Enumeration of micronucleated reticulocytes in rat peripheral blood: a flow cytometric study

Micronuclei (MN) are routinely enumerated in mouse peripheral blood to index genotoxicity. Recent data from the Collaborative Study Group for the Micronucleus Test (CSGMT) [CSGMT (The Collaborative Study Group for the Micronucleus Test), Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS MMS, Environ. Mol. Mutagen. 32 (1998) 84-100] suggest that rat peripheral blood may also be appropriate for the enumeration of MN, if scoring is limited to the youngest fraction of reticulocytes. The experiments described herein were designed to test whether modifications to a flow cytometric scoring procedure for measuring micronucleated reticulocytes (MN-RET) in mouse peripheral blood could be extended to accurately enumerate MN in rat peripheral blood. Rats were treated with saline or one of three genotoxic agents (6-mercaptopurine, ethyl methanesulfonate or propane sultone) in an acute dosing protocol. Peripheral blood samples were subsequently collected for both microscopic and flow cytometric analysis. Micronucleus frequencies were scored in the youngest fraction of reticulocytes: scoring by microscopy was restricted to the types I and II reticulocytes based on RNA content utilizing acridine orange supravital staining; flow cytometric measurements were restricted to the youngest fraction of reticulocytes based on transferrin receptor (CD71) staining. A statistically significant dose-related increase in the incidence of MN was observed, irrespective of scoring method. A higher level of statistical discrimination between control and genotoxin-treated groups was observed for the flow cytometric data and can most likely be explained by the increased number of cells scored (10x more than microscopy) and the lower scoring variability. Together, these data suggest that (i) rat peripheral blood represents an appropriate compartment for evaluating genotoxin-induced MN when the analysis is restricted to young reticulocytes, and (ii) the measurement of MN in rat peripheral blood reticulocytes benefits from the high throughput methodology of flow cytometry.     

Methionine and neural tube closure in cultured rat embryos: morphological and biochemical analyses

When headfold-stage rat embryos were cultured on cow serum, their neural tubes failed to close unless the serum was supplemented with methionine. Methionine deficiency did not appear to affect the ability of the neural epithelium to fuse as a type of fusion was observed between anterior and posterior regions of the open neural tube in methionine-deficient embryos. Although methionine deficiency reduced the cell density and mitotic indices of cranial mesenchyme and neural epithelial cells, this did not appear to be a factor in failure of the neural tube to close. For example, embryos cultured on diluted cow serum also had fewer mesenchymal cells yet could complete neural tube closure if provided with methionine. Examination of the tips of the neural folds suggested that microfilament contraction could be involved; in the absence of methionine the neural folds failed to turn in. This possibility was supported by the reductions in neurite extension of isolated neural tubes cultured without methionine and by the reductions in microfilament associated methylated amino acids contained in embryo neural tube proteins.     

Variability in molluscan hemocytes: a flow cytometric study

Reported variability in numbers and relative proportions of hemocytes in marine bivalves may be related to environmental conditions and laboratory method differences. An automated identification assay, flow cytometry, removes much laboratory bias, but its usefulness is limited because the putative cell types in delineated subpopulations have never been confirmed. The present study was designed to: (1) confirm the identity of oyster hemocyte subpopulations described by flow cytometry, and (2) use flow cytometry in an experimental analysis of potential causes of variation. Light-scatter flow cytometry consistently differentiated three subpopulations in oysters from two mid-Atlantic (USA) sites. Cell sorting and microscopy identified them as granular, small granular, and agranular (hyalinocytes and apparently degranulated) hemocytes. Subpopulation proportions estimated by microscopy and by flow cytometry were significantly correlated (r(2) = 0.27 to 0.50). In a 4-week laboratory experiment, neither temperature (12 vs. 22 degrees C) nor food (fed vs. not fed) had a statistically significant effect on total or differential counts, or on hemocyte viability. Most of the variability was attributable to individual differences and was very similar to that reported for vertebrates. We hypothesize that variability in molluscan hemocytes may be more immediately linked to individual metabolic condition than to ambient changes.     

