Clinical and acquired immunologic responses to West Nile virus infection of domestic chickens (Gallus gallus domesticus).

Numerous bird species are highly susceptible to North American strains of West Nile virus (WNV), and although domestic chickens are relatively resistant to WNV-associated disease, this species currently represents the most practical avian model for immune responses to WNV infection. Knowledge of the immunomodulation of susceptibility to WNV in birds is important for understanding taxonomic differences in infection outcomes. While focusing on immunophenotyping of CD3(+), CD4(+), CD8(+), and CD45(+) lymphocyte subpopulations, we compared lymphocyte subpopulations, blood chemistries, cloacal temperatures, IgM and IgG antibody titers, and differential whole-blood cell counts of WNV-infected and uninfected hens. Total blood calcium and lymphocyte numbers were lower in WNV-infected chickens compared with uninfected chickens. The heterophil-to-lymphocyte ratio increased over time from 2 to 22 d postinoculation (DPI) in uninfected chickens and from 2 to 8 DPI in WNV-infected chickens, although levels declined from 8 to 22 DPI in the latter group. No significant differences were found in the remaining immunological and hematological variables of the WNV-infected and uninfected groups. Our results reaffirm that chickens are resistant to WNV infection, and demonstrated that the heterophil-to-lymphocyte ratio differed between groups, allowing for sorting of infection status. Similar patterns in immune responses over time in both infected and uninfected hens may be related to age (i.e., 10 wk) and associated immune development.

Evaluation of Bacillus anthracis and Yersinia pestis sample collection from nonporous surfaces by quantitative real-time PCR.

AIM: We will validate sample collection methods for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. METHODS AND RESULTS: We evaluated the sample recovery efficiencies of two collection methods - swabs and wipes - for both nonvirulent and virulent strains of Bacillus anthracis and Yersinia pestis from four types of nonporous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than nonvirulent strains. For the two nonvirulent strains, collection efficiency was similar between all four surfaces, albeit B. anthracis Sterne exhibited higher levels of recovery compared to Y. pestis A1122. In contrast, recovery of B. anthracis Ames spores and Y. pestis CO92 from the hydrophilic glass or stainless steel surfaces was generally more efficient compared to collection from the hydrophobic vinyl and plastic surfaces. CONCLUSIONS: Our results suggest that surface hydrophobicity may play a role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings contribute to the validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.

Inhibitory action of epidermal growth factor on progesterone biosynthesis in hen granulosa cells during short term culture: two sites of action.

The acute effect of epidermal growth factor (EGF) on progesterone biosynthesis by hen granulosa cells in short term culture was investigated. Pretreatment of cells for 5 h with EGF at concentrations of 1000-4000 ng/ml inhibited LH-stimulated progesterone production by 54%. Shorter EGF pretreatment times of 1 and 3 h caused 25% and 35% inhibition of LH-stimulated progesterone production, respectively. In additional experiments, EGF was found to inhibit progesterone production in response to 8-bromo-cAMP (1 mM) and forskolin (100 microM) by 34% and 35%, respectively. EGF had no effect on the conversion of 25-hydroxy-cholesterol or pregnenolone to progesterone, indicating that one site at which EGF inhibits progesterone biosynthesis is distal to cAMP generation, but before the side-chain cleavage step. EGF also inhibited LH-stimulated cAMP production by 32%, but had no effect on forskolin-stimulated cAMP accumulation. This indicated that there was a second site of EGF action in these cells, probably at the level of LH receptor coupling to the adenylate cyclase. Nerve growth factor (4000 ng/ml) had no effect on progesterone production, but fibroblast growth factor (4000 ng/ml) facilitated LH-stimulated progesterone production. The results demonstrate that the acute inhibitory effect of EGF on LH-stimulated progesterone biosynthesis in hen granulosa cells is due to its action at two sites: one at a site before the production of cAMP and the other at a step beyond cAMP generation.

Characterization of granulosa cell subpopulations from avian preovulatory follicles by multiparameter flow cytometry.

