Quiescent Form Research Articles

Identification of a novel E2F3 product suggests a mechanism for determining specificity of repression by Rb proteins.

The tumor suppressor function of Rb is intimately related to its ability to interact with E2F and repress the transcription of E2F target genes. Here we describe a novel E2F product that specifically interacts with Rb in quiescent cells. This novel E2F, which we term E2F3b, is encoded by a unique mRNA transcribed from an intronic promoter within the E2F3 locus. The E2F3b RNA differs from the previously characterized E2F3 RNA, which we now term E2F3a, by the utilization of a unique coding exon. In contrast to the E2F3a product that is tightly regulated by cell growth, the E2F3b product is expressed equivalently in quiescent and proliferating cells. But, unlike the E2F4 and E2F5 proteins, which are also expressed in quiescent cells and form complexes with the p130 protein, the E2F3b protein associates with Rb and represents the predominant E2F-Rb complex in quiescent cells. Thus, the previously described specificity of Rb function as a transcriptional repressor in quiescent cells coincides with the association of Rb with this novel E2F product.

A surprising role for the proteasome in the regulation of herpesvirus infection

Recurrent herpes simplex virus (HSV) cold sores in response to cold winter winds or hot summer sunshine are a mere annoyance for most of us, but the ability of the virus to establish a life-long latent infection can have serious clinical consequences. During latency, the viral genome resides in neuronal cell nuclei in a transcriptionally quiescent form, except for the production of a specific set of latency-associated transcripts. Reactivation of the viral genome is characterized by lytic infection of epithelial tissues enervated by the latently infected ganglia1. The mechanisms that govern HSV-1 gene expression, latency and reactivation have intrigued virologists for years. However, studies on viruses have a habit of coming up with surprises that can be relevant to a wider spectrum of molecular and cell biologists. Two topical issues of significant general interest are the functions of the discrete nuclear structures known as ND10 or PML nuclear bodies (reviewed in Ref. 2), and regulation via specific proteasome-mediated proteolysis. Recently, it has been shown that HSV-1 associates with ND10 and then induces proteasome-mediated degradation of component proteins. In addition, proteasome activity is thought to be required for efficient viral lytic infection and reactivation from quiescence. These findings demonstrate another example of the relevance of virology to general biology.

Rectal endosonography in inflammatory bowel disease: differential diagnosis and prediction of remission.

There are few and conflicting data on EUS features of the rectal wall in Crohn's disease (CD) and ulcerative colitis (UC). The aim of our study was to determine whether rectal EUS could first differentiate between CD and UC, and secondly predict remission in CD. During a 14 month period we prospectively and blindly studied several parameters on rectal EUS (total wall thickness, mucosal appearance, submucosal thickness, number of enlarged vessels in the submucosa and number of pathological lymph nodes around the rectum and sigmoid colon) in 20 normal subjects, 26 patients with UC, 39 patients with CD, and four with infectious colitis. Comparisons were made between normal controls, patients with acute UC and those with acute CD, as well as between acute flare-ups and quiescent forms of CD and UC in the same patients. Normal subjects showed some features which were significantly different from those in CD or UC patients. A greater number of pathological lymph nodes was characteristic for acute UC, whereas the number of enlarged vessels was increased in acute CD. Quiescent CD showed a lower amount of wall thickening than acute CD. No significant alterations of the five parameters were found in quiescent UC compared to acute UC. This study suggests that EUS could be helpful in differentiating acute UC from CD, and to predict remission in CD.

Binding of Long-Term Glycated Low Density Lipoprotein and Age- Albumin by Peripheral Monocytes and Endothelial Cells

Modification of low density lipoprotein (LDL) and plasma or tissue proteins by non-enzymatic glycation culminating in the formation of advanced glycation endproducts (AGEs) is one of the essential pathomechanisms leading to diabetes-associated long-term complications. We compared binding of glycated, glycoxidated and oxidated LDL by peripheral monocytes in activated and quiescent form. Interaction via specific receptors was different for glycated as compared to (glyc)oxidated LDL-modifications. In addition, binding of glycated LDL to quiescent and activated human umbilical vein endothelial cells was studied. In patients with insulin-dependent diabetes mellitus (IDDM), AGE-binding was significantly increased as compared to healthy individuals. Specific and non-specific monocyte binding mechanisms were detected, and both were significantly increased in IDDM patients. Specific and non-specific binding strategies possibly act in concert to eliminate circulating AGEs, which are instrumental in the development and progress of microangiopathic and macroangiopathic complications of diabetes mellitus.

