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PubMed1998

Human immunodeficiency virus replication and genotypic resistance in blood and lymph nodes after a year of potent antiretroviral therapy.

Potent antiretroviral therapy can reduce human immunodeficiency virus (HIV) in plasma to levels below the limit of detection for up to 2 years, but the extent to which viral replication is suppressed is unknown. To search for ongoing viral replication in 10 patients on combination antiretroviral therapy for up to 1 year, the emergence of genotypic drug resistance across different compartments was studied and correlated with plasma viral RNA levels. In addition, lymph node (LN) mononuclear cells were assayed for the presence of multiply spliced RNA. Population sequencing of HIV-1 pol was done on plasma RNA, peripheral blood mononuclear cell (PBMC) RNA, PBMC DNA, LN RNA, LN DNA, and RNA from virus isolated from PBMCs or LNs. A special effort was made to obtain sequences from patients with undetectable plasma RNA, emphasizing the rapidly emerging lamivudine-associated M184V mutation. Furthermore, concordance of drug resistance mutations across compartments was investigated. No evidence for viral replication was found in patients with plasma HIV RNA levels of <20 copies/ml. In contrast, evolving genotypic drug resistance or the presence of multiply spliced RNA provided evidence for low-level replication in subjects with plasma HIV RNA levels between 20 and 400 copies/ml. All patients failing therapy showed multiple drug resistance mutations in different compartments, and multiply spliced RNA was present upon examination. Concordance of nucleotide sequences from different tissue compartments obtained concurrently from individual patients was high: 98% in the protease and 94% in the reverse transcriptase regions. These findings argue that HIV replication differs significantly between patients on potent antiretroviral therapy with low but detectable viral loads and those with undetectable viral loads.

Amino Acid Sequence↗

PubMed1999

Thymocyte-thymic epithelial cell interaction leads to high-level replication of human immunodeficiency virus exclusively in mature CD4(+) CD8(-) CD3(+) thymocytes: a critical role for tumor necrosis factor and interleukin-7.

This work aims at identifying the thymocyte subpopulation able to support human immunodeficiency virus (HIV) replication under the biological stimuli of the thymic microenvironment. In this report we demonstrate that interaction with thymic epithelial cells (TEC) induces a high-level replication of the T-tropic primary isolate HIV-1(B-LAIp) exclusively in the mature CD4(+) CD8(-) CD3(+) thymocytes. Tumor necrosis factor (TNF) and interleukin-7 (IL-7), secreted during this interaction, are critical cytokines for HIV long terminal repeat transactivation through NF-kappaB-dependent activation. TNF is the major inducer of NF-kappaB and particularly of the p50-p65 complex, whereas IL-7 acts as a cofactor by sustaining the expression of the p75 TNF receptor. The requirement for TNF is further confirmed by the observation that the inability of the intermediate CD4(+) CD8(-) CD3(-) thymocytes to replicate the virus is associated with a defect in TNF production during their interaction with TEC and correlates with the absence of nuclear NF-kappaB activity in these freshly isolated thymocytes. Addition of exogenous TNF to the intermediate thymocyte cultures induces NF-kappaB activity and is sufficient to promote HIV replication in the cocultures with TEC. The other major subpopulation expressing the CD4 receptor, namely, the double-positive (DP) CD4(+) CD8(+) CD3(+/-) thymocytes, despite the entry of the virus, do not produce a significant level of virus, presumably because they are unresponsive to TNF and IL-7. Together, these data suggest that in vivo, despite an efficient entry of the virus in all the CD4(+) subpopulations, a high viral load may be generated exclusively within the mature CD4(+) CD8(-) CD3(+) subset of thymocytes. However, under conditions of inflammatory response after infection, TNF might also be present in the intermediate thymocyte compartment, leading to efficient HIV replication in these cells.

Antigens, CD↗

PubMed1995

Polymerase chain reaction in liposomes.

BACKGROUND: Compartmentalization of biochemical reactions within a spherically closed bilayer is an important step in the molecular evolution of cells. Liposomes are the most suitable structures to model this kind of chemistry. We have used the polymerase chain reaction (PCR) to demonstrate that complex biochemical reactions such as DNA replication can be carried out inside these compartments. RESULTS: We describe the first example of DNA amplification by the PCR occurring inside liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or of a mixture of POPC and phosphatidylserine. We show that these liposomes are stable even under the high temperature conditions used for PCR. Although only a very small fraction of liposomes contains all eight different reagents together, a significant amount of DNA is produced which can be observed by polyacrylamide gel electrophoresis. CONCLUSIONS: This work shows that it is possible to carry out complex biochemical reactions within liposomes, which may be germane to the question of the origin of living cells. We have established the parameters and conditions that are critical for carrying out this complex reaction within the liposome compartment.

