A new paradigm in respiratory hygiene: increasing the cohesivity of airway secretions to improve cough interaction and reduce aerosol dispersion

Background

Infectious respiratory diseases are transmitted to non-infected subjects when an infected person expels pathogenic microorganisms to the surrounding environment when coughing or sneezing. When the airway mucus layer interacts with high-speed airflow, droplets are expelled as aerosol; their concentration and size distribution may each play an important role in disease transmission. Our goal is to reduce the aerosolizability of respiratory secretions while interfering only minimally with normal mucus clearance using agents capable of increasing crosslinking in the mucin glycoprotein network.

Methods

We exposed mucus simulants (MS) to airflow in a simulated cough machine (SCM). The MS ranged from non-viscous, non-elastic substances (water) to MS of varying degrees of viscosity and elasticity. Mucociliary clearance of the MS was assessed on the frog palate, elasticity in the Filancemeter and the aerosol pattern in a "bulls-eye" target. The sample loaded was weighed before and after each cough maneuver. We tested two mucomodulators: sodium tetraborate (XL"B") and calcium chloride (XL "C").

Results

Mucociliary transport was close to normal speed in viscoelastic samples compared to non-elastic, non-viscous or viscous-only samples. Spinnability ranged from 2.5 ± 0.6 to 50.9 ± 6.9 cm, and the amount of MS expelled from the SCM increased from 47 % to 96 % adding 1.5 μL to 150 μL of XL "B". Concurrently, particles were inversely reduced to almost disappear from the aerosolization pattern.

Conclusion

The aerosolizability of MS was modified by increasing its cohesivity, thereby reducing the number of particles expelled from the SCM while interfering minimally with its clearance on the frog palate. An unexpected finding is that MS crosslinking increased "expectoration".     

Evaluation of reactive oxygen metabolites in frozen serum samples. Effect of storage and repeated thawing

Measuring the free radical activity in serum samples from prospective studies is the best way to investigate the association between oxidative stress and human diseases. Prospective studies require the analysis of serum samples that have often been stored for a long time. Our study was designed to determine the effect of storage at -30 degrees C and -80 degrees C for two years on free radical activity. We analyzed the free radical activity by measuring circulating hydroperoxides in a pool of sera at baseline and after one day, one week, one month and 25 months of storage, using a photometric method (d-ROMs test). Measurements were performed in aliquots thawed only once at each time point and in aliquots frozen and thawed repeatedly over the study period. After two years we observed a small but statistically significant 4% decrease in the hydroperoxide concentration, which was substantially unaffected by storage temperatures and repeated freeze-thaw cycles. We also carried out the d-ROMs test in sera from ten apparently healthy volunteers at 2, 8, 24, and 48 hours after collection and storage at 4 degrees C and did not observe any significant variation. In conclusion, the d-ROMs test is a simple method suitable to evaluate the free radical activity in frozen serum samples after long-term storage.     

Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores

The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30 min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin-nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of -30, -80 or -80 degrees C and immediately stored in liquid nitrogen (cooling rates of -2, -4, -6, -8, -10, -12, -14 or -16 degrees C/min). Frozen spermatophores were thawed (2 min) at 30, 60, 70, or 90 degrees C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of -2 degrees C/min between 25 and -80 degrees C before storing in liquid nitrogen. Optimal thawing was in a 30 degrees C water bath for 2 min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8+/-2.9%) and cryopreserved spermatophores held for less than 60 days (87.3+/-4.1%) did not differ (P>0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P<0.05) and varied from 27.3+/-3.4 to 53.3+/-4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6-72.2% and 63.6-64.1%, respectively) at rates comparable to fresh spermatophores (70.8-78.2% and 66.3-67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days.     

