Fresh and vitrified bovine preantral follicles have different nutritional requirements during in vitro culture

The aim of this study was to compare the efficiency of different media for the in vitro culturing of fresh and vitrified bovine ovarian tissues. Fragments of the ovarian cortex were subjected to vitrification and histological and viability analyses or were immediately cultured in vitro using the alfa minimum essential medium, McCoy’s 5A medium (McCoy), or medium 199 (M199). Samples of different culture media were collected on days 1 (D1) and 5 (D5) for quantification of reactive oxygen species and for hormonal assays. In non-vitrified (i.e., fresh) ovarian tissue cultures, the percentage of morphologically normal follicles was significantly greater than that recorded for the other media (e.g., M199). In the case of previously vitrified tissues, the McCoy medium was significantly superior to the other media in preserving follicular morphology up until the last culture day (i.e., D5), thus maintaining a similar percentage from D1 to D5. Reactive oxygen species levels were higher in D1 vitrified cultured tissues, but there were no differences in the levels among the three media after 5 days. The hormonal assays showed that in the case of previously vitrified tissues, at D5, progesterone levels increased on culture in the M199 medium and estradiol levels increased on culture in the McCoy medium. In conclusion, our results indicate that the use of M199 would be recommended for fresh tissue cultures and of McCoy for vitrified tissue cultures.     

A comparison of Menezo's B2 and tissue culture Medium-199 for in vitro production of bovine blastocysts

The objectives of this study were, first, to evaluate the effectiveness of 2 culture media, Menezo's B2 (B2) and Tissue Culture Medium-199 (M-199), for the production of bovine blastocysts in a commercial embryo transfer program; and, second, to characterize the stage of development, quality grade and cell number of blastocysts produced in each medium. One-cell bovine embryos were produced using in vitro maturation and fertilization procedures. After fertilization, the embryos were co-cultured on Buffalo rat liver (BRL) cell monolayers in either B2 or M-199+1% BSA (M-199) medium. Both media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. Embryo cultures were continued undisturbed to either Day 7 or Day 8 post-insemination. In the Day 7 cultures, all blastocysts were removed for evaluation on Day 7, and the remaining embryos were cultured for a further 24 h. Any additional blastocysts that formed were removed for evaluation and designated as Day 8 disturbed embryos. All blastocysts were classified for stage and quality grade. Embryos were fixed and stained for determination of cell number. Overall, the proportion of blastocysts was greater (P = 0.0003) with B2 medium (46%) than with M-199 (33%). This was due to a larger (P = 0.0001) proportion of blastocysts produced in B2 medium when cultures were left undisturbed for 8 d (50 vs 28% for B2 vs M-199). The proportion of blastocysts on Day 7 of culture tended to differ (P = 0.073) between media (33 vs 24% for B2 vs M-199). In addition, there were more (P = 0.007) blastocysts at advanced stages of development in B2 medium on Day 7. There was no effect of type of medium on the distribution of embryo quality grades on any day examined. The number of cells per blastocyst did not differ between media but did vary significantly (P < .05) with both stage and grade. In conclusion, B2 medium was superior to M-199 medium when used in a co-culture system with BRL cells for the production of bovine blastocysts.     

Luteinizing hormone-enhanced in vitro maturation of bovine oocytes with and without protein supplementation

Luteinizing hormone was shown to enhance maturation of immature oocytes obtained from slaughtered cattle as reflected by elevated proportions of oocytes that fertilized and reached blastocyst stages in vitro after in vitro fertilization (IVF). Higher proportions of ova were fertilized in vitro after in vitro maturation (IVM) in modified TCM-199 (TCM-199 + BSA + LH [USDA-bLH-B-5, 100 micrograms/ml]) than in TCM-199 alone (p less than 0.01). Further improvement in IVF (p less than 0.005) followed IVM when 20% proestrous (Day 20) bovine serum replaced the BSA, but similar proportions of inseminated ova (22.2% and 22.6%) developed into blastocysts. The positive LH effect was verified in defined conditions for IVM. Exposure of oocytes to the purified LH preparation (without any other added protein or biological substances) during IVM improved IVF (39.7% in TCM-199 vs. 73.5% in TCM-199 + LH; p less than 0.001) and blastocyst development (7.9% vs. 28.2%; p less than 0.005), respectively. Efforts to better define effective concentrations of LH revealed no difference in viability after IVM with 50 micrograms LH/ml vs. 100 micrograms LH/ml (27.0% vs. 28.3%, respectively); 10 micrograms LH/ml did not enhance viability when compared to TCM-199 alone (10.8% vs. 9.9%). Results demonstrate potential utility of this approach for investigation of factors influencing mammalian development by specific effects initiated during the interval of oocyte maturation.     