Animals↗

PubMed2006

Post-translational modification of delta antigen of hepatitis D virus.

The hepatitis delta virus (HDV) genome has only one open reading frame, which encodes the viral small delta antigen. After RNA editing, the same open reading frame is extended 19 amino acids at the carboxyl terminus and encodes the large delta antigen. These two viral proteins escort the HDV genome through different cellular compartments for the complicated phases of replication, transcription and, eventually, the formation of progeny virions. To orchestrate these events, the delta antigens have to take distinct cues to traffic to the right compartments and make correct molecular contacts. In eukaryotes, post-translational modification (PTM) is a major mechanism of dictating the multiple functions of a single protein. Multiple PTMs, including phosphorylation, isoprenylation, acetylation, and methylation, have been identified on hepatitis delta antigens. In this chapter we review these PTMs and discuss their functions in regulating and coordinating the life cycle of HDV.

Acetylation↗

PubMedDate not supplied

Growth potential and differentiation capacity of adult rat hepatocytes in vitro.

We have previously reported a medium that supports the continuous growth of hepatocytes without their losing replicative potential and differentiation capacity for an extended period. The medium contains four key substances in addition to fetal bovine serum, that is, epidermal growth factor, nicotinamide, ascorbic acid 2-phosphate, and dimethyl sulfoxide. When a nonparenchymal cell fraction containing small hepatocytes and nonparenchymal cells was cultured in this medium, small hepatocytes grew clonally and differentiated into cells expressing either mature hepatocyte marker proteins or biliary cell marker proteins. The growth potential of small hepatocytes was variable among the cells, the highest case being that of a single cell that produced a colony containing over 100 cells in 10 days. When a hepatocyte was allowed to divide for 105 days, it produced a colony of approximately 0.2 mm2, which contained approximately 1,700 hepatocytes, indicating that the cell divided more than 10 times. Thus, for the first time, we showed the presence of a small compartment of bipotent and highly replicative clonogenic hepatocytes in the rat adult liver in vitro.

Animals↗

PubMed2004

A survey of genes differentially expressed in subcutaneous and visceral adipose tissue in men.

Adipose tissue located within the abdominal cavity has been suggested to be functionally and metabolically distinct from that of the subcutaneous compartment. These differences could play a role in obesity-related complications. The aim of this study was to compare gene expression profiles of subcutaneous and visceral adipose tissues of 10 nondiabetic, normolipidemic obese men. Affymetrix human U133A arrays (10 arrays for subcutaneous fat samples and 10 arrays for visceral fat samples) were used. Differential gene expression was confirmed by real-time polymerase chain reaction in a subset of genes. A total of 5894 transcripts were detected in both depots in all 10 subjects, and 409 transcripts representing 347 encoded genes were differentially expressed. Of these, 131 genes were expressed at higher levels in subcutaneous adipose tissue, and 216 were expressed more abundantly in visceral fat. Differentially expressed profiles included genes of the Wnt signaling pathway, as well as CEPBA and HOX genes. In addition, genes involved in lipolytic stimuli and cytokine secretion were differentially expressed. The identification of a consistent and rather uniform pattern of differentially expressed genes between the two fat depots using multiple array replicates (10 arrays per fat compartment) generated new perspectives for future research on regional differences in adipose tissue biology.

Abdomen↗

PubMed1989

Cyclic adenosine monophosphate acts synergistically with dexamethasone to inhibit the entrance of cultured adult rat hepatocytes into S-phase: with a note on the use of nucleolar and extranucleolar [3H]-thymidine labelling patterns to determine rapid changes in the rate of onset of DNA replication.