Immunochemical Determination of DNA in Immune Complexes Present in the Circulation of Patients with Systemic Lupus Erythematosus

The presence of DNA antigen in circulating immune complexes formed with anti-DNA antibodies of IgG class was determined in sera of 138 SLE patients and 23 healthy donors, employing monoclonal anti-dsDNA antibodies in a quantitative dot blot assay. About half of the SLE patients had elevated amounts of DNA antigen in the immune complexes (presumably as nucleosomes). The number of patients with the SLE manifestations was not higher in the group with the high amount of DNA in immune complexes. Elevated levels of DNA in immune complexes was found only in sera of SLE patients with the active, as well as quiescent form, of the disease and not in sera of healthy donors. The presence of increased amounts of DNA antigen in circulating immune complexes could indicate the presence of SLE pathology even if no manifestations of SLE are found. The level of the circulating anti-DNA antibodies was not correlated with the amount of DNA in immune complexes.

Advances in soil microbial ecology and the biodiversity.

Recent studies on the colony formation of soil bacteria opened the way to categorize soil bacteria into colony forming curve (CFC) groups of different growth rates. A bacterial culture collection comprising organisms from every CFC group is called an ecocollection (EC). Outlines of ECs of paddy soil 1992 and grassland soil 1987 and 1992 were described. Phylogenetic studies by 16S rDNA sequencing showed a great diversity of culture strains of the ecocollections. A set of alternative concepts was proposed; the active and the quiescent forms of bacterial cells in soil. The former is able to be cultivated and thus counted by the plate method, while the latter is not unless it transforms into the former. Based on the results several points required for extensive cataloguing of soil bacteria were noted.

Potato blackleg: Epidemiology, host-pathogen interaction and control

Blackleg is caused byErwinia carotovora subsp.atroseptica (Eca) andE. chrysanthemi (Echr) in cool and hot climates respectively. The bacteria are opportunistic pathogens and rely on their strong pectolytic character to infect plants when conditions favor their multiplication. Blackleg is a seedborne disease and the bacteria can survive in a quiescent form in lenticels and wounds during storage. The contaminated mother tuber and not the blackleg plant is the main source of progeny tuber contamination. Other sources of the pathogen are airborne (insects and aerosols) erwinias deposited on leaves and from there to the soil and progeny tubers, and erwinias in rotting tubers smeared into wounds incurred during mechanical crop handling. Most seed tubers are contaminated but blackleg incidence is related to seed contamination level modulated by soil water status. Competitiveness of the erwinias in the rotting mother tuber is affected by temperature, Eca is favored at <25°C and Echr at higher temperatures. The ubiquitousE. carotovora subsp.carotovora apparently fails to compete successfully with the other erwinias and saprophytic pectolytic bacteria in mother tubers and therefore does not cause blackleg. Disease control measures are based on avoiding tuber contamination by cultural means (early harvesting), reducing tuber contamination level (dry storage and hot water treatment) and planting ‘clean’ seed identified by quantifying tuber contamination rather than by visual crop inspection. Finally, recently identified highly resistant, even under anaerobic conditions, wildSolanum spp. could be used to breed for resistant cultivars by conventional methods or by genetic engineering.

Preliminary study on vertical distribution of microorganisms in the Bannock Basin waters (Eastern Mediterranean Sea)

Preliminary study on vertical distribution of microorganisms in the Bannock Basin waters (Eastern Mediterranean Sea)

PREDATORY BEHAVIOR AND PREY-STAGE PREFERENCES OF STIGMAEID AND PHYTOSEIID MITES AND THEIR POTENTIAL COMPATIBILITY IN BIOLOGICAL CONTROL

AbstractAlthough the role of phytoseiid mites as predators of the European red mite, Panonychus ulmi (Koch), on apple is well established, the role of another family of European red mite predators, the stigmaeids, is not as clearly understood. We compared predatory behavior and prey-stage preferences of the stigmaeid Zetzellia mali (Ewing) and the phytoseiid Typhlodromus caudiglans (Schuster) in the laboratory. The only predator–predator interaction we found to be potentially important was consumption of phytoseiid eggs by stigmaeids. Given a choice of equal numbers of both European red mite eggs and phytoseiid eggs, Z. mali consumed 38% phytoseiid eggs. Eggs and quiescent larval stages of European red mite were preferred by Z. mali, but active larval stages also were consumed. By contrast T. caudiglans preferred active forms to quiescent forms and eggs, and was able to consume adult forms. Although Z. mali was found to be slower, less active, and less voracious than T. caudiglans, Z. mali produced more eggs for a given number of prey consumed than did T. caudiglans. These behavioral and prey-stage preference differences should enable phytoseiids and stigmaeids to be compatible in the short term (within one generation).