Mucociliary clearance in chronic sinusitis: related human nasal clearance and in vitro bullfrog palate clearance

Nasal mucociliary clearance was measured in both healthy subjects and patients with chronic sinusitis using saccharin granule technique. Nasal mucociliary transit time (ST) was significantly slower in the patients with chronic sinusitis compared with that in controls (p less than 0.005). Nasal mucus collected from each nasal cavity was used for in vitro bullfrog palate clearance studies and compared to the in vivo nasal ST. Mucociliary clearance rate (MTR) on frog palate was 12.5 +/- 2.5 mm/min in the mucus from control subjects, 6.1 +/- 1.5 mm/min in the mucus from the patients. The difference was statistically significant (p less than 0.005). The MTR on frog palate in the patients whose nasal ST was within normal range was significantly slower than that in controls (p less than 0.005), but not significantly different from that in the patients whose nasal ST was over the normal range. These results suggest that the nasal mucous properties which decreased the mucociliary clearance on frog palate did not contribute to the mucociliary clearance of the patients who had a normal one. No significant correlation existed between MTR on frog palate and nasal ST in both control and chronic sinusitis. In chronic sinusitis patients, decelerated nasal ST was recovered significantly by normal saline nebulization compared with the value before the nebulization (p less than 0.01). None of the significant change of ST was observed in control before and after the nebulization.     

Changes of viability and composition of the Escherichia coli flora in faecal samples during long time storage

Long-time storage of faecal samples is necessary for investigations of intestinal microfloras. The aim of the present study was to evaluate how the viability and the composition of the Escherichia coli flora are affected in faecal samples during different storage conditions. Four fresh faecal samples (two from calves and two from infants) were divided into sub-samples and stored in four different ways: with and without addition of glycerol broth at -20 degrees C and at -70 degrees C. The viability and the phenotypic diversity of the E. coli flora in the sub-samples were evaluated after repeated thawings and after storage during 1 year. The samples stored for 1 year without thawing were also kept at room temperature for 5 days and subsequently analysed. According to phenotyping (PhP analysis) of 32 isolates per sample on day 0, all four samples contained two dominating strains of E. coli each, and between one and eight less common strains. Samples that were stored at -70 degrees C in glycerol broth showed equal or even higher bacterial numbers as the original samples, even after repeated thawings, whereas samples stored at -20 degrees C showed a considerably lower survival rate, also with addition of glycerol. Sub-samples containing glycerol broth that were kept at room temperature after storage for 1 year showed a clear increase in the number of viable cells as well as in diversity. The diversities in each sub-sample showed a tendency to decrease after several thawings as well as after storage. Generally, the E. coli populations in samples stored at -20 degrees C were less similar to the population of the original sample than that in samples stored at -70 degrees C. Samples that had been mixed with glycerol broth had an E. coli flora more similar to that in the original sample than those without glycerol broth. Furthermore, the sub-samples that were kept at room temperature after storage for 1 year generally were more similar to the original samples than if they were processed directly. We conclude that for long time storage of faecal samples, storage at -70 degrees C is preferable. If samples have to be thawed repeatedly, addition of glycerol is preferable both for samples stored at -70 degrees C and for samples stored at -20 degrees C. Our data also have indicated that when E. coli isolates from faecal samples are selected for, e.g. analysis of virulence factors, it is necessary to pick several isolates per sample in order to obtain at least one isolate representing the dominating strain(s).     

Tracheal mucus rheology and epithelial potential difference in two day old puppies