Analogs of cyclic adenosine monophosphate (cAMP) (N6benzoyl cAMP and N6monobutyryl cAMP) as well as agents that increased the intracellular level of cAMP (glucagon and isobutylmethylxanthine) inhibited the EGF-stimulated DNA replication of adult rat hepatocytes in primary culture independently of cell density. This inhibition was strongly potentiated by the glucocorticoid dexamethasone. The effect of cAMP (and dexamethasone) was not due to toxicity, because the inhibition was reversible and the cell ultrastructure preserved. cAMP acted by decreasing the rate of transition from G1- to S-phase, the duration of G2- and S-phase of the hepatocyte cell cycle being unaffected. DNA replication started in the extranucleolar compartment of the nucleus and ended in the nucleolar compartment as described earlier for cells grown in the absence of cAMP (O.K. Vintermyr and S.O. Døskeland, J. Cell. Physiol., 1987, 132:12-21). The action of cAMP was very rapid: significant inhibition of the transition was noted 2 hr after the addition of glucagon/IBMX and half-maximal inhibition after 4 hours. The determination of extranucleolarly labelled nuclei in cells pulse-labelled with [3H]thymidine allowed precise analysis of rapid changes in the probability of transition from G1- to S-phase. The extranucleolar labelling index could also be determined in cells continuously exposed to [3H]thymidine.

Animals↗

PubMed2007

Compartmentalized self-replication: a novel method for the directed evolution of polymerases and other enzymes.

Compartmentalized self-replication (CSR) is a novel method for the directed evolution of enzymes and, in particular, polymerases. In its simplest form, CSR consists of a simple feedback loop involving a polymerase that replicates only its own encoding gene (self-replication). Self-replication occurs in discrete, spatially separate, noncommunicating compartments formed by a heat-stable water-in-oil emulsion. Compartmentalization ensures the linkage of phenotype and genotype (i.e., it ensures that each polymerase replicates only its own encoding gene to the exclusion of those in the other compartments). As a result, adaptive gains by the polymerase directly (and proportionally) translate into genetic amplification of the encoding polymerase gene. CSR has proven to be a useful strategy for the directed evolution of polymerases directly from diverse repertoires of polymerase genes. In this chapter, we describe some of the CSR protocols used successfully to evolve variants of T. aquaticus Pol I (Taq) polymerase with novel and useful properties, such as increased thermostability or resistance to the potent inhibitor, heparin, from a repertoire of randomly mutated Taq polymerase genes.

DNA Replication↗

PubMed1999

High Epstein-Barr virus serum load and elevated titers of anti-ZEBRA antibodies in patients with EBV-harboring tumor cells of Hodgkin's disease.

Hodgkin's disease is commonly associated with EBV latent infection. The incidence of EBV reactivation (active infection or EBV infection with replicative cycle) was evaluated in a series of 30 patients with untreated Hodgkin's disease (except for one case with chronic lymphocytic leukemia) by quantitation of EBV DNA and titration of anti-ZEBRA antibodies in serum samples. DNA was detected in serum (>2.5 x 10(2) genomes/ml) in 15 of 30 patients and was more frequent in Hodgkin's disease with EBV-positive Reed-Sternberg cells (10/12) than in EBV-negative cases (5/18), (P< 0.01). Of interest was the demonstration that viremia correlated well with increased titers of anti-ZEBRA IgG and/or standard serological profiles of EBV reactivation (12/15), (P < 0.05). However the lack of EBV replicative cycle in Reed-Sternberg cells (negative for ZEBRA antigen and early antigen BHLF1) suggests that the viral replication occurs in a nonneoplastic cell compartment rather than in tumor cells. The measurement of EBV DNA loads and the titration of anti-ZEBRA antibodies shed new lights on the link between activation of EBV replication and Hodgkin's disease: these serological markers together with the determination of the EBV status of the tumor suggest that replication of the viral genome occurs with a decreased efficiency of the immune system, thus allowing progression of the tumor.

Adolescent↗

PubMed1998

Theta oscillations in somata and dendrites of hippocampal pyramidal cells in vivo: activity-dependent phase-precession of action potentials.