The objective of the present study was to characterize the granulosa cell populations from individual hen (Gallus domesticus) preovulatory follicles at defined stages of follicular maturation using multiparameter flow cytometry. Granulosa cells were fixed and stained with three fluorochromes that selectively bind to DNA (Hoechst 33342, blue), RNA (pyronin Y, red), or protein (fluorescein isothiocyanate, green). A flow cytometer equipped with a three-laser excitation system was used to analyze three colors of fluorescence from stained cells. Forward angle light scatter and axial light loss measurements were made on each cell to determine relative cell size. In addition, the ratios of RNA to protein and DNA to protein were measured. The major findings obtained from correlated measurements of cell cycle (DNA), protein, RNA, cell size, and ratios were: 1) the percentage of proliferating cells decreased while cell size increased during follicular maturation; 2) two subpopulations of granulosa cells were identified within each follicle based on relatively high and low protein contents; the fraction of cells in the high protein subpopulation increased, and the fraction of cells in the low protein subpopulation decreased during follicular maturation; 3) the high and low protein subpopulations also differed in cell cycle distribution, RNA content, and cell size; and 4) the distribution of cells into the two subpopulations and the degree of proliferation were influenced by stage of the ovulatory cycle, primarily in the most mature follicles. The results demonstrate the dynamic heterogeneity of the granulosa cell populations from individual ovarian follicles and show the influences of follicular maturation and stage of the ovulatory cycle on cell growth and metabolism.

Progesterone metabolism by the hypothalamus, pituitary, and uterus of the aged rat.

Progesterone metabolism was examined in tissues of rats in three stages of reproductive senescence (constant estrus, repeated pseudopregnancies, and anestrus) and in young rats. Metabolites were quantitated by reverse isotopic dilution analysis after incubation of the hypothalamus, pituitary, and uterus with [3H]progesterone. The metabolism of progesterone to 5 alpha-dihydroprogesterone and to 20 alpha-hydroxy-5 alpha-pregnan-3-one and the formation of total 5 alpha-reduced products was significantly reduced (by half) in pituitaries of constant estrous rats compared to rats in all other stages. The formation of 3 alpha-hydroxy-5 alpha-pregnan-20-one and total 3 alpha-reduced products was about 2-fold higher in pituitaries and hypothalami of pseudopregnant and anestrous rats than in constant estrous and young rats, but these differences were statistically significant only in the pituitary samples. In the uterus, progesterone metabolism to 20 alpha-dihydroprogesterone was significantly increased in anestrous rats compared to that in constant estrous and pseudopregnant rats. The results indicate that progesterone metabolism by target tissues, particularly the pituitary, is altered during reproductive senescence. They suggest the possibility that changes in the tissue metabolism of progesterone may be one means by which the effectiveness of progesterone is decreased during aging.

Progesterone metabolism by the hypothalamus, pituitary, and uterus of the rat during pregnancy.

Metabolites of [3H]progesterone were quantitated from incubations of hypothalamus, pituitary, and uterus of rats during different stages of pregnancy. The hypothalamus, anterior pituitary, and a section of uterus from five rats on Days 1, 8, 15, and 21 of pregnancy were incubated individually with [3H]progesterone and analyzed for metabolite formation by reverse isotopic dilution analysis. The radioactive metabolites present were 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 alpha-hydroxy-5 alpha-pregnan-20-one, 20 alpha-hydroxy-4-pregnen-3-one, 20 alpha-hydroxy-5 alpha-pregnan-3-one, and 5 alpha-pregnane-3 alpha, 20 alpha-diol. The major metabolite formed by the hypothalamus and pituitary was 5 alpha-DHP. In the pituitary samples, formation of 5 alpha-DHP was decreased on Days 15 and 21 of pregnancy compared to Day 1, and formation of 20 alpha-hydroxy-5 alpha-pregnan-3-one was decreased on Day 21 compared to Day 1. In the uterine samples, 3 alpha-hydroxy-5 alpha-pregnan-20-one was the major metabolite formed at all stages of pregnancy. The formation of all metabolic products of progesterone by the uterus was increased on Day 21 compared to Days 1, 8, and 15 of pregnancy. No changes in the formation of progesterone metabolites were observed in the hypothalamic samples during pregnancy. It is concluded that there are different profiles in the in vitro metabolism of [3H]progesterone by the hypothalamus, pituitary, and uterus of the rat during the course of pregnancy.

Single cell endocrinology: analysis of P-450scc activity by fluorescence detection methods.