The transepithelial potential difference (PD) value represents an integral of ion fluxes across the epithelium, and relates to the regulation of airway fluid. We studied six healthy two day old husky puppies for their tracheal mucus rheology and bioelectric properties, since this data in newborns are still unknown. PD (-mV, epithelium vs. subcutaneous space) was measured using the agar bridge technique in two locations - lower trachea and subglottic region. For the rheological analysis, the magnetic microrheometer was employed; data are presented as mechanical impedance log G* and loss tangent tan delta (1 rad/s). The mucus collection rate (mg/min) and the solid content (%) were determined by gravimetry. Mucociliary clearability, normalized to frog mucus, (MCFP) was determined directly by the frog palate method; a cough clearability index (CCI) was computed from simulated cough machine data obtained with mucus-like gels. The mucus collection rates and PD values were considerably lower than those observed in adult dogs; the mechanical impedance values were also reduced in comparison with adult data. The PD profile within the trachea (-13.9 +/- 1.2 mV lower trachea vs. -18.4 +/- 1.4 mV subglottical, i.e. more negative subglottically), however, is similar to that observed in adult dogs. Intratracheal profiles in mucus collection rate and mucus rheology were also comparable between puppies and adult dogs. The low collection rates in puppies, particularly in lower trachea, could indicate either reduced mucus volume or slower clearance. PD and collection rate correlated very strongly (r = 0.82, p = 0.0003). PD also correlated negatively with log G* (r = 0.73, p = 0.003) and positively with tan delta (r = 0.58, p = 0.03). MCFP and % solids correlated positively (r = 0.84, p = 0.0012), in contradistinction to the usual relationship, perhaps due to the presence of non-glycoprotein components that do not contribute to crosslink formation. The apparent maturation of airway bioelectric properties, mucus collection rate and mucus viscoelasticity are all consistent with the maturation of mucociliary clearance, which has previously been reported.     

Preservation of rainbow trout (Oncorhynchus mykiss) eyed eggs using a perfluorochemical as an oxygen carrier

The objective of this study was to maintain the viability of chilled rainbow trout (Oncorhynchus mykiss) eyed eggs during storage using oxygenated perfluorochemical (PFC). Three trials were conducted using eggs at 161, 180 or 217 degree days (days from fertilization x incubation temperature in degrees C). A separate trial was conducted for 147 degree day eggs that were not at the eyed stage. For each trial, eggs were stored in a moisture-saturated atmosphere at 1 degrees C in PFC, water, and 1:1 combinations of PFC and PBS, PFC and 0.3 M glucose, PFC and mineral oil, or PFC and water. The PFC was oxygenated before each trial and all media were oxygenated at weekly intervals during the storage period. Eggs from each trial were also incubated without storage to provide Day 0 results. After 3 and 5 weeks of storage, eggs from each medium were incubated at 10 degrees C until hatch. Hatching percentage was expressed as a percentage of Day 0 results. The percentage of normal alevins that hatched was also determined. There were interactions (P < 0.01) between stage of development and treatment for hatching percentage after 3 and 5 weeks of storage. After 3 weeks of storage, eggs stored at 161, 180, or 217 degree days without PFC had hatching rates of 0-14.3% but eggs stored in any medium with PFC had hatching percentages from 75.1 to 106.4% of Day 0 values. After 5 weeks of storage, eggs stored at 161 degree days in PFC plus PBS or PFC plus water, and eggs stored at 217 degree days in PFC or PFC plus water, had higher (P < 0.05) hatching percentages than eggs stored in any of the other media. Eggs stored at 161 degree days for 5 weeks in PFC and water had a higher (P < 0.05) percentage of normal alevins hatching than eggs stored in PFC and PBS. Because of their early developmental stage, eggs stored at 147 degree days had low hatching percentages, except eggs stored for 3 weeks in PFC or PFC plus PBS. Chilling eyed eggs of rainbow trout to 1 degrees C and storing them in water with PFC as an oxygen carrier can preserve their viability for 5 weeks.     

Correlation between rheologic properties and in vitro ciliary transport of rat nasal mucus.