Theta frequency field oscillation reflects synchronized synaptic potentials that entrain the discharge of neuronal populations within the approximately 100-200 ms range. The cellular-synaptic generation of theta activity in the hippocampus was investigated by intracellular recordings from the somata and dendrites of CA1 pyramidal cells in urethane-anesthetized rats. The recorded neurons were verified by intracellular injection of biocytin. Transition from non-theta to theta state was characterized by a large decrease in the input resistance of the neuron (39% in the soma), tonic somatic hyperpolarization and dendritic depolarization. The probability of pyramidal cell discharge, as measured in single cells and from a population of extracellularly recorded units, was highest at or slightly after the negative peak of the field theta recorded from the pyramidal layer. In contrast, cyclic depolarizations in dendrites corresponded to the positive phase of the pyramidal layer field theta (i.e. the hyperpolarizing phase of somatic theta). Current-induced depolarization of the dendrite triggered large amplitude slow spikes (putative Ca2+ spikes) which were phase-locked to the positive phase of field theta. In the absence of background theta, strong dendritic depolarization by current injection led to large amplitude, self-sustained oscillation in the theta frequency range. Depolarization of the neuron resulted in a voltage-dependent phase precession of the action potentials. The voltage-dependent phase-precession was replicated by a two-compartment conductance model. Using an active (bursting) dendritic compartment spike phase advancement of action potentials, relative to the somatic theta rhythm, occurred up to 360 degrees. These data indicate that distal dendritic depolarization of the pyramidal cell by the entorhinal input during theta overlaps in time with somatic hyperpolarization. As a result, most pyramidal cells are either silent or discharge with single spikes on the negative portion of local field theta (i.e., when the somatic region is least polarized). However, strong dendritic excitation may overcome perisomatic inhibition and the large depolarizing theta rhythm in the dendrites may induce spike bursts at an earlier phase of the extracellular theta cycle. The magnitude of dendritic depolarization is reflected by the timing of action potentials within the theta cycle. We hypothesize that the competition between the out-of-phase theta oscillation in the soma and dendrite is responsible for the advancement of spike discharges observed in the behaving animal.

Action Potentials↗

PubMed1995

Immunocytochemistry, autoradiography, in situ hybridization, selective stains: complementary tools for ultrastructural study of structure-function relationships in the nucleus. Applications to adenovirus-infected cells.

A significant amount of new information on structure-function relationships in nuclei of adenovirus-infected cells has accumulated during the last decade as a result of the combined use of several new cytochemical techniques. Localization of viral DNA on ultrathin sections of infected cells has been investigated at the ultrastructural level by using specific DNA staining and immunocytochemistry with monoclonal anti-DNA antibodies. Both techniques, however, concomitantly visualize cellular and viral DNA. The specific stain for DNA reveals the configuration of the DNA molecules in the different nuclear substructures, whatever their synthetic activities. The immunodetection of DNA reveals that specific antibodies strongly bind to DNA of condensed host chromatin and to both encapsidated and nonencapsidated inactive viral genomes. However, the observation of an abnormally low level of labeling over the substructures in which synthetic activities of viral genomes are known to be intense demonstrates a serious limitation of this technique for the detection of active DNA. Postembedding in situ hybridization is the most useful method for identifying with certainty the structures containing defined nucleic acid sequences. By using a biotinylated viral DNA probe, in situ hybridization provides specific identification of structures containing either viral DNA or viral RNA molecules. In addition, with appropriate pretreatment of the sections, it is possible to reveal either all the viral DNA--that is, both double- and single-stranded DNA molecules (dsDNA, ssDNA)--or more specific species such as only ssDNA or only dsDNA molecules. The replicative and transcriptional activities of viral genomes are determined by high-resolution autoradiography. Autoradiography after a short pulse incorporation of appropriate radioactive precursors by infected cells reveals the sites of cellular and viral DNA replication or transcription. A short pulse followed by chase periods of different durations reveals the progressive migration of the cellular and viral synthesized products. The in situ distribution of the viral 72 kDa DNA-binding protein, a highly phosphorylated protein which protects the viral ssDNA, is revealed either by immunocytochemistry with specific antibodies or by the bismuth staining method which stains all highly phosphorylated proteins, including both cellular and viral proteins. The combined results of all these cytochemical procedures reveal the composition and functions of some of the structures induced by adenovirus infection. They demonstrate that viral genomes engaged in replication lead to the formation of the replicative foci in which two compartments rapidly develop, one of which results from the aggregation of single strands of viral DNA and their accompanying 72 kDa protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviruses, Human↗

PubMed2002

Semiquantitative detection of JCV-DNA in peripheral blood leukocytes from HIV-1-infected patients with or without progressive multifocal leukoencephalopathy.