Detection of infectious Cryptosporidium oocysts by cell culture immunofluorescence assay: applicability to environmental samples

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.     

In-vitro development and degeneration of stromal cells from the uterus of the rat at three stages of pregnancy

On Days 6, 11 or 16 of pregnancy, endometrial tissue (Day 6) or decidual tissue (Days 11 and 16) were removed from rat uteri, dissociated into single cells and grown in culture. At intervals during the culture period, the cells were examined and the cell density was calculated. The cells from Days 11 and 16 of pregnancy were similar in appearance, being very large and flattened. Initially, cells from Day 6 were much smaller, but showed an increase in size and came to resemble cells from the later stages of pregnancy. Growth curves for cells from each of the three stages of pregnancy over the first 16 days of culture differed. Cells from Day 6 of pregnancy increased exponentially in number. In cultures of cells from Day 11, cell numbers were stable, and in cultures of cells from Day 16, an exponential decline was seen. Cells from Days 6 and 11 of pregnancy were cultured for 42 days, by which time only a few cells remained viable. Cultures of cells from Day 16 degenerated within 3 weeks. These results indicate that, in vivo, decidual cells are not controlled solely by a 'programmed lifespan'. Changes occurring during pregnancy, however, limit the potential of the cells for division and survival in culture.     

Enumeration of Viable Bacteria in the Marine Pelagic Environment

The low percentage of living bacteria commonly obtained when comparing viable counts with total direct counts in seawater could be due more to inappropriate techniques for appreciating the growth ability of living cells than to unadapted culture conditions. The most-probable-number counts in filtered seawater cultures and the microscopic counts of 4(prm1),6-diamidino-2-phenylindole (DAPI)-stained aggregate-forming units grown on black polycarbonate filters appeared significantly correlated to the direct counts. Both these techniques show that in the superficial and intermediate water masses, the living cells may constitute an important (frequently higher than 20%) but highly variable part of the total populations. These viable counts appear more realistic than the conventional CFU counts, which provide only 0.001 to 0.2% of the total counts.     

Assessment of Cell Viability in Intact Glandular Tissue in <Emphasis Type="Italic">Chironomus ramosus</Emphasis> using Dye-exclusion and Colorimetric Assays

Conventionally, dye-exclusion test for determining cell viability has been restricted only for cells in suspension in tissue culture. In this paper, salivary gland of Chironomus has been proposed as a simple tissue model system where dye-exclusion test can be reliably employed for the intact gland. We have compared suitability of commonly used vital dyes and nigrosin was found suitable for the salivary gland cells. Biochemical tests using tetrazolium salts are also commonly used for determining quantitative indices of cell viability in metabolically active cells. Ours is the first attempt to extend the same technique for the whole tissue. We standardized the conditions and prepared a protocol for MTT-based colorimetric assay suitable for the salivary gland of Chironomus. A strong correlation (r² = 0.9893) was obtained where increasing O.D. correlated linearly with the number of live glands. We concluded that nigrosin dye-exclusion and MTT metabolic inclusion assays are suitable methods for the viability test of metabolically active intact salivary gland of Chironomus which can serve as a potential model for the assessment of cytotoxicity in future.     

Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing

Factors affecting viability of IVF-derived bovine blastocysts after freezing and thawing were investigated. A total of 1,101 ova matured and fertilized in vitro were cultured under 2 different conditions, 1) in TCM-199 on granulosa cell monolayers at 5% CO(2) in air and 2) in synthetic oviduct fluid (SOF) medium without somatic cell support at 5% CO(2), 5% O(2), 90% N(2). All blastocysts that developed from the 2 different culture systems were individually classified into 4 grades of embryo quality and were then frozen by conventional slow freezing. Developmental rates of the IVF-derived ova to blastocysts and the survival rates of the frozen-thawed blastocysts were not different between the SOF medium (16 and 49%) and the co-culture system (13 and 61%, respectively). Survival of frozen-thawed blastocysts was affected by embryo quality in both the SOF and co-culture systems (P<0.001). Blastocysts produced in vitro were also individually classified into 3 developmental stages and were then cultured for 3 d in the co-culture system with granulosa cells after freezing and thawing. There was a difference in the survival rate of frozen-thawed embryos between blastocyst developmental stages (early vs mid, P<0.05; mid vs expanded, P<0.01; early vs expanded, P<0.001). The post-thawing survival rate of blastocysts frozen at Day 7 (62%) of culture was higher compared with that of Day 8 (45%), but there was no difference in survival rate between Day 7 and 8 of culture. The results indicate that the quality and developmental stage of blastocysts are important factors influencing their survival after freezing and thawing.     

Detection of Infectious Cryptosporidium Oocysts by Cell Culture Immunofluorescence Assay: Applicability to Environmental Samples

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.     

Luteotropic and luteolytic responsiveness of ovine luteal cells in long-term culture

Ovine luteal cells were collected and plated 36 h (Day 2) after injection of human chorionic gonadotropin (Day 0) to induce ovulation. Cells were maintained (Days 2-12) in Medium 199 containing 5% calf serum, which was replaced daily. Progesterone secretion was not stimulated (p greater than 0.05) by luteinizing hormone (LH, 10 ng/ml or 100 ng/ml) at any time during culture. However, it was enhanced (p less than 0.05) with a 24-h pulse of dibutyryl adenosine 3', 5'-monophosphate (dbcAMP) during early (2.2-fold stimulation over basal; Days 5,6) or mid- (1.7-fold stimulation over basal: Days 8,9) culture if the pulsing medium contained serum, but not if serum had been withdrawn for 24 h. Continuous exposure of cultures to dbcAMP (2 mM, Days 3-12) resulted in continuously stimulated (p less than 0.05) progesterone secretion (range 1.8- to 4.1-fold stimulation). An increased (p less than 0.05) percentage of cells staining positive for 3 beta-hydroxy-delta 5-steroid dehydrogenase-delta 5, delta 4-isomerase (3 beta HSD) activity were recovered on Day 12 in cultures incubated (Days 3-12) with dbcAMP. Incubation of cultures continuously with prostaglandin F2 alpha (PGF2 alpha) produced dose-dependent inhibition (p less than 0.05) of progesterone secretion. Reduced numbers of 3 beta HSD-positive cells were recovered from these incubations. These experiments demonstrate luteotropic (dbcAMP) as well as luteolytic (PGF2 alpha) effects on ovine luteal cells in long-term culture. This study provides evidence that these cultures will be useful for investigating the development of hormonal regulation of luteal function.     

Steroidogenic capacity and ultrastructural morphology of cultured ovine luteal cells

Corpora lutea were surgically collected from superovulated ewes 36 h post-injection of human chorionic gonadotropin (hCG) (Day 2), dissociated (0.2% collagenase), plated, and maintained in culture Days 2-10 in Medium 199 supplemented with 5% calf serum. Accumulation of progesterone in the cultures did not decrease (p greater than 0.05) from Day 3 (17.5 +/- 5.1 nmol/10(6) cells) to Day 10 (4.8 +/- 1.7 nmol/10(6) cells). Calf serum (5%) in the medium supported greater (p less than 0.05) progesterone production than fetal calf serum (5%) or medium without added serum. Steroidogenic cells did not increase (Days 2-10) in numbers, but increased (p less than 0.01) in mean cell diameter (Day 2, 11.7 +/- 0.4 micron; Day 10, 24.5 +/- 1.6 micron). Steroidogenic capacity on Day 10 of cells cultured Days 2-10 (in vitro) was not different (p greater than 0.05) from that of cells collected from the ovary on Day 10 (in vivo); however, steroidogenic cells recovered from plates had greater (p less than 0.01) mean cell diameters (24.5 +/- 1.6 micron, in vitro, compared to 15.2 +/- 1.0 micron, in vivo). Transmission electron microscopy revealed that cultured cells (Days 5, 10) possessed less smooth endoplasmic reticulum but more lipid droplet inclusions, ribosomes, and rough endoplasmic reticulum than cells obtained in situ (Day 10). Electron-dense secretory granules were rarely seen. Although subcellular morphology of ovine luteal cells in culture was altered, these changes did not appear to significantly affect the ability of these cells to produce progesterone.     