A mechanism-based, fluorogenic probe for the cytochrome P-450scc (cholesterol side chain cleavage) enzyme, rate-limiting for the conversion of cholesterol to steroid hormones, is introduced and its application to the study of enzyme activity and regulation in single steroidogenic cells by several fluorescence detection methods is demonstrated. Reaction of the probe with P-450scc gives pregnenolone and the highly fluorescent resorufin anion. Spectroscopic changes in probe fluorescence, indicative of P-450scc activity, were monitored by steady-state fluorescence spectroscopy, flow cytometry, and microspectrofluorometry. This unique probe provides a nonradiometric indicator for real-time measurement of P-450scc activity in single living cells.

Suppression of luteal steroidogenesis by an LHRH antagonist (Nal-Lys antagonist: antide) in vitro during early pregnancy in the rat.

LHRH and its analogues are known to exert direct effects on the ovary. Herein we have described a direct inhibitory effect of an LHRH antagonist (Nal-Lys antagonist: antide) on the basal progesterone (P4) and pregnenolone (P5) production by luteal cells obtained from the day-8 pregnant rat. Luteal cells incubated with two doses of antide (10(-4) and 10(-7) M) for 24 or 48 h showed suppression of P4 production. P5 production was suppressed by both doses of antide within 12 h of incubation. Neither dose of antide interfered with P5 production when the duration of incubation was extended beyond 12 h. The 20 alpha-dihydroprogesterone yield from the luteal cells treated with these doses of antide remained unaffected. We estimated the activities of the cholesterol side-chain cleavage (P450scc) enzyme (which is a key enzyme involved in the conversion of cholesterol to P5) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) (which catalyses the conversion of P5 to P4) in the luteal cells treated with different doses of antide. Both doses of antide suppressed the activity of the P450scc enzyme after 12 h of incubation and the 3 beta-HSD content of the luteal cells after 48 h of incubation. These observations indicate that antide exerts a direct inhibitory effect at the level of the corpus luteum, that differential suppression of P5 and P4 during different periods of incubation with antide is due to a defect in either the P450scc or the 3 beta-HSD enzyme system, or both.

Alpha-particle-induced sister chromatid exchange in normal human lung fibroblasts: evidence for an extranuclear target.

We investigated the relationship between nuclear hits by alpha particles and the subsequent occurrence of sister chromatid exchanges (SCEs) in normal human diploid lung fibroblasts (HFL1). Cells were exposed to 238Pu alpha particles at doses ranging from 0.4-12.9 cGy and subsequently analyzed for SCEs. A significant increase in SCE frequency was observed even at the lowest dose examined. The extent of induction of SCEs in the HFL1 cells showed dose dependency in the very low dose range, i.e. 0.4-2.0 cGy. Thereafter, induction of SCEs was independent of dose. Based on measurements of the nuclear areas of the HFL1 cells in conjunction with target theory calculations, the lowest dose resulted in an approximately 8.6-fold increase in the percentage of cells showing excessive SCEs over the theoretically expected percentage of cells whose nuclei were calculated to be traversed by one or more alpha particles. The extent of the discrepancies between theoretically expected and experimentally observed frequencies of SCEs became progressively reduced with increasing radiation dose. We additionally determined that SCEs induced by the alpha particles have no significant dependency on the time of cell collection after exposure to a selected dose of alpha particles, thereby confirming that the differences between the theoretically predicted and observed SCE frequencies were not due to an artifact of the time of cell sampling for the SCE measurements. These results obtained with normal human cells are similar to those of other investigators who observed excessive SCEs in immortalized rodent cells beyond that which could be attributed exclusively to nuclear traversals by alpha particles. Such consistent findings point to the existence of an alternative, extranuclear target through which alpha particles cause DNA damage, as detected by SCE analysis. The existence of an extranuclear compartment as a target for alpha particles may have important implications for the susceptibility of lung cells to the DNA-damaging effects of alpha-particle exposure due to the inhalation of radon progeny.

Hormone stimulated steroid biosynthesis in granulosa cells studied with a fluorogenic probe for cytochrome P-450SCC.