Progressive Multifocal Leukoencephalopathy (PML) is a severe and fatal demyelinating disease that occurs especially in HIV-infected patients. It has been suggested that JC virus (JCV) migrates in peripheral blood leukocytes from the kidney to the central nervous system where it initiates demyelination. To investigate the physiopathological role of the peripheral blood virus in the development of PML, the prevalence of JCV infection and the levels of JCV DNA load were evaluated in peripheral blood leukocytes or mononuclear cells of 10 AIDS patients at the time of onset of PML symptoms, and in 150 non-PML HIV-1-infected patients using a semiquantitative PCR and ELISA-hybridization assay. In PML-AIDS patients, 60% (6/10) were positive for JCV-DNA detection in peripheral blood cells compared with 26% (13/50) and 18% (18/100) positive for non-PML HIV-infected control patients with CD4+ T lymphocyte counts below and above 200.10(6) /l, respectively (60 vs. 26%, P = 0.06; 60 vs. 18%; P = 0.007). The prevalence of JCV infection in the peripheral blood cells taken from controls appeared to be independent of the CDC stage of infection and CD4+ T lymphocyte counts. The predictive positive value of a positive JCV DNA PCR in peripheral blood cells for the diagnosis of PML in an HIV-infected patient was 16% whereas the predictive negative value was 96%. The levels of circulating JCV DNA load, ranging from 1.69 to 2.53 log of copies per 10(6) cells, did not differ between patients at time of PML symptoms onset and controls, and appeared to be independent of the clinical and the biological status in control patients. The findings do not indicate any significant JCV genomic replication activity in peripheral blood cells at the onset of PML disease, and suggest that JCV replication markers in the systemic compartment would not be valuable for predicting the development of PML in AIDS patients.

AIDS-Related Opportunistic Infections↗

PubMed2001

Purification, crystallization, and preliminary X-ray analysis of L-A: a dsRNA yeast virus.

TheL-A virus (LAV) particle is a specialized compartment for the transcription and replication of double-stranded RNA. It is 390 A in diameter and infects yeast. The particle is formed by a capsid containing 120 copies of a 680-residue gene product arranged with T = 1 icosahedral symmetry, approximately two copies of an RNA-directed RNA polymerase, and a 4.6-kb linear, duplex RNA. LAV crystals diffracting to at least 4.5-A resolution were grown in a combination of polyethylene glycol 8000, ethylene glycol, and lithium chloride. Following crystallization the reservoir solution was replaced by a 2x concentrated reservoir solution in order for ethylene glycol to function as a cryoprotectant even though initial crystals would not grow at sufficiently high concentrations of ethylene glycol for cryoprotection. A complete data set was collected to 6-A resolution from a frozen crystal obtained with this procedure. The crystals belong to space group P2(1). The unit cell dimensions are a = 406.7 A, b = 403.3 A, c = 572.5 A, beta = 90.3 degrees with two virus particles in the unit cell. The particle orientation was determined with the rotation function and the particle center was estimated on the basis of packing considerations.

Capsid↗

PubMed1993

Epstein-Barr virus and carcinomas.

The Epstein-Barr virus, a human B lymphotropic herpes virus, is strongly associated with undifferentiated nasopharyngeal carcinoma, which is endemic in Southern China and North Africa. More recently, an association of the virus with certain other epithelial malignancies has been described, some of which are more common in Western countries. Also, the observation that oral hairy leukoplakia, an acquired immunodeficiency syndrome-related lesion of the tongue, supports Epstein-Barr virus replication in the epithelial cell compartment has further strengthened the notion that Epstein-Barr virus infection of epithelial cells may be an important phenomenon in vivo. The purpose of this article is to review the association of Epstein-Barr virus with nasopharyngeal carcinomas, to examine the evidence suggesting an association of the virus with other epithelial neoplasias and to discuss Epstein-Barr virus infection of non-neoplastic epithelial cells.

Epithelial Cells↗

PubMed2002

Type IV secretion and Brucella virulence.

The type IV secretion system, encoded by the virB region, is a key virulence factor for Brucella. The 12 genes of the region form an operon that is specifically induced by phagosome acidification in cells after phagocytosis. We speculate that the system serves to secrete unknown effector molecules, which allow Brucella to pervert the host cell endosomal pathways and to create a novel intracellular compartment in which it can replicate.

Animals↗

PubMed2006

[The life of human hair follicle revealed].

