adapted eel kidney, particularly at the level of both proximal and distal tubules. Quantitation of tubular cell Na(+)/K(+)ATPase activity, by imaging, gave values in SW-adapted eels which were double those found in FW-adapted eels (Student's t-test: P<0.0001). This was due to a reduced number of positive tubules present in FW-adapted eels compared with SW-adapted eels. By conventional enzymatic assay, the Na(+)/K(+)ATPase activity in isolated tubular cells from SW-adapted eels showed values 1.85-fold higher those found in FW-adapted eels (Student's ttest: P<0.0001). Perfusion of kidney for 20 min with 100 nM Ang II provoked a significant increase (1.8-fold) in Na(+)/K(+)ATPase activity in FW, due to up-regulation of Na(+)/K(+)ATPase activity in a significantly larger number of tubules (Student's t-test: P<0.0001). The effect of 100 nM Ang II in SW-adapted kidneys was not significant. Stimulation with increasing Ang II concentrations was performed on isolated kidney tubule cells: Ang II provoked a dose-dependent stimulation of the Na(+)/K(+)ATPase activity in FW-adapted eels, reaching a maximum at 100 nM (1.82-fold stimulation), but no significant effect was found in SW-adapted eels (ANOVA: P<0.001 and P>0.05 respectively). Isolated tubule cells stimulated with 100 nM Ang II showed a significant generation of inositol trisphosphate (InsP(3)) and an increment in calcium release from intracellular stores. In conclusion, our results suggest that tubular Na(+)/K(+)ATPase is modulated by environmental salinity, and that Ang II has a role in regulating its activity in FW-adapted eels, probably through an InsP(3)-dependent mechanism.

Exogeneous factors are not necessary in attachment, spreading and polarization of hela cells

1978 ◽  
Vol 36 (2) ◽  
pp. 310-311
Author(s):  
W. Liebrich
Keyword(s):  
Hela Cells ◽  
Calf Serum ◽  
Glass Tube ◽  
Serum Free Medium ◽  
Nacl Solution ◽  
Free Medium ◽  
Minimum Essential Medium ◽  
Serum Free ◽  
All Solutions ◽  
Glass Support

HeLa cells were grown for 2-3 days in EAGLE'S minimum essential medium with 10% calf serum (S-MEM; Seromed, München) and then incubated for 24 hours in serum free medium (MEM). After detaching the cells with a solution of 0. 14 % EDTA and 0. 07 % trypsin (Difco, 1 : 250) they were suspended in various solutions (S-MEM = control, MEM, buffered salt solutions with or without Me++ions, 0. 9 % NaCl solution) and allowed to settle on glass tube slips (Leighton-tubes). After 5, 10, 15, 20, 25, 30, 1 45, 60 minutes 2, 3, 4, 5 hours cells were prepared for scanning electron microscopy as described by Paweletz and Schroeter. The preparations were examined in a Jeol SEM (JSM-U3) at 25 KV without tilting.The suspended spherical HeLa cells are able to adhere to the glass support in all solutions. The rate of attachment, however, is faster in solutions without serum than in the control. The latter is in agreement with the findings of other authors.

Serosal and cutaneous recordings of gastric myoelectrical activity in patients with gastroparesis

1994 ◽  
Vol 266 (1) ◽  
pp. G90-G98 ◽  
Author(s):  
J. D. Chen ◽  
B. D. Schirmer ◽  
R. W. McCallum
Keyword(s):  
Electrical Stimulation ◽  
Slow Wave ◽  
Test Meal ◽  
Frequency Change ◽  
Fasting State ◽  
Gastric Myoelectrical Activity ◽  
Myoelectrical Activity ◽  
Gastric Slow Wave ◽  
Student’S T ◽  
Student’S T Test

The aims of this study were to 1) investigate gastric myoelectrical activity in patients with gastroparesis, 2) validate the cutaneous electrogastrogram (EGG) in tracking the frequency change of the gastric slow wave, and 3) investigate the effect of electrical stimulation on gastric myoelectrical activity. Gastric myoelectrical activity was recorded in 12 patients with documented gastroparesis using serosal electrodes for > 200 min in each subject. All recordings were made at least 4 days after surgery. Each session consisted of a 30-min recording in the fasting state and a 30-min recording after a test meal. The test meal (liquid or mixed) was selected according to patient's tolerance. Electrical stimulation was performed in three subjects via the serosal electrodes at a frequency of 3 cycles/min. Gastric myoelectrical activity was recorded using serosal electrodes in each session. The serosal recording showed slow waves of 2.5 to 4.0 cycles/min in all 12 subjects. Absence of spikes was noted in 11 of the 12 subjects. The simultaneous serosal and cutaneous recording of gastric myoelectrical activity showed that the frequency of the EGG was exactly the same as that of the serosal recording. Liquid meals resulted in a significant decrease in slow-wave frequency (Student's t test, P = 0.006), and the EGG accurately reflected this change. Electrical stimulation had no effect on the frequency of the gastric slow wave and did not induce spikes.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effect of Changes in Dialysate Volume on Glucose and Urea Equilibration