The regulation of steroidogenesis by luteinizing hormone (LH) was studied in granulosa cells during follicular development using a fluorescent reporter assay based on the metabolism of a fluorescent probe specific for cytochrome P-450SCC (cholesterol side-chain cleavage enzyme). Intact granulosa cells or mitochondria were obtained from the first (F1) second (F2) and third (F3) largest preovulatory follicles of the hen ovary and incubated with the fluorogenic substrate. Metabolism of this substrate by cytochrome P-450SCC generates the highly fluorescent resorufin anion (the fluorescent reporter). In both mitochondria and intact granulosa cells, incubated with the fluorescent substrate, an increase in resorufin fluorescence was observed and the increase was greater in samples derived from F1 than in samples from F2 or F3. In cells, LH added simultaneously with the P-450SCC substrate significantly increased resorufin fluorescence above control values in a time- and dose-dependent manner up to 2-3 h after the incubation was initiated. Forskolin and 8-bromo-cAMP also stimulated metabolism of the P-450SCC substrate significantly by 15 min. When granulosa cells were preincubated with LH before exposure to the P-450SCC substrate resorufin fluorescence was significantly attenuated compared to controls (not exposed to LH in the preincubation period). The decrease in resorufin fluorescence observed when cells were pretreated with LH, may be due to the release of cholesterol from endogenous pools and its competition with the exogenous fluorogenic for the substrate P-450SCC enzyme. In granulosa cells that were preloaded with the P-450SCC substrate, the stimulatory effect of LH treatment remained constant from 30 min to 2 h after hormone addition. The results show that this fluorescent probe can be used in a rapid assay for the continuous measurement of the acute effects of hormone agonists on cholesterol conversion to pregnenolone in steroidogenic cells.

Inhibition of steroidogenesis by luteal cells of early pregnancy in the rat in response to in vitro administration of a gonadotropin-releasing hormone agonist.

Previous studies from this laboratory have demonstrated that the administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo in early or mid-pregnancy to rats induces antifertility effects by suppressing the luteal production of progesterone (P4) within 24h with a concomitant increase in luteal lipid droplets and decreases in the luteal cytochrome P450 side chain cleavage (P450scc) enzyme and its mRNA content. These observations suggest a direct inhibitory effect of GnRH-Ag on the corpus luteum. Here we demonstrate a suppressive effect of GnRH-Ag in vitro on the basal P4, pregnenolone (P5) and 20 alpha-dihydroprogesterone (20 alpha-DHP) production by luteal cells obtained during early pregnancy in rats. We further studied its effect on two key enzymes, namely P450scc and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), which participate in the conversion of cholesterol to P5 and conversion of P5 to P4, respectively. We observed that two doses of GnRH-Ag, 10(-4) and 10-7 M, suppress the basal P4 production in vitro after 12 h of incubation by luteal cells; P4 remained suppressed after 48 h of incubation. Basal P5 production was also suppressed after luteal cells were incubated for 12 h with 10(-4) M and 10(-7) M GnRH-Ag, but incubation for 48 h with GnRH-Ag failed to alter P5 production by these cells. 20 alpha-DHP production was suppressed after incubating the luteal cells with both doses of GnRH-Ag for 12 h. GnRH-Ag inhibited P450scc activity after 12 h of incubation and 3 beta-HSD protein content at all time periods measured. These results suggest that GnRH exerts a direct inhibitory effect on luteal steroidogenesis. This inhibition is due to its suppressive effect on P450scc and/or 3 beta-HSD and not due to an increase in P4 metabolites.

Rapid identification of pathogenic bacteria by single-enzyme amplified fragment length polymorphism analysis.

Despite major progress in their treatment and prevention, bacterial infections remain a significant cause of morbidity and mortality worldwide. In responding to a disease outbreak, rapid and accurate identification of the bacterial species involved is of paramount importance. Strain level discrimination is desirable to allow selection of treatment modalities, and in the case of a deliberate release, for identification of the source. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis was used to perform species and strain identification of subgroup I Bacilli, Yersinia, Staphylococci and Escherichia coli. By careful selection of AFLP primers, it was possible to obtain reproducible and sensitive identification to strain level, even within the highly monomorphic species Bacillus anthracis. SE-AFLP fragments can be analyzed using standard gel electrophoresis, and can be easily scored by visual inspection, due to the low complexity of the fingerprint obtained by this method. These features make SE-AFLP suitable for use in either field or laboratory applications.

Inhibition of normal human lung fibroblast growth by beryllium.