Short term hypothyroidism affects ovarian function in the cycling rat

Background  

Rats made hypothyroid with propilthyouracil start showing abnormal cycling on the second cycle after the start of the treatment, with a high proportion of spontaneous pseudopregnancies and reduced fertility.     

Heat shock proteins: a history of study in Russia

This review describes a brief history of the discovery and studies in Russia and associated countries of the main stress protein (Hsp70) that plays important roles both in the normal function of the cell and body as well as under various stressful stimuli. Research on this protein at the Institute of Molecular Biology (Moscow) began with the elucidation of its adaptive functions at the cellular level and at the level of the whole organism. These studies examined the function of Hsp70 under normal and extreme conditions using a wide range of model and non-model animal species, from Leishmania and Drosophila to camels and humans. These analyses made it possible to elucidate the primary regulations in the evolution and function of heat shock (HS) genes in the studied organisms. Next, we studied the structure and characteristic features of heat shock genes and proteins in species with contrasting habitat temperatures. The systems of Hsp70 expression and isolation we developed using various research objects allowed us to proceed to study the protective properties of human recombinant Hsp70 in normal-aging animal models as well as animal models experiencing sepsis, Alzheimer’s disease, and stroke. The results obtained open the prospects of using recombinant Hsp70 for the treatment of various neuropathologies in humans. This review describes the logic and history of investigation of Hsp70 performed by one group of scientists from Engelhardt Institute of Molecular Biology, Russian Academy of Sciences. It was not the goal of this paper to give a comprehensive general picture of other similar studies carried out in Russia during this period.     

Kinetics of DNase I digestion of interphase chromatin in differentiated cell nuclei of the mouse: a flow cytometric study

The process of DNA digestion with DNase I was monitored in interphase chromatin of differentiated cells by flow cytometry after DNA staining with either the intercalating dye propidium iodide (PI) or the AT specific dye Hoechst 33258 (HO). Nuclei from the liver, kidney and spleen of the mouse were studied after different digestion times (0 to 120 min). During the first 30 min of treatment, a tissue specific digestion pattern was found after PI staining; from 60 min onward, the digestion curves ran parallel, with minor quantitative differences among the cell types. After HO staining, the digestion kinetics appeared to be similar for all the cell types; this is likely due to the peculiar base composition of the mouse genome, where inactive c-heterochromatin is exceptionally AT-rich. No quantitative correlation was found between interphase "heterochromatin" and chromatin DNA which is resistant to DNase I cleavage, while the amount of DNase-I-sensitive DNA does not correspond to the interphase "euchromatic" component. It was confirmed that the flow cytometric approach is a tool for quantifying relative changes in the functional state of chromatin in differentiated cell systems.     

Automatic identification of algae: neural network analysis of flow cytometric data

The performance of an artificial neural network for automaticidentification of phytoplankton was investigated with data fromalgal laboratory cultures, analysed on the Optical PlanktonAnalyser (OPA), a flow cytometer especially developed for theanalysis of phytoplankton. Data from monocultures of eight algalspecies were used to train a neural network. The performanceof the trained network was tested with OPA data from mixturesof laboratory cultures. The network could distinguish Cyanobacteriafrom other algae with 99% accuracy. The identification of specieswas performed with less accuracy, but was generally >90%.This indicates that a neural network under supervised learningcan be used for automatic identification of species in relativelycomplex mixtures. Incorporation of such a system may also increasethe operational size range of a flow cytometer. The combinationof the OPA and neural network data analysis offers the elementsto build an operational automatic algal identification system.     