The relationship between mucus rheologic variables and in vitro ciliary transport was investigated in mucus samples collected from the upper airways of 30 Wistar rats. In vitro mucus transportability was determined by means of the frog palate preparation. Rheologic evaluation was done by measuring the rigidity modulus (log G*, representing the vectorial sum of viscosity and elasticity) and the loss tangent (tan delta, i.e. the ratio between viscosity and elasticity) at 1 and 100 radian/s using a magnetic microrheometer. The correlation between the rheologic variables and in vitro mucus transportability was made by stepwise multiple linear regression analysis, with frog palate transport rate considered as the dependent variable. A significant relationship was obtained between the rheologic parameters (log G* and tan delta) measured at 1 radian/s and the frog palate transport ratio. The relative speed of mucus samples was related to rheology according to the following relationship: rat/frog speed ratio = 1.666-0.434 log G*-0.331 tan delta, for G* and delta determined at 1 radian/s (multiple r = 0.666, p < 0.001). Transport rates predicted from the above formula gave a satisfactory fit to those observed in a second set of 30 rats. The present results indicate that the overall mucus impedance, as well as the ratio between viscosity and elasticity, are important in determining the efficiency of clearance. In addition, it was shown that measurements performed by applying relatively low frequency deformations are preferable for predicting ciliary transport.     

Mucus-depleted frog palate as a model for the study of mucociliary clearance

To better understand the frog palate model of mucociliary transport, we measured the transport rate of mucus (MTR) from the leopard frog, Rana pipiens, and from the bullfrog, R. catesbeiana, recorded the stability of the MTR over a period of hours and days and over the course of 1 yr, and measured the viscoelasticity, percent solid composition, and spinnability (filance) of mucus from both species. Bullfrog mucus was less rigid than leopard frog mucus (log G* at 1 rad/s 2.09 vs. 2.61; P less than 0.01) and had a higher viscosity-to-elasticity ratio (tan delta at 1 rad/s 0.36 vs. 0.26; P less than 0.05). It also had a lower solids content (8.71 vs. 13.72%; P = 0.02), and there was a trend to lower spinnability for bullfrog mucus (filance 26.7 vs. 33.5 mm). These data suggest that bullfrog mucus has viscoelastic properties similar to normal mammalian respiratory mucus and leopard frog mucus has viscoelasticity similar to sputum samples. MTR was significantly slower in the winter than in the summer months (17 vs. 30 mm/min; P less than 0.0001). Although the leopard frog palate could be used for at least 7 consecutive days without exhaustion, bullfrog palates could be used for only 5 days. Palates of either species could generally be tested for 6 h/day without a significant decrease in MTR. These data clarify some of the sources of variability in the use of this system and suggest methods of standardization.     

Effect of rapid addition and dilution of dimethyl sulfoxide and 37 degrees C equilibration on viability of rabbit morulae thawed rapidly

The aim of the present study was to examine the effects of various conditions of addition and dilution of dimethyl sulfoxide (Me2SO) and 37 degrees C equilibration, and also the effects of freezing in the solution which was prepared in advance and stored in plastic straws at -20 degrees C on the viability of rabbit morulae thawed rapidly. The embryos were cooled from room temperature to -30 degrees C at 1 degree C/min in the presence of 1.5 M Me2SO using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, then cooled rapidly, and stored in liquid nitrogen. The frozen straws were thawed rapidly (greater than 1000 degrees C/min). When Me2SO was added in a single step, equilibrated with embryos at 37 degrees C for 15 min and diluted out in a single step, a very high survival was obtained: transferable/recovered, 90%: developed/recovered, 96%. When embryos were pipetted into 1.5 M Me2SO that was prepared in advance, stocked in straws at -20 degrees C, and cooled, the proportions of transferable and developed embryos were equivalent to those of embryos frozen in the solution that was prepared immediately before use.     

Effect of storage temperature and duration on viability of eggs of Helicoverpa armigera (Lepidoptera: Noctuidae)