Inhalation of particulate beryllium (Be) and its compounds causes chronic Be disease (CBD) in a relatively small subset ( approximately 1-6%) of exposed individuals. Hallmarks of this pulmonary disease include increases in several cell types, including lung fibroblasts, that contribute to the fibrotic component of the disorder. In this regard, enhancements in cell proliferation appear to play a fundamental role in CBD development and progression. Paradoxically, however, some existing evidence suggests that Be actually has antiproliferative effects. In order to gain further information about the effects of Be on cell growth, we: (1) assessed cell proliferation and cell cycle effects of low concentrations of Be in normal human diploid fibroblasts, and (2) investigated the molecular pathway(s) by which the cell cycle disturbing effects of Be may be mediated. Treatment of human lung and skin fibroblasts with Be added in the soluble form of BeSO(4) (0.1-100 microM) caused inhibitions of their growth in culture in a concentration-dependent manner. Such growth inhibition was found to persist, even after cells were further cultured in Be(2+)-free medium. Flow cytometric analyses of cellular DNA labeled with the DNA-binding fluorochrome DAPI revealed that Be causes a G(0)-G(1)/pre-S phase arrest. Western blot analyses indicated that the Be-induced G(0)-G(1)/pre-S phase arrest involves elevations in TP53 (p53) and the cyclin-dependent kinase inhibitor CDKN1A (p21(Waf-1,Cip1)). That Be at low concentrations inhibits the growth of normal human fibroblasts suggests the possibility of the existence of abnormal cell cycle inhibitory responses to Be in individuals who are sensitive to the metal and ultimately develop CBD.

Detection of beryllium sensitivity using a flow cytometric lymphocyte proliferation test: the Immuno-Be-LPT.

Measurement of lymphocyte proliferation to detect hypersensitivity to beryllium (Be-LPT) in vitro is done presently using a method based on tritiated thymidine incorporation. Although this method is sensitive it gives no information on cell viability or responding lymphocyte subsets. We have developed reliable and simple flow cytometric assays for lymphocyte proliferation testing (Immuno-Be-LPT) by combining immunophenotyping with bromodeoxyuridine (BrdU) incorporation or DNA content using propidium iodide (PI) or 4'6'-diimidazolin-2-phenylindole (DAPI). Evaluation of beryllium-induced lymphocyte proliferation in blood cells from seven patients with chronic beryllium disease (CBD) and 120 beryllium workers by both the Bc-LPT and the Immuno-Be-LPT showed agreement between the tests. The Immuno-Bc-LPT provided additional information about the specific type of lymphocytes responding. CD4+ lymphocytes proliferated in response to beryllium in blood samples from all seven CBD individuals and CD8+ lymphocytes proliferated in six of the seven. Four beryllium workers without CBD had positive responses to beryllium primarily in the CD8+ cells. The use of the individual's own plasma supported a greater beryllium or tetanus-induced proliferation of CD4+ lymphocytes when compared to commercial human serum. The response of CD4+ lymphocytes measured in the Immuno-Be-LPT may provide a new marker for the diagnosis of CBD.

Beryllium sensitivity is linked to HLA-DP genotype.

Chronic beryllium disease (CBD) appears to arise from a combination of both exposure and genetic risk factors. A distinguishing feature of CBD is beryllium hypersensitivity, which can be measured in vitro by a lymphocyte proliferation test. The objective of this study was to determine whether certain allelic variations of the HLA-DPB1 gene, which had been observed previously in CBD, could be found in a group of individuals having beryllium hypersensitivity, but no symptoms of CBD. A flow cytometry-based Lymphocyte Proliferation Test combined with immunophenotyping (Immuno-LPT) was used to detect CD4+ and CD8+ T cell proliferation in response to in vitro stimulation with beryllium. The HLA-DPB1 haplotypes of the same individuals were determined by automated DNA sequencing. Twenty-two out of 25 beryllium-sensitive, non-CBD individuals were found to be carriers of the HLA-DPB1 gene having a substitution of a glutamic acid at position 69 in Exon 2 (Glu69), and a significantly high percentage (24%) were Glu69 homozygotes. Most of the CD4+ responders on the Immuno-LPT (10/14) carried rare, non-*0201 Glu69 DPB1 alleles; while most of the non-CD4+ responders (9/11) were common Glu69 carriers (*0201 or *0202) or non-Glu69 individuals (non-Glu69/non-Glu69). This is the first direct evidence that HLA-DP genotype is linked to a phenotypic response that occurs in beryllium sensitization in the absence of clinical CBD.