The mechanism of cytosine arabinoside toxicity on quiescent astrocytes in vitro appears to be analogous to in vivo brain injury

Neuronal cultures derived from the septal diagonal band region of the embryonic rat brain and grown in a chemically defined medium contained a very small number of contaminating astroglial cells. During the first week in culture, these cells were well dispersed in the form of a single isolated cell with fine fibrous branched processes. Treatment with 4 μM cytosine arabinoside for 24 h failed to kill these astrocytes (most probably present in quiescent form), as judged by glutamine synthetase activity and glial fibrillary acidic protein-positive cell count. On the other hand, the exposure of cultures to cytosine arabinoside resulted in a marked increase in choline acetyltransferase enzyme activity. The overall results, together with our previous findings, are consistent with the proposal that a brief exposure to a relatively low concentration of cytosine arabinoside induces quiescent astrocytes to produce a large quantity of a neurotrophic factor that is involved in the regulation of cholinergic cells.

Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells.

The site of the reaction between plasminogen activators and plasminogen activator inhibitor 1 (PAI-1) was investigated in cultures of human umbilical vein endothelial cells. In conditioned medium from endothelial cells, two forms of a plasminogen activator-specific inhibitor can be demonstrated: an active form that readily binds to and inhibits plasminogen activators and an immunologically related quiescent form which has no anti-activator activity but which can be activated by denaturation. In conditioned medium, only a few percent of PAI-1 is the active form. However, the addition of increasing concentrations of tissue-type plasminogen activator (t-PA) or urokinase to confluent endothelial cells produced a saturable (3.0 pmol/5 x 10(5) cells), dose-dependent increase of the activator-PAI-1 complex in the conditioned medium even in the presence of actinomycin D or cycloheximide. This resulted also in a dose-dependent decrease of the residual PAI activity measured by reverse fibrin autography both in the conditioned medium and cell extracts. Short-time exposure of endothelial cells to a large amount of t-PA caused almost complete depletion of all cell-associated PAI activity. Although there was no detectable PAI activity even after activation of PAI by denaturants or antigen in the culture medium at 4 degrees C without the addition of t-PA, the addition of t-PA at 4 degrees C not only resulted in the formation of 70% of the amount of the t-PA.PAI complex in conditioned medium at 37 degrees C, but also induced PAI-1 antigen in a time and dose-dependent manner in the conditioned medium. Moreover, 125I-labeled t-PA immobilized on Sepharose added directly to endothelial cells formed a complex with PAI-1 in a dose-dependent manner. On the other hand, no detectable complex was formed with PAI-1 when Sepharose-immobilized 125I-labeled t-PA was added to endothelial cells under conditions in which the added t-PA could not contact the cells directly but other proteins could pass freely by the use of a Transwell. All these results suggest that a "storage pool" on the surface of endothelial cells or the extracellular matrix produced by endothelial cells contains almost all the active PAI-1, and reaction between PA and PAI-1 mainly occurs on the endothelial cell membranes, resulting in a decrease of the conversion of active PAI-1 to the quiescent form.

Phosphorylation of polyomavirus large T antigen: effects of viral mutations and cell growth state.

Phosphorylation is responsible for the shift in electrophoretic mobility of polyomavirus large T antigen observed in pulse-chase or continuous-labeling experiments. Phosphorylated forms migrated more slowly than newly synthesized [35S]methionine large T antigen, and alkaline phosphatase treatment reversed the mobility shift. Analysis of phosphopeptides with Staphylococcus aureus V8 protease showed that large T antigen forms of intermediate mobility were enriched in peptides 1 to 4, 8, and 9, while the slower migrating species had all nine phosphopeptides, including peptides 5 and 7. The phosphorylations represented by phosphopeptides 5 and 7 were of particular interest. These phosphopeptides were entirely lacking in large T antigen from tsa mutants such as ts616 labeled at the nonpermissive temperature. Also, the phosphorylation of peptides 5 and 7 depends on the growth state of the cell. Early in infection of quiescent cells intermediate mobility forms of large T antigen with little or no phosphorylation, particularly of peptides 5 and 7, were seen, whereas peptides 5 and 7 were well represented at the same time in patterns from growing cells. Later in infection of growth-arrested cells, these phosphorylations were observed, suggesting that infection stimulates the relevant kinase. Because large T antigen of hrt mutants, which lack middle and small T antigens, showed phosphorylation of peptides 5 and 7, large T antigen was apparently responsible for the stimulation. Because some differences in the distribution of phosphopeptides were noted between hrt mutants and the wild type, middle T antigen, small T antigen, or both may play a modulating role in large T antigen phosphorylation.