The human hair follicle is a unique appendage which results from epithelio-mesenchymal interactions initiated around the 3rd month of development. This appendage has a very complex structure, with a dermal compartment and an epithelial compartment. The dermal compartment comprises the connective tissue sheath and the dermal papilla, both of which are irrigated by microvessels. The epithelial compartment is made of highly replicating matrix cells giving rise to three concentrical domains, namely the outer root sheath, the inner root sheath and the hair shaft. The pigmentation unit, responsible for hair color, is made of fully active melanocytes located on top of the dermal papilla. Altogether a hair follicle contains more than 20 different cell types, engaged in different differentiation pathways and/or interacting with each other. This complex appendage has a unique behavior in mammals since, after a hair production phase, it involutes in place before entering a resting phase after which it renews itself under a cyclical but stochastic way, out of a double reservoir of pluripotent stem cells able to also regenerate epidermis. For yet unknown reasons, this well ordered process can be disturbed, provoking alopecia. The pigmentation unit also renews itself under a cyclical way, out of a melanocyte progenitor reservoir which progressively declines with time, provoking the hair whitening process. Finally, the shape of the hair shaft is programmed from the bulb. What makes this appendage unique and fascinating is its high degree of autonomy, its incredibly complex though stable structure, the number of different cell types interacting under an equilibrated way and its potential of regeneration. It represents a true paradigm of tissue homeostasis, exemplifying in a small living cylinder all the fundamental laws of cell-cell and tissue interactions. This life is revealed in this short synthesis.

Growth Substances↗

PubMed2005

Presence of hepatitis C virus (HCV) RNA in the genital tracts of HCV/HIV-1-coinfected women.

BACKGROUND: Hepatitis C virus (HCV)-infected women--in particular, those coinfected with human immunodeficiency virus type 1 (HIV-1)--can transmit infection to their children and sex partners. METHODS: The present study was conducted to analyze the presence of HCV RNA in cervicovaginal lavage (CVL) fluid from 71 women (58 HCV/HIV-1-coinfected women and 13 HCV-infected, HIV-1-uninfected women) enrolled in the Women's Interagency HIV Study. RESULTS: HCV RNA was detected (by a commercial polymerase chain reaction assay) in CVL fluid from 18 (29%) of the HIV-1-infected women and from none of the HIV-1-uninfected women (P<.05). Multivariate analysis revealed that risk factors for the presence of HCV RNA in CVL fluid were HCV viremia (odds ratio [OR], 16.81; P=.02) and HIV-1 RNA in CVL fluid (OR, 19.87; P=.02). This observation suggests local interactions between HIV-1 and HCV in the genital tract compartment. There was no correlation between HCV RNA in CVL fluid and CD4, CD8, or CD3 cell counts, HIV-1 RNA viremia, the number of leukocytes in CVL fluid, or HIV-1 therapy. Furthermore, in 3 of 5 analyzed patients who had a detectable CVL HCV RNA load, we found viral variants differing in the 5' untranslated region that were present neither in plasma nor in peripheral-blood mononuclear cells. CONCLUSIONS: Our observations point to the importance of the genital tract compartment, in which local HCV replication could be facilitated by local HIV-1 replication.

5' Untranslated Regions↗

PubMed1986

Two-color flow-cytometric analysis of the growth cycle of Plasmodium falciparum in vitro: identification of cell cycle compartments.

A previous study (Hare JD, Bahler DW: J Histochem Cytochem 34:215, 1986) has shown that the flow cytometric analysis of acridine-orange-stained Plasmodium falciparum growing in vitro generates a complex two-color display, regions of which correlate with the major morphological stages. In this report, four cell cycle compartments (A-D) are defined by characteristic ratios of red and green fluorescence of cells distributed throughout the erythrocytic cycle as well as by the differential effects of several metabolic inhibitors. The primary characteristic of cells in compartment A is the significant increase in red fluorescence. Inhibition of DNA synthesis by either aphidicolin or hydroxyurea causes the accumulation of cells at the interface between compartments A and B, whereas n-butyrate prevents cells in compartment A from reaching the A-B interface. Cells in compartment A display a small increase in green fluorescence which is independent of DNA synthesis but is enhanced by n-butyrate treatment. Cells in compartment B display a continued increase in red fluorescence coupled with a significant increase in green fluorescence, reflecting the onset of DNA synthesis in compartment B. The transition to compartment C is more abrupt and is associated with a marked increase in green fluorescence and little increase in red fluorescence. Compartment D is characterized by an increase in red fluorescence and a continued rise in green fluorescence. It is postulated that these discontinuities in the two-color display reflect not only changes in the rates of RNA and DNA synthesis but also decondensation of parasite chromatin in compartment A as the organism prepares for DNA synthesis, and re-condensation in compartment D as the newly replicated chromatin prepares for segregation into merozoites. The method described promises to provide a sensitive and rapid technique to study the effects of various factors on the growth cycle of the parasite.

Animals↗