Heat shock response of the rat lens

The sequence relationship between the small heat shock proteins and the eye lens protein alpha-crystallin (Ingolia, T. D., and E. E. Craig, 1982, Proc. Natl. Acad. Sci. USA, 79: 2360-2364) prompted us to subject rat lenses in organ culture to heat shock and other forms of stress. The effects on protein synthesis were followed by labeling with [35S]methionine and analysis by one- and two-dimensional gel electrophoresis and fluorography. Heat shock gave a pronounced induction of a protein that could be characterized as the stress protein SP71. This protein probably corresponds to the major mammalian heat shock protein hsp70. Also two minor proteins of 16 and 85 kD were induced, while the synthesis of a constitutive heat shock-related protein, P73, was considerably increased. The synthesis of SP71 started between 30 and 60 min after heat shock, reached its highest level after 3 h, and had stopped again after 8 h. In rat lenses that were preconditioned by an initial mild heat shock, a subsequent shock did not cause renewed synthesis of SP71. This effect resembles the thermotolerance phenomenon observed in cultured cells. The proline analogue azetidine-2-carboxylic acid, zinc chloride, ethanol, and calcium chloride did not, under the conditions used, induce stress proteins in the rat lens. Sodium arsenite, however, had very much the same effects as heat shock. Calcium ionophore A23187 specifically and effectively induced the synthesis of the glucose-regulated protein GRP78. No special response to stress on crystallin synthesis was noticed.     

Heat shock proteins and thermotolerance in a cultured cell line from the Mediterranean fruit fly, Ceratitis capitata.

Heat shock proteins (hsps) were identified in a cell line from the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae) exposed to elevated temperatures. Cells produced three hsps (Mr 87,000, 69,000, and 34,000) in response to a temperature shift from 26 degrees C to 37 degrees C (30-60 min) with a concomitant decrease in synthesis of most other cellular proteins. Synthesis of low Mr hsps was not evident. The heat shock response is triggered within 30 min at temperatures from 33 degrees C to 41 degrees C. At temperatures greater than 41 degrees C protein synthesis was shut down. Within 2-3 h after return to 26 degrees C, synthesis of proteins repressed at the higher temperatures resumed production while the major hsps disappear. Heat shock proteins were not produced in the presence of actinomycin D. Evaluations on the role of hsps in conferring thermotolerance to the cells showed an increase in cell viability in heat-shocked cells over non-heat-shocked cells (after 3 and 10 days) when subsequently placed at 45 degrees C for 1 h, a normally lethal temperature. Heat shock alone had little effect on subsequent cell viability or growth at 26 degrees C. These results suggest that hsps produced by these cells may aid in the maintenance of cell integrity and thus play a transitory role in thermotolerance.     

Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos

Heat shock has a dramatic effect on the organization of the cytoplasm, causing the intermediate filament cytoskeleton to aggregate at the nucleus. This has previously been shown in cultured Drosophila and mammalian cells. In this paper we analyze the heat lability of the intermediate filament cytoskeleton in early Drosophila embryos by indirect immunofluorescence. At all stages of embryogenesis tested, the intermediate filament cytoskeleton, which is maternally provided, is severely disturbed by 30 min heat shock at 37 degrees C. After the nuclei have migrated to the subcortical cytoplasm, it collapses around them. Nuclei in all heat-shocked embryos are considerably enlarged and become displaced. Embryos before cellular blastoderm stage, in which heat shock protein synthesis is not inducible, are irreversibly arrested in development by heat shock. Embryos at or after cellular blastoderm, which do synthesize heat shock proteins in response to stress, are also immediately arrested in development but continue development when returned to 25 degrees C. We discuss the possibility that cytoplasmic events such as the intermediate filament cytoskeleton rearrangement may be involved in heat shock-mediated phenocopy induction.     

Discrimination of cycling and non-cycling lymphocytes by BUdR-suppressed acridine orange fluorescence in a flow cytometric system.

Stimulated lymphocytes which pass through the cell cycle may be distinguished from dormant G0 lymphocytes rapidly by flow cytometry. The method is based on cell incubation with 5-bromodeoxyuridine (BUdR) and their subsequent staining with acridine orange under conditions in which cellular DNA and RNA stain differentially. The DNA-specific green fluorescence of stimulated, cycling cells is suppressed while RNA-specific red fluorescence is affected only minimally. It is possible, therefore, to distinguish cycling vs non-cycling cells based on two entirely different parameters, i.e. BUdR incorporation and RNA content.     