Study of the properties of MgATP2--induced stationary bends in demembranated sea urchin sperm.

Methods of demembranation and reactivation of Lytechinus pictus sperm were developed that result in non-motile sperm which take on a stable bend of about 3.5 radians at the proximal end of the cell. The middle and distal portion of the flagellum is relatively straight or slightly bent in the same direction forming a somewhat "C" shaped sperm cell. In these studies, we refer to this characteristic shape as the quiescent form, and as opposed to "rigor wave" sperm, the quiescent form is induced and maintained by a relatively high concentration of MgATP2- (greater than 0.2 mM). Other conditions important to the production and maintenance of the quiescent form in demembranated sperm include: starting with concentrated, undiluted sperm, maintaining low Ca++ in the demembranation buffer, using a minimum of 0.2 mM MgATP2- and pH of 7.9-8.1 in the reactivation buffer. Deviation from some of these conditions results in a dramatic increase in motile, asymmetrically beating sperm. Addition of 0.4 mM CaCl2 to the reactivation buffer increased the proximal bend angle to 5 radians. The induction and maintenance of the stationary bend is mediated by dynein activity: "rigor wave" sperm were transformed to the quiescent form upon 0.2 mM ATP addition; micromolar vanadate abolished the quiescent form by "relaxation" of the proximal bend; and the vanadate relaxed sperm were restored to quiescent form by catechol. Importantly, 20 microM cAMP activated motility of the otherwise quiescent-form sperm. Quiescent-form, demembranated sperm were also activated by mild trypsin digestion. These and other data suggest that the quiescent-form sperm are trapped at the end of the principal bend, and these data are consistent with the proposal that the single stationary bend results from asymmetry of active microtubule sliding [Gibbons and Gibbons, (1980): J. Cell Biol. 84:13-27].

Glyoxylate metabolism and adaptation of Mycobacterium tuberculosis to survival under anaerobic conditions.

Tuberculosis is characterized by periods in which the disease may be quiescent or even clinically inapparent, but in which tubercle bacilli persist and retain the potential to reactivate the disease. The present study was carried out in pursuit of an in vitro model which might contribute to the understanding of the physiology of nonreplicating persisters, with oxygen limitation used as the means of inducing this state. When actively growing aerated cultures of Mycobacterium tuberculosis were suddenly placed under anaerobic conditions the bacilli died rapidly, with a half-life of 10 h. When the bacilli were grown in liquid medium without agitation, they adapted to the microaerophilic conditions encountered in the sediment; the adapted bacilli in the sediment did not replicate there but were tolerant of anaerobiosis, exhibiting a half-life of 116 h. Among the early events associated with the adaptation were the synthesis of an antigen designated URB, the function of which is not known, and a fourfold increase in isocitrate lyase activity. The bacilli later exhibited a 10-fold increase in synthesis of a glycine dehydrogenase that catalyzes the reductive amination of glyoxylate, concomitantly oxidizing NADH to NAD. Specific activities of other enzymes studied were either not affected or moderately diminished in the sedimented bacilli. It is proposed that the glyoxylate synthesis in this model serves mainly to provide a substrate for the regeneration of NAD that may be required for the orderly completion of the final cycle of bacillary replication before oxygen limitation stops growth completely. This orderly shutdown is essential to continued survival of M. tuberculosis in a quiescent form.

Epidemiology of Chronic Congenital and Perinatal Infections of Man

Rubella vims, cytomegaloviruses, herpes simplex, hepatitis B virus, and Toxoplasma gondii all commonly produce acute disease in the pregnant mother and can result in insidious disease in the infected baby which may not be manifest for years after delivery. Some of these infections may be truly chronic, in that there is continuing replication of the organism; some may be latent with periodic reactivations; and some may be persistent in some other quiescent form capable of recrudescent activity. Widespread application of laboratory detection techniques and longitudinal clinical studies have permitted more precise definition of the public health significance of these infections.

Temporal fluctuations in the luminosity of diffuse aurora

Using low light level television observations of the diffuse aurora, frequency spectra have been derived from time series data obtained by digitizing segments of a recorded video signal. The results suggest that temporal fluctuations having periods in the range 2–40 s are common to both quiescent and pulsating forms of the diffuse aurora and are the dominant fluctuation period. A representation of the data in the form of dynamic spectra, which shows the evolution of the temporal fluctuations at intervals of 20–30 s, has been derived by using maximum entropy spectral analysis. In one of the examples the 2 to 4‐Hz modulation often observed during pulsating aurora is evident in the dynamic spectrum.