In vitro evaluation of alginate encapsulated adipose-tissue stromal cells for use as injectable bone graft substitute

This study aims to investigate the survival and osteogenic behavior of murine-derived adipose-tissue stromal cells (ATSCs) encapsulated in alginate microcapsules thereby instigating further studies in this cell delivery strategy for in vivo osteogenesis. Cell viability was quantified using a tetrazolium-based assay and osteogenic differentiation was evaluated by both alkaline-phosphatase (ALP) histochemistry and osteocalcin mRNA analysis. Following microencapsulation, cell numbers increased from 3.9 x 10(3) on day 1 to 7.8 x 10(3) on day 7 and maintained excellent viability in the course of 21-day culture. ALP was 6.9, 5.5, and 3.2 times higher than monolayer cultures on days 7, 14, and 21, respectively. In addition, osteocalcin mRNA was detectable in encapsulated cultures earlier (day 14) than monolayer cultures. We conclude that alginate microcapsules can act as three-dimensional matrix for ATSC proliferation and has potential for use as injectable, biodegradable scaffold in bone tissue engineering.     

Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state

The viabilities of five strains of Vibrio vulnificus were evaluated during the storage of the organisms in sterile seawater at 5 degrees C. The number of CFU was measured by plate count methods on rich media. The total cell numbers were determined by direct microscopic count methods. The titer of CFU declined logarithmically to undetectable levels over a period of 2 to 3 weeks, while the total cell numbers were unchanged. Midway through each study, higher culturable cell counts began to be observed on plates containing catalase or sodium pyruvate; during the latter stages of the study, the plate counts on such media were up to 1,000-fold higher than those on unsupplemented plates. Because autoclaving is known to generate hydrogen peroxide in rich media, and because catalase and sodium pyruvate are known to eliminate hydrogen peroxide, it appears that the conditions of the experiments led to the selection of a hydrogen peroxide-sensitive culturable cell subpopulation. At the time of the final stage of the decline in viability of each culture, hydrogen peroxide-sensitive cells were the only culturable cells present. Warming samples of the cultures to room temperature led to the growth of these residual culturable cells, utilizing nutrients provided by the nonculturable cells. The cells that grew recovered hydrogen peroxide resistance. When mixtures of culturable and nonculturable cells were diluted to the point where only nonculturable cells were present, or when the hydrogen peroxide-sensitive culturable cells had declined to undetectable levels, warming had no effect; no culturable cells were recovered. Warming has been reported to "resuscitate" nonculturable cells. Recognition of the existence of hydrogen peroxide-sensitive culturable cell populations, as well as their ability to grow to high levels in the warmed seawater microcosms, leads instead to the conclusion that while warming permits culturable cells to grow, it has no effect on nonculturable cells.     

Diagnostic polymerase chain reaction assay to detect Kudoa neurophila (Myxozoa: Multivalvulida) in a marine finfish hatchery

A single-round polymerase chain reaction (PCR) diagnostic assay was developed from a small subunit ribosomal DNA (SSU rDNA) gene sequence to detect the myxozoan parasite Kudoa neurophila, the causative agent of myxozoan disease in the hatchery reared marine finfish, striped trumpeter Latris lineata (Forster). The assay was developed for use as a disease control management tool in a hatchery system specifically designed to research and produce marine finfish such as striped trumpeter juveniles for aquaculture. The assay is sufficiently species specific and sensitive enough to detect a small fragment of the parasite's SSU rDNA. At the lower limits of detection, the test is consistently positive to an estimated 0.1 spore or 60 fg of parasite DNA per 25 microl PCR reaction in serial dilution and positive to an estimated 0.1 spore in 25 mg of infected fish CNS tissue (4 spores g(-1). Specifically, the test is capable of detecting early stages of the life cycle within the fish host and consequently diagnosing an infection not normally detected using traditional histological techniques. The test is also effective for screening water supplies and prey species cultures throughout the hatchery system to determine bio-security efficacy, to assist in identification of an alternate or other primary fish host, to indicate the location of potential disease reservoirs, and to enable a targetted approach to disease prevention.     