Interactions of cadmium and zinc in cultured rat embryos

Rat embryos were cultured in serum taken from animals dosed with cadmium, or serum with cadmium added $\text{in}$$\text{vitro}$ in the presence or absence of additional zinc. Embryos explanted at day ten and grown in serum taken from animals sooner than 4 h after dosing had a reduced DNA content after 24 h culture. In one-hour serum, the yolk sac had become thick and brittle. Zinc ameliorated the effects but had no stimulatory effect on post eight-hour serum when serum zinc levels were at their lowest. The hypothesis that cadmium induces a maternal zinc deficiency sufficient to cause teratogenic changes could not be sustained. Embryos explanted at nine days were much more susceptible to cadmium added $\text{in}$$\text{vitro}$ than ten-day embryos. The principal anomaly, apart from a reduced DNA content, was a thickening of the yolk sac similar to that seen in embryos grown in serum taken from animals one hour after cadmium dosing. Addition of zinc to the medium prevented both of these effects. The suggestion is made that the cadmium-induced dysgenesis of the yolk sac precludes appropriate embryonic nutrition.     

Hormonal inducibility of liver-specific enzymes in cultured rat embryos

In monolayer cultures, hepatocyte-specific enzymes are inducible by hormones as soon as hepatocytes differentiate from the embryonic foregut (15-somite stage). Though offering an excellent opportunity for quantitative studies, several features of a normal cell environment are lost in such a model system. To determine the inducibility of such tissue-specific enzymes in intact organisms, rat embryos were cultured in vitro for 48 h and exposed to the hormonal factors that had been found effective in monolayer culture, viz. dexamethasone, triiodothyronine and dibutyryl cyclic AMP. Normal development of the embryos during culture in vitro was assessed by general criteria reflecting growth, morphogenesis and cytodifferentiation. Development of external features, organogenesis, the distribution of cell divisions and the appearance of tissue-specific proteins such as alpha-fetoprotein and glutamate dehydrogenase served as parameters. Despite undisturbed development of the embryos as judged by these criteria, irrespective of whether the culture was started at day 10 or at day 11 of gestation (just before, respectively after the appearance of the liver primordium), induction of hepatocyte-specific enzymes like carbamoylphosphate synthetase by hormones could not be demonstrated immunohistochemically. However, induction of this enzyme by hormones could be demonstrated in monolayers of hepatocytes isolated from such embryos after 48 h of culture, providing yet another demonstration of the adequate culture conditions. In addition, an adequate uptake of hormones by the embryo during culture could be shown with radio-actively labeled dexamethasone and triiodothyronine and with a radioreceptor assay for cyclic AMP. Therefore, the presence of factors in young embryos that inhibit tissue-specific enzyme synthesis has to be postulated.     

A flow cytometric study of chromosomes from rat kangaroo and chinese hamster cells

Summary Chromosomes from rat kangaroo (PTK) and chinese hamster (CHV 79) cells have been prepared for quantitative flow-cytometric analysis. The preparation time was optimized down to 30 (PTK) and 40 min (CHV 79). DAPI was used as a AT-sensitive fluorescent dye to stain for monoparameter DNA measurements. Simultaneous two-parameter DNA-protein analysis was carried out with DAPI and SR 101 (as a general protein fluorochrome) in combination. The karyotype of the PTK cells with 13 (14) chromosomes was separated into 10 DNA peaks. The X-chromosome bearing the nucleolus organizer region generates a distinct peak. The karyotype of the CHV 79 cells with 22 chromosomes was separated into 15 peaks. The DNA profile obtained indicates a geometric grading of the chromosomal amount of AT components in the karyotype of this particular cell line. The simultaneous DNA-protein analysis performed show enough sensitivity of the instrument utilizing high power UV excitation illumination to discriminate the two color emission consisting of blue (DAPI) and red (SR 101) fluorescence. Color overlapping could be completely avoided. Additionally, the quality (number, location, and resolution of peaks) of the DNA distribution was not influenced by the simultaneous application of a second fluorescent stain. Fluorescence activated electronic sorting applied on chromosomal fluorescence distributions providing purified fractions of chromosomes for subsequent biochemical and biological determinations is discussed.     