Auroral spirals, 1. Observations

Auroral spirals of 20- to 1300-km diameter are a common part of the active auroral display. They appear most frequently in the evening and midnight sectors, where they appear to be produced as a consequence of magnetic field line distortion caused by field-aligned currents. More quiescent spiral forms exhibiting somewhat different characteristics occur during quiet times in the polar cap near the dawn portion of the auroral oval. (AIP)

REGENERATION OF THE CAP OF PRIMARY ROOTS OF ZEA MAYS

SummaryThe entire cap can be removed from the apex of the primary root of Zea mays. When this is done the nuclei of cells of the quiescent centre are stimulated to synthesize DNA and subsequently enter mitosis; the labelling and mitotic indices reach maximum values 12 and 18 h, respectively, after decapping. Division of cells of the quiescent centre and their descendants leads to the regeneration of a new cap which is completed about 3‐4 days after decapping. At this time a new quiescent centre is also established. The development of a new cap is a consequence of the changes in the polarity of growth and division of cells at the apex. Quantitative data are presented of the changes in rates and planes of division of the cells involved in cap regeneration. The data suggest that cells of the quiescent centre, stimulated to divide by de‐capping, complete about four mitotic cycles before a new quiescent centre forms. Experiments in which decapped roots were grown in a solution of colchicine, to induce polyploid nuclei, also showed that cells of the quiescent centre have the potential to enter four or five successive mitotic cycles as, in the quiescent centre of roots so treated, nuclei with up to 64C DNA content were found after 4 days.Mature cells of the cap of intact roots contain nuclei with an 8‐16C DNA content. In regenerating caps nuclei with an 8C DNA content were frequent on the fourth day after decapping. The pattern of [3H]glucose incorporation, as judged by quantitative autoradiography, by regenerating caps was compared with that by normal caps. On the fourth day of cap regeneration the patterns were similar. These two sets of results suggest that differentiation of the regenerating cap proceeds normally at the levels of both nucleus and cytoplasm.It is suggested that the initials of the cap collumella constrain the growth of the cells that constitute the quiescent centre and this in some way slows their rate of mitosis. Removing the cap relieves the constraint to cell growth and allows cell division, thus providing the trigger for regeneration. The changes in the pattern of cells in the regenerating apex, that accompany the reorganization of a new cap and quiescent centre, are explained in terms of the stresses that the cells impose upon each other.

The fine structure and method of feeding of the tissue parasitizing stages of Histomonas meleagridis.

The structure, method of feeding and relationship with the host cells of the tissue parasitizing stages ofHistomonas meleagridishave been studied by means of light microscopy, cytochemistry and electron microscopy. The invasive stage is amoeboid, lacks flagella but has what appears to be remnants of the flagella apparatus, and feeds by phagocytosis. The vegetative stage is a round quiescent form. It apparently feeds by secreting proteolytic enzymes, which carry out extra-corporeal digestion of the host's tissues, and then taking up small particles of disrupted host cells by means of pinocytosis and probably by diffusion. The vegetative stage also contains remnants of flagella apparatus. Neither the invasive stage nor the vegetative stage contains mitochondria and succinic dehydrogenase is absent. It is concluded that there is no resistant stage.The reaction of the host's tissues to the parasites has been briefly described.The authors are grateful to Dr C. C. D. Shute for permission to use the electron microscope, to Mrs B. Fisher and Mr M. Shirley for technical assistance and to Mr P. Rogers for assistance with the photography.

FINE‐STRUCTURAL CHANGES IN THE CYTOPLASM OF GERMINATING POLLEN

Quiescent and germinated pollen grains of Parkinsonia aculeata, Hymenocallis occidentalis, Hippeastrum belladonna, and Ranunculus macranthus were studied electron microscopically following KMnO4‐fixation, and comparisons between the 2 developmental stages were made. In the process of germination, mitochondrial structure characteristically remains unchanged from the quiescent stage form, while other organelles undergo significant modifications. In all 4 species, the Golgi apparatus becomes secretory, producing large vesicles which migrate to the cell surface and contribute substrate(s) to the forming pollen tube wall. In Hymenocallis and Hippeastrum, plastids initiate starch storage only after germination begins; this appears to involve an increase in plastid lamellae. Vacuoles arise in germinating pollen of P. aculeata from elements of the ER, while in the other 3 species vacuoles develop from “prevacuolar bodies.” Generative cell structure, vegetative nuclear structure and a unique plasma membrane‐ER relationship in H. belladonna are briefly described.