The ability to store different insect stadia for prolonged periods provides considerable flexibility and ability to conduct experiments properly. Therefore, studies were undertaken to determine the effect of storage temperature and duration on viability of eggs of Helicoverpa armigera (Hübner). The percentage egg hatch and incubation period were significantly (P=0.01) influenced by egg age, storage temperature, and storage duration. Egg hatch ranged from 0.0 to 96.8% across temperatures and storage durations. None of the eggs hatched when stored at -20 and 0 degrees C. The regression model with the optimum Mallow Cp statistic for any of the identified linear and quadratic terms did not improve the precision of prediction in egg hatch beyond 67.0%. Forecasting of incubation period based on egg age, storage duration, and durationxtemperature was quite effective (R2=84.2%). Day degrees required for egg hatching decreased with an increase in temperature from 10 to 27 degrees C, and egg age from 0 to 3 days. The day degree requirements were highest for 0-day-old eggs at 10 degrees C, and lowest at 27 degrees C. Although the incubation period was higher, the hatchability was lower for 0- and 1-day-old eggs stored at constant 10 degrees C, these eggs can be stored for 10 days at 10 degrees C, with a hatchability of >75.0%. It was safer to store the H. armigera eggs for 10 days at 10 degrees C, which will hatch within 1.6 to 2.0 days after restoration at 27 degrees C with a hatchability of >75.0%. This information will be useful in planning and execution of experiments involving H. armigera on various aspects of research in entomology.     

Infection of llamas with stored Eimeria macusaniensis oocysts obtained from guanaco and alpaca feces

Oocysts obtained from a guanaco and an alpaca with natural infections were identified as Eimeria macusaniensis and evaluated for host specificity and infectivity over time. In 3 separate trials conducted over 4 yr, 4 adult llamas were fed 500-5,000 sporulated oocysts obtained from guanaco feces stored under laboratory conditions for 41-84 mo. Infections with prepatent periods of 36-41 days and patent periods of 38-55 days developed in 4/4 llamas. In a fourth trial, 3 adult llamas and 1 alpaca were each fed 1,000 sporulated E. macusaniensis oocysts obtained from alpaca feces stored in the laboratory for 3 mo. Infections with prepatent periods of 33-34 days and patent periods of 14-20 days developed in 3/3 llamas. Infection in the alpaca had a prepatent period of 58 days and a patent period of 1 day. Clinical signs associated with infection, if any, were minimal and included increased fecal mucus and occasional soft feces. These results provide evidence that E. macusaniensis is a single species transmissible amongst alpacas, llamas, and guanacos and that oocysts of this species can remain infective for many years.     

Effect of storage on the measurement of apolipoproteins A-I and A-II by radial immunodiffusion

We studied the effect of storage time and conditions on the measurement of apolipoprotein A-I and A-II by radial immunodiffusion. Purified A-I and A-II standards were stable for at least 6 months before any change in immunoreactivity was detected if stored at 4 degrees C at concentrations of 0.06-0.24 mg/ml for A-I and 0.016-0.064 mg/dl for A-II in 0.84 M tetramethylurea, 6.4 M urea, and 8 mM Tris-hydrocholoride, pH 8.0. Purified A-I (0.8-1.6 mg/ml) and A-II (0.5-1.0 mg/ml) were stable for 1 year if stored at -60 degrees C in 5 mM NH4HCO3 with or without 4.2 M tetramethylurea. Serum or plasma could be stored at 4 degrees C (under conditions where evaporation and bacterial growth were minimized) for at least 46 days or at -20 degrees C for up to 3 years without any change in A-I or A-II levels. For four serum samples stored at -20 degrees C for 2 to 3 years, the coefficient of variation of measurement ranged from 6.3 to 9.8% for A-I and from 6.7 to 10.6% for A-II. Samples stored at 4 degrees C had comparable apolipoprotein levels to those stored at -20 degrees C. However, apolipoprotein levels in serum samples were 3-5% higher than those obtained on plasma samples. We conclude that purified A-I or A-II and serum and plasma can be stored for long periods without any change in the measurement of the A-I or A-II by radial immunodiffusion.     