Dynamics of testicular germ cell proliferation in normal mice and transgenic mice overexpressing rat androgen-binding protein: a flow cytometric evaluation

Transgenic mice carrying rat androgen-binding protein (ABP) genomic DNA express high amounts of testicular ABP and develop a progressive impairment of spermatogenesis. To understand the mechanism of these changes, we have studied the pattern of testicular germ cell proliferation from 7 to 360 days of age in wild-type (WT) control and transgenic homozygous (ABP-TG) mice by flow cytometry after labeling DNA in isolated germ cells with propidium iodide. At all ages studied, the body weight of the ABP-TG mice was lower than that of age-matched WT controls. Significantly reduced testicular weight and total germ cell number in the ABP-TG mice were evident from Day 30 and Day 60, respectively. Flow cytometric analysis of isolated germ cells revealed that the number of germ cells undergoing proliferation (S-phase cells) was identical in WT control and ABP-TG mice up to Day 14. Subsequently, the number of germ cells in S-phase was consistently higher in ABP-TG than in WT mice. The number of primary spermatocytes was significantly increased starting from Day 60, and the numbers of round and elongated spermatids were significantly reduced in the ABP-TG animals from Day 21 and Day 60 onwards, respectively. Immunocytometry for intracellular ABP at 90 days of age revealed that the percentage of ABP-containing germ cells was greater in ABP-TG than in WT mice. The continuous presence of ABP in mouse seminiferous tubules at greater than physiological concentrations facilitates the formation of primary spermatocytes but impairs subsequent transformation to round and elongated spermatids. Based on our observations and the analysis of the available literature, the most likely mechanism for production of these effects is sustained reduction in the bioavailability of androgens.     

Variation of mitochondrial size during the cell cycle: A multiparameter flow cytometric and microscopic study.

BACKGROUND: Changes in mitochondrial structure and size are observed in response to alterations in cell physiology. Flow cytometry provides a useful tool to study these changes in intact cells. We have used flow cytometry and digital fluorescence microscopy to analyze the variations in mitochondrial size in relation to specific phases of the cell cycle. METHODS: Supravital staining of rat fibroblasts was done with Hoechst 33342 and rhodamine 123, and cells were analyzed in a dual-laser flow cytometer. Synchronized cells at various stages of the cell cycle were analyzed for changes in mitochondrial size. These cells were also examined by electron microscopy, digital fluorescence microscopy and computerized image analysis to compare the lengths of the mitochondria. RESULTS: By using fluorescence pulse width analysis, we observed two populations of mitochondria in intact cells. The percentage of cells with small and large mitochondria at specific stages of the cell cycle indicated that mitochondrial size increases during the cell cycle; early G1 phase cells had the smallest mitochondria and the mitotic phase cells had the largest mitochondria. These results were confirmed by microscopic analysis of cells. CONCLUSIONS: Flow cytometry can distinguish the relative mitochondrial size in intact cells, and in combination with digital microscopy it can be used to study mitochondrial variation during the cell cycle.     

The polarization of neuroepithelial cells when a portion of the neural plate is inserted into the neural tube cavity in amphibian embryos

Portions of homologous neuroepithelium of various length were introduced into the neural tube cavity in Rana arvalis and Pleurodeles waltlii late neurula. The neural tube in R. arvalis was destroyed independently of the transplant length. In P. waltlii neural primordia were deformed if a large transplant prevented closing of the neural tube. Redundant increase in the host cells length (polarization) was observed during the small transplant-induced retardation of neurulation. Recipient cells elongated 1.5-2 fold as compared to the normal ones. Zones of contact polarization were detected. They arise in slowly relaxing layer under conditions during the transplant-induced retardation of neurulation. The labelled TRITC cells introduced into the neural tube cavity integrated into the wall of the neural tube and retarded polarization of its cells.