1994 ◽  
Vol 14 (3) ◽  
pp. 236-239 ◽  
Author(s):  
Edward C. Kohaut ◽  
F. Bryson Waldo ◽  
Mark R. Benfield
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Prospective Study ◽  
Body Surface ◽  
Patient Population ◽  
Different Ages ◽  
Student’S T ◽  
A Prospective Study ◽  
Dialysate Volume ◽  
Student’S T Test

Objectives To determine the effect of changing dialysate volume on urea and glucoseequilibration curves and to determine, if dialysate volume is prescribed on the basis of body surface area, whether equilibration curves will be consistent in patients of different sizes and ages. Design A prospective study wherein children with acute or chronic renal failure had peritoneal equilibration studies done with dwell volumes of 30 mL/kg, 40 mL/kg, and 1200 mL/m2. Patient Population Twenty-two children: 7 under 3 years of age; 8 between 3 and 10 years of age; 7 older than 10 years of age. Statistics Student's t-test. Results Urea and glucose equilibrated rapidly at dwell volumes of 30 mL/kg, slower at dwell volumes of 40 mL/kg, and slowest at dwell volumes of 1200 mL/m2. Equilibration curves were similar in children of different ages when dialysate volumes of 1200 mL/m2 were infused. Conclusion Dialysate volumes of 1200 mL/m2 should be used when equilibration studies are being done to compare individuals of different ages and sizes.

scholarly journals Development and Validation of 2-Azaspiro [4,5] Decan-3-One (Impurity A) in Gabapentin Determination Method Using qNMR Spectroscopy

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1656
Author(s):  
Nataliya E. Kuz’mina ◽  
Sergey V. Moiseev ◽  
Mikhail D. Khorolskiy ◽  
Anna I. Lutceva
Keyword(s):  
Test Procedure ◽  
Active Pharmaceutical Ingredient ◽  
Test Methods ◽  
Intermediate Precision ◽  
Coefficients Of Variation ◽  
Validation Parameters ◽  
Limit Of Quantitation ◽  
Student’S T ◽  
Development And Validation ◽  
Student’S T Test

The authors developed a 1H qNMR test procedure for identification and quantification of impurity A present in gabapentin active pharmaceutical ingredient (API) and gabapentin products. The validation studies helped to determine the limit of quantitation and assess linearity, accuracy, repeatability, intermediate precision, specificity, and robustness of the procedure. Spike-and-recovery assays were used to calculate standard deviations, coefficients of variation, confidence intervals, bias, Fisher’s F test, and Student’s t-test for assay results. The obtained statistical values satisfy the acceptance criteria for the validation parameters. The authors compared the results of impurity A quantification in gabapentin APIs and capsules by using the 1H qNMR and HPLC test methods.

scholarly journals Potential intravenous drug incompatibilities in a pediatric unit

2016 ◽  
Vol 14 (2) ◽  
pp. 185-189 ◽  
Author(s):  
Karla Dalliane Batista Leal ◽  
Ramon Weyler Duarte Leopoldino ◽  
Rand Randall Martins ◽  
Lourena Mafra Veríssimo
Keyword(s):  
Risk Factors ◽  
University Hospital ◽  
Intravenous Drug ◽  
Penicillin G ◽  
Cross Sectional ◽  
Relative Risks ◽  
Related Risk ◽  
Student’S T ◽  
Level Of Significance ◽  
Student’S T Test

ABSTRACT Objective To investigate potential intravenous drug incompatibilities and related risk factors in a pediatric unit. Methods A cross-sectional analytical study conducted in the pediatric unit of a university hospital in Brazil. Data on prescriptions given to children aged 0-15 years from June to October 2014 were collected. Prescriptions that did not include intravenous drugs and prescriptions with incomplete dosage regimen or written in poor handwriting were excluded. Associations between variables and the risk of potential incompatibility were investigated using the Student’s t test and ANOVA; the level of significance was set at 5% (p<0.05). Relative risks were calculated for each drug involved in potential incompatibility with 95% confidence interval. Results A total of 222 children participated in the study; 132 (59.5%) children were male and 118 (53.2%) were aged between 0 and 2 years. The mean length of stay was 7.7±2.3 days. Dipyrone, penicillin G and ceftriaxona were the most commonly prescribed drugs. At least one potential incompatibility was detected in about 85% of children (1.2 incompatibility/patient ratio). Most incompatibilities detected fell into the non-tested (93.4%), precipitation (5.5%), turbidity (0.7%) or chemical decomposition (0.4%) categories. The number of drugs and prescription of diazepam, phenytoin, phenobarbital or metronidazole were risk factors for potential incompatibility. Conclusion Most pediatric prescriptions involved potential incompatibilities, with higher prevalence of non-tested incompatibilities. The number of drugs and prescription of diazepam, phenobarbital, phenytoin or metronidazole were risk factors for potential incompatibilities.

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