Effect of long-term lead exposure on the seawater and sediment bacteria from heterogeneous continuous flow cultures

Lead-influenced changes of the composition of seawater and sediment bacteria were studied in two flow cultures run with lead-contaminated artificial seawater (1 mg Pb²⁺1^–1) and one control culture. During the experiment viable counts of physiological groups of bacteria from the control culture were not significantly different from that of the lead-contaminated cultures. Lead tolerance of seawater and sediment bacteria strains was investigated. Comparisons of growth yields showed that lead tolerance of seawater and sediment bacteria was lost again if the bacteria were cultivated in a medium without lead. Lead tolerance could not be demonstrated for the sediment bacteria of one lead-contaminated culture. Heterotrophic uptake measurements with radioactive glucose indicated that seawater bacteria from the lead-contaminated cultures became adapted to lead pollution. The sediment bacteria, however, did not reveal lead tolerance by this method. Fluctuations in lead content of the sediment as well as of the overlying seawater gave indications of adsorption-desorption processes between seawater and sediment. Lead was not homogeneously distributed at the sediment surface.     

miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines

Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines. To determine if miR-199a-3p has a tumor suppressive role, pre-miR-199a-3p oligonucleotides were transfected into the HCC cell lines. Pre-miR-199a-3p oligonucleotide reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two cell lines (SNU449 and SNU423); the proliferation of the other 5 treated cell lines was similar to control oligonucleotide. A pre-miR-199a-3p oligonucleotide formulated with chemical modifications to enhance stability while preserving processing, reduced cell proliferation in SNU449 and SNU423 to the same extent as the commercially available pre-miR-199a-3p oligonucleotide. Furthermore, only the duplex miR-199a-3p oligonucleotide, and not the guide strand alone, was effective at reducing cell viability. Since a CD44 variant was essential for c-Met signaling [V. Orian-Rousseau, L. Chen, J.P. Sleeman, P. Herrlich, H. Ponta, CD44 is required for two consecutive steps in HGF/c-Met signaling, Genes Dev. 16 (2002) 3074-3086] and c-Met is a known miR-199a-3p target, we hypothesized that miR-199a-3p may also target CD44. Immunoblotting confirmed that only the two HCC lines that were sensitive to the effects of pre-miR-199a-3p were CD44+. Direct targeting of CD44 by miR-199a-3p was confirmed using luciferase reporter assays and immunoblotting. Transfection of miR-199a-3p into SNU449 cells reduced in vitro invasion and sensitized the cells to doxorubicin; both effects were enhanced when hyaluronic acid (HA) was added to the cell cultures. An inverse correlation between the expression of miR-199a-3p and CD44 protein was noted in primary HCC specimens. The ability of miR-199a-3p to selectively kill CD44+ HCC may be a useful targeted therapy for CD44+ HCC.     

An in vitro method for detecting infectious Cryptosporidium oocysts with cell culture.