Development and ciliation of the palate in two frogs, Bombina and Xenopus; a comparative study

The morphological changes that occur during metamorphosis in the palates of two types of anuran larvae (a discoglossid, Bombina orientalis, and a pipid, Xenopus laevis) are compared. In B. orientalis the structural changes are accompanied by the ciliation of the palate epithelium. Ciliation begins in the anterior region of the palate and continues in a posterior direction throughout metamorphosis. By contrast, the palate of X. laevis never becomes ciliated during its development. Instead, two ciliated grooves develop between the choanae (nasal openings) and the esophageal opening. The grooves transport mucus and trapped objects out of the internal nares and toward the esophagus. These grooves are compared to similar structures on the palate of adult B. orientalis. The timing and pattern of ciliogenesis during metamorphosis in each of these anurans is also described relative to well-established staging series for external frog development. We show that the onset and location of ciliogenesis are consistent and predictable in these anurans and, therefore, make the frog palate an excellent system for the study of ciliogenesis.     

Effects of frozen storage on the structure of sarcocysts in pig muscle and implications in taxonomic studies

The morphology of the cyst wall of Sarcocystis has unique characteristics that can be used in species identification. To find a suitable way to preserve Sarcocystis cyst samples for species identification, by light microscopy and electron microscopy, we recorded the morphological changes in the cysts of Sarcocystis suihominis and Sarcocystis miescheriana from pig muscle, induced by storage at -20 degrees C. Comparisons were made between fresh cysts and those subjected to frozen storage for periods of 3 days, 20 days and 30 days. Results: cyst wall of the two Sarcocystis species appeared unaffected by storage. There was no obvious change in the length, nor in the width of the protrusions after storage (P>0.05), but the structure of the bradyzoite in the sarcocyst was in many cases disintegrated at -20 degrees C in 20 days for S. miescheriana and 30 days for S. suihominis. To our knowledge this is the first report that Sarcocystis cyst in muscle can be stored at -20 degrees C before and remain suitable for ultrastructural morphological study. Consequently, this paper proposes freezing as a convenient storage method for samples used in taxonomic studies of Sarcocystis.     

Impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 populations to undercooking and simulated exposure to gastric fluid

This study evaluated the impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 exposed to consumer-induced stresses simulating undercooking and digestion. Lean beef tissue samples were inoculated with E. coli O157:H7 cultures prepared in tryptic soy broth or meat decontamination runoff fluids (WASH) or detached from moist biofilms or dried biofilms formed on stainless steel coupons immersed in inoculated WASH. After inoculation, the samples were left untreated or dipped for 30 s each in hot (75 degrees C) water followed by lactic acid (2%, 55 degrees C), vacuum packaged, stored at 4 (28 days) or 12 degrees C (16 days), and periodically transferred to aerobic storage (7 degrees C for 5 days). During storage, samples were exposed to sequential heat (55 degrees C; 20 min) and simulated gastric fluid (adjusted to pH 1.0 with HCl; 90 min) stresses simulating consumption of undercooked beef. Under the conditions of this study, cells originating from inocula of planktonic cells were, in general, more resistant to heat and acid than cells from cultures grown as biofilms and detached prior to meat inoculation. Heat and acid tolerance of cells on meat stored at 4 degrees C was lower than that of cells on nondecontaminated meat stored at 12 degrees C, where growth occurred during storage. Decontamination of fresh beef resulted in injury that inhibited subsequent growth of surviving cells at 12 degrees C, as well as in decreases in resistance to subsequent heat and acid stresses. The shift of pathogen cells on beef stored under vacuum at 4 degrees C to aerobic storage did not affect cell populations or subsequent survival after sequential exposure to heat and simulated gastric fluid. However, the transfer of meat stored under vacuum at 12 degrees C to aerobic storage resulted in reduction in pathogen counts during aerobic storage and sensitization of survivors to the effects of sequential heat and acid exposure.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.     

Effect of phosphoenolpyruvate on metabolic and morphological recovery of red cells after prolonged liquid storage and subsequent freezing in glycerol medium.