High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules.

We are developing a laser based technique for the rapid sequencing of large fragments (approximately 40 kb) of DNA based upon the detection of single, fluorescently tagged nucleotides cleaved from a single DNA fragment. We have demonstrated significant progress on several of the important steps of this technique. The projected rate of sequencing is several hundred bases per second which is orders of magnitude faster than existing methods. Once developed, this technology could be utilized by investigators for rapid sequencing of genetic material from virtually any source.

Multiple nuclear localization signals in XPG nuclease.

We report here evidence for the mechanism of nuclear localization of XPG nuclease in human cells. Several candidate nuclear localization signal (NLS) peptides have been proposed for XPG protein. We have identified XPG peptides containing functional NLS and a potential nuclear retention signal (NRS) using in situ immunofluorescene localization of transiently expressed beta-galactosidase fusion proteins. Two XPG regions with putative NLS [amino acid (AA) coordinates: NLS-B (AA 1057-1074) and NLS-C (AA 1171-1185)] were each shown to independently localize the beta-gal extensively (> 80%) to the nucleus of HeLa cells. The C-terminus peptide containing NLS-C, an NLS conserved evolutionarily between yeasts and humans, also directed sub-localization of beta-galactosidase to intranuclear foci reminiscent of native XPG protein, as well as to peri-nucleolar regions. Peptides in the putative XPG 'NLS domain' (AA approximately 1051-1185) apparently function in concert for nuclear localization and also for retention of XPG in nuclear matrix-associated foci. Evidence presented elsewhere (Park et al., 1995) indicates that the peptide containing NLS-C (AA 1146-1185) also regulates the dynamic localization of XPG in the nucleus following UV-irradiation.

Role of catechol estrogens in activation of lordosis in female rats and guinea pigs.

In a variety of experiments, we tested the effectiveness of the 2-hydroxylated estrogen in facilitating sexual receptivity. A single injection of 2-hydroxy-estradiol-17beta (2-OHE2) to ovariectomized rats or 2-hydroxy-estrone (2-OHE1) to ovariectomized guinea pigs was ineffective in priming animals for facilitation of sexual receptivity even when a subsequent injection of progesterone was administered. The only facilitatory effect of catechol estrogens on lordosis that was demonstrated in this study occurred when 2-OHE2 was injected in combination with E2 and a subsequent injection of progesterone was given to rats. These results suggest a cooperatively between catechol estrogen and E2, but they also indicate that catechol estrogens, by themselves, do not play a crucial role in mediating sexual receptivity in rodents.

New flow cytometric technologies for the 21st century.

The envelope that defines the limits within which flow cytometry was developed is being rapidly expanded. For example: detection sensitivity has been extended to single molecules, the size range of "particle" analysis now extends from DNA fragments to plankton (1,000.+ microns), cell and chromosome sorting rates are being increased dramatically by using inactivation procedures (50,000 per second versus 2,000 per second), rapid kinetic flow cytometry enables real-time analysis of molecular assembly and cell function in the sub-second time domain, the lifetime of a fluorochrome bound to a single cell can be measured with nsec precision, and classical karyotype information (cell to cell heterogeneity) can be determined in a flow based system. These frontiers have greatly expanded the range of new and exciting flow cytometric based biomedical applications. New enabling technologies have provided the means to measure DNA cleavage by the structure-specific nuclease, human Flap Endonuclease (FEN-1), in the 300 msec time frame. Phase sensitive measurements and fluorescence lifetime are proving to be major advances for understanding molecular environments that change with, for example, the process of apoptosis. The ability to detect single fluorescent molecules has been applied to the analysis of DNA fragments obtained from enzymatic digestion of lambda DNA. This technology is being used to rapidly and very accurately size DNA fragments for the human genome project. Optical chromosome selection is a faster, better, less complex approach to chromosome sorting. This method is based on the induction of specific damage to the DNA of selected chromosomes. Lastly, the miniaturization of a single cell fractionator has made it possible to perform single cell flow cytogenetics.