Current assay methods to detect Cryptosporidium oocysts in water are generally not able to evaluate viability or infectivity. A method was developed for low-level detection of infective oocysts by using HCT-8 cells in culture as hosts to C. parvum reproductive stages. The infective foci were detected by labeling intracellular developmental stages of the parasite in an indirect-antibody assay with a primary antibody specific for reproductive stages and a secondary fluorescein isothiocyanate-conjugated antibody. The complete assay was named the focus detection method (FDM). The infectious foci (indicating that at least one of the four sporozoites released from a viable oocyst had infected a cell) were enumerated by epifluorescence microscopy and confirmed under Nomarski differential interference contrast microscopy. Time series experiments demonstrated that the autoreinfective life cycle in host HCT-8 cells began after 12 h of incubation. Through dilution studies, levels as low as one infectious oocyst were detected. The cell culture FDM compared well to other viability assays. Vital stains and excystation demonstrated that oocyst populations less than 1% viable (by vital dyes) and having a low sporozoite yield following excystation could not infect host cells. Until now, the water industry has relied on an oocyst detection method (under an information collection regulation) that is unable to determine viability. The quantifiable results of the cell culture method described demonstrate two important applications: (i) an infectivity assay that may be used in conjunction with current U.S. Environmental Protection Agency-mandated detection methodologies, and (ii) a method to evaluate oocyst infectivity in survival and disinfection studies.     

Defining viability in mammalian cell cultures

A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure membrane integrity (a measure of necrotic cell death) and assays that measure apoptotic cells, and show that discrepancies in the measurement of culture viability have a significant impact on the calculation of cell culture parameters and lead to skewed experimental data.     

Schistosoma mansoni: protein composition and synthesis during early development; evidence for early synthesis of heat shock proteins

There are alterations in the proteins synthesized during different stages of development of Schistosoma mansoni. The protein profiles at different stages were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When stained by Coomassie blue, no significant differences were seen in protein profiles derived from cultured schistosomula from Days 0 to 6 and from adults. Newly synthesized proteins were detected by [35S]methionine incorporation. There were only a few differences in the protein profiles of schistosomula from Days 1 to 6 and from adults. Profiles derived from Day 0 schistosomula showed striking differences. Only a few proteins appear to be synthesized on Day 0 under these conditions. Schistosomula on Day 0 synthesized several minor proteins as well as a major protein of approximately 69,000 Da. This protein was immunoprecipitated by rabbit antiserum against bovine uncoating adenosine triphosphatase which recognizes the constitutive and induced 70,000 Da heat shock proteins in a wide variety of eukaryotic cells. More significant differences were observed when the newly synthesized proteins were analyzed by two dimensional gel electrophoresis. The profiles of newly synthesized proteins showed a specific repertoire of expression during the early stages of development in the parasite. A shift in temperature and medium during transformation from aquarium water to isotonic medium may initiate the synthesis of heat shock protein in these parasites.     

Kinetic differences between fed and starved Chinese hamster ovary cells

When Chinese hamster (CHO-K1) cells are grown as monolayer cultures, they eventually reach a population-density plateau after which no net increase in cell numbers occurs. The kinetics of aged cells in nutritionally deprived (starved) or density-inhibited (fed) late plateau-phase cultures were studied by four methods: (i) Reproductive integrity and cell viability were monitored daily by clonogenic-cell assay and erythrosin-b dye-exclusion techniques. (ii) Mitotic frequencies of cells from 18 day old cultures were determined during regrowth by analysing time-lapse video microscope records of dividing cells. (iii) Tritiated-thymidine ([3H]TdR) autoradiography was used to determine the fractions of DNA-synthesizing cells in cultures entering plateau phase and during regrowth after harvest. (iv) The rate of labelled nucleoside uptake and incorporation into DNA was measured using liquid scintillation or sodium iodide crystal counters after labelling with [3H]TdR or [125I]UdR. Non-cycling cells in starved cultures accumulate primarily as G1 phase cells. Most cells not in G1 phase had stopped in G2 phase. Very few cells (less than 2%) were found in S phase. In contrast, about half of the cells in periodically fed cultures were found to be in DNA-synthetic phase, and the percentage of these S phase cells fluctuated in a manner reflecting the frequency of medium replacement. Populations of both types of plateau-phase cultures demonstrate extremely coherent cyclic patterns of DNA synthesis upon harvest and reculturing. They retain this high degree of synchrony for more than three generations after the resumption of growth. From these data it is concluded that nutritionally deprived (starved) late plateau-phase cells generally stop in either G1 or G2 phase, whereas periodically fed late plateau-phase cultures contain a very large fraction of cycling cells. Populations of cells from these two types of non-expanding cultures are kinetically dissimilar, and should not be expected to respond to extracellular stimuli in the same manner.