The present study was designed to determine the effects of (i) phosphoenolpyruvate (PEP) treatment of red blood cells (RBCs) previously cold stored for a prolonged period in a liquid medium and (ii) the freezing of these treated cells in glycerol. RBCs stored for 21 days at 4 degrees C were incubated for 30 min at 37 degrees C with rejuvenant solution containing 50 mM PEP, 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, and 1 mM adenine, pH 6.0, and then frozen at -80 degrees C for 4 weeks. Red cell recovery as frozen and thawed red cells (FTRCs) after deglycerolization was increased to 80 +/- 4% compared to 43 +/- 9% in units without rejuvenation; the percentage of PEP-treated FTRCs was similar to the percentage of FTRCs recovered from fresh RBCs within 5 days after donation. Incubation of RBCs with PEP solution restored ATP and 2,3-DPG to levels seen in fresh RBCs, and also facilitated transformation of crenated RBCs to discocytes. These results indicate that maximum recovery of viable RBCs can be attained when FTRCs are processed from cells stored in the frozen state after they had been rejuvenated with PEP even after prolonged liquid storage.     

The effects of 1,2-propanediol as a cryoprotectant on the freezing of mouse oocytes

Cryopreservation of mammalian eggs has been successfully accomplished using 1,2-propanediol (PG). Effects of holding times of 0 and 30 min at -40 degrees C and storage times of 1 d and 1 mo at -196 degrees C were investigated in combination with various concentrations of PG (1.0, 1.5, and 2.0M) to determine the survival and fertilizability of mouse oocytes rapidly frozen and thawed in straws. A rapid one-step dilution using 0.5 M sucrose solution inside the straws was used following the thawing of oocytes. A significant effect of PG concentration was found between 1.0 M and 1.5 or 2.0 M (P<0.01), but no significance was discovered between 1.5 M and 2.0 M (P>0.05) on subsequent survival and fertilizability of frozen and thawed mouse oocytes. With 2.0 M PG, the best survival rate (58.3%) and fertilizability rate (19.0%) were obtained by holding at -40 degrees C for 30 min and by storage at -196 degrees C for 1 d. Thirty minutes of holding at -40 degrees C reduced oocyte damage during the procedure but not significantly (P>0.05). In addition, there was no significant difference in the various storage periods (P>0.05). This study demonstrated that mammalian oocytes can be cryopreserved in the presence of 1,2-propanediol by utilizing a rapid freezing and thawing procedure.     

Drug metabolism and viability studies in cryopreserved rat hepatocytes

Rat hepatocytes were cryopreserved optimally by freezing them at 1 degrees C/min to -80 degrees C in cryoprotectant medium containing either 20% (v/v) dimethylsulfoxide (Me2SO) and 25% (v/v) fetal calf serum in Leibowitz L15 medium (Me2SO cryoprotectant) or 25% (v/v) vitrification solution (containing Me2SO, acetamide, propylene glycol and polyethylene glycol) in Leibowitz L15 medium (VS25). The VS25 solution was superior for maintaining viability during short-term storage (24-48 hr) but was slightly toxic during longer storage periods (7 days). Although thawed cells were 40-50% viable on ice after cryopreservation, their viability fell rapidly during incubation in suspension at 37 degrees C. This decline in viability occurred more rapidly after freezing in Me2SO cryoprotectant than in VS25 and was associated with extensive intracellular damage and cell swelling. The loss in viability at 37 degrees C does not appear to be due to ice-crystal damage as it occurred in cells stored at -10 degrees C (above the freezing point of the cryoprotectants) and it may be due to temperature/osmotic shock. Both cryoprotectant media were equally efficient at preserving enzyme activities in the hepatocytes over 7 days at -80 degrees C. Cytochrome P450 and reduced glutathione content and the activities of the microsomal enzymes responsible for aminopyrine N-demethylation and epoxide hydrolysis were well maintained over 7 days storage. In contrast, the cytosolic enzymes glutathione-S-transferase and glutathione reductase were markedly labile during cryopreservation. Cytosolic enzymes may be more susceptible to ice-crystal damage, whereas the microsomal membrane may protect the enzymes which are embedded in it.