Non-exponential growth by mammalian cells in culture

The concept of exponential growth by mammalian cells in culture is based upon the apparent linearity of semilogarithmic data plots. This method of graphical analysis is known to be an unreliable test of the exponential hypothesis. We have re-examined the question of growth exponentiality using the more sensitive method of Smith plots, in which specific growth rate is plotted against either time or density on transformed graphical coordinates which linearize the mathematical expression of the growth hypothesis being tested. With exponential growth, data points fall on a horizontal straight line when specific growth rate is plotted against time or density. Using both our own and literature data, we have performed Smith plot analyses on the growth of 125 different mammalian and avian cell lines. Of these, only eleven exhibited an exponential phase. The remaining cell lines all had non-exponential growth patterns. The most common of these consisted of an initial period of growth acceleration followed by a later phase of deceleratory growth. A smaller number of lines exhibited deceleratory kinetics at all times after plating. We conclude that mammalian cell growth in culture is predominantly non-exponential, and that the apparent exponentiality of semilogarithmic data plots is usually a methodological artifact.     

O6-3-[131I]iodobenzylguanine: improved synthesis and further evaluation of a potential agent for imaging of alkylguanine-DNA alkyltransferase

O(6)-Benzylguanine derivatives with suitable radionuclides attached to the benzyl ring are potentially useful in the noninvasive imaging of the DNA repair protein, alkylguanine-DNA alkyltransferase (AGT). Previously, O(6)-3-[(131)I]iodobenzylguanine ([(131)I]IBG) was prepared using a two-step approach; we now report its synthesis in a single step by the radioiododestannylation of O(6)-3-(trimethylstannyl)benzylguanine in 85-95% radiochemical yield. The in vitro specific uptake of [(131)I]IBG in DAOY human medulloblastoma cells, in TE-671 human rhabdomyosarcoma cells and a CHO cell line transfected to express AGT was linear (r(2) = 0.9-1.0) as a function of cell density. After intravenous injection of [(131)I]IBG in athymic mice bearing TE-671 xenografts, tumor uptake was 1.38 +/- 0.34% ID/g at 0.5 h and declined at 2 and 4 h. Preadministration of O(6)-(3-iodobenzyl)guanine (IBG) at 0.5 h increased uptake not only in tumor but also in several normal tissues. Notable exceptions were thyroid (p < 0.05), lung (p <0.05) and stomach. After intratumoral injection of [(131)I]IBG in the same xenograft model, the uptake in tumors that were depleted of AGT by BG treatment (165.8 +/- 27.5% ID/g) was about 60% of that in control mice (272.4 +/- 48.2% ID/g; p < 0.05).     

Phage regulatory circuits and virulence gene expression

In many pathogenic bacteria, genes that encode virulence factors are located in the genomes of prophages. Clearly bacteriophages are important vectors for disseminating virulence genes, but, in addition, do phage regulatory circuits contribute to expression of these genes? Phages of the lambda family that have genes encoding Shiga toxin are found in certain pathogenic Escherichia coli (known as Shiga toxin producing E. coli) and the filamentous phage CTXphi, that carries genes encoding cholera toxin (CTX), is found in Vibrio cholerae. Both the lambda and CTXphi phages have repressor systems that maintain their respective prophages in a quiescent state, and in both types of prophages this repressed state is abolished when the host cell SOS response is activated. In the lambda type of prophages, only binding of the phage-encoded repressor is involved in repression and this repressor ultimately controls Shiga toxin production and/or release. In the CTXphi prophage, binding of LexA, the bacterial regulator of SOS, in addition to binding of the repressor is involved in repression; the repressor has only limited control over CTX production and has no influence on its release.     

NOX1-induced accumulation of reactive oxygen species in abdominal fat-derived mesenchymal stromal cells impinges on long-term proliferation

Mesenchymal stromal cells (MSCs) are multipotent and can be derived from different adult tissues including fat. Our repeated attempts to produce long-term proliferative cultures of rat abdominal adipose stem cells (aASCs) under normal oxygen concentration (21%) were unsuccessful. We set to examine the events controlling this cytostasis of aASCs and found that it resulted from overproduction of reactive oxygen species (ROS) that led to apoptosis. ROS overproduction in aASCs was accompanied by increased expression of NOX1 but not of NOX2 or NOX4. NOX family members are an important source of intracellular ROS pointing to NOX1 involvement in ROS accumulation. This was verified when aASCs that were grown under 3% oxygen conditions expanded long term, displaying reduced NOX1 expression and decreased ROS accumulation. NOX1 involvement in aASC cytostasis was reaffirmed when cells that were expanded under normoxic conditions in the presence of a specific NOX1 inhibitor, ML171, demonstrated reduced ROS accumulation, reduced apoptosis and long-term expansion. aASC expansion arrest was accompanied also by a weak fat differentiation and migratory potential, which was enhanced by NOX1 inhibition. This suggests an inhibitory role for NOX1-induced ROS overproduction on aASCs, their fat differentiation and migratory potential. In contrast to aASCs, similar cells produced from subcutaneous fat were easily expanded in normoxic cultures, exhibiting low ROS concentrations, a low number of apoptotic cells and improved fat differentiation and migration. Taken together, our results show, for the first time, that NOX1-induced ROS accumulation halts ASC expansion and reduces their differentiation and migratory potential under normoxic conditions. Importantly, this phenotype comprises a tissue-specific signature as it was evident in aASCs but not in subcutaneous ASCs. NOX-induced ROS accumulation and cytokine production by fat are part of the metabolic syndrome. The similarity of this phenomenon to aASC phenotype may indicate that they arise from similar molecular mechanisms.Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are produced and propagated from a wide range of adult tissues.¹ MSCs were suggested to originate from a perivascular source in various adult tissues.² Although they were originally derived mainly from bone marrow, adipose-derived MSCs (ASCs) were recently demonstrated to harbor properties similar to bone marrow-derived MSCs.³ MSCs that were expanded under ‘hypoxic'' conditions (1–5% oxygen) demonstrated improved culture expansion, differentiation and genomic stability compared with MSCs that were grown under ‘normoxic'' conditions (atmospheric oxygen level).^{4, 5, 6} Reduced reactive oxygen species (ROS) accumulation was suggested as a possible explanation to the improved expansion of MSCs under low oxygen conditions.⁷ROS are produced in cells mostly by the mitochondrial oxidative phosphorylation process or as cellular signaling molecules mainly by the family of NOX NADPH oxidases.⁸ NOX family members generate superoxides and other downstream ROS products.^{8, 9} NOX1 the first NOX2 homolog to be described^{10, 11} is highly expressed in colon epithelium, and expressed in many other tissues and cells, including fibroblasts.^{9, 12, 13} ROS overproduction leads to many destructive cellular processes, such as aging, DNA damage and apoptosis.^{14, 15} Importantly, NOX-induced ROS accumulation in fat tissue during obesity was shown to be the cause of the deregulated production of adipocytokines and the induction of the metabolic syndrome.^{16, 17, 18} Development of the metabolic syndrome was correlated with the accumulation of abdominal/visceral fat rather than the accumulation of total body fat, indicating the importance of abdominal fat in the development of this syndrome.¹⁹Here we show that the inability of abdominal rat ASCs to reach long-term culture expansion (i.e expansion arrest), their weak fat differentiation and migratory potential and their increased cytokine expression results from NOX1-induced ROS accumulation that leads to their apoptotic death. Specific inhibition of NOX1 enabled long-term propagation of abdominal ASCs (aASCs) and their improved fat differentiation and migration. The role of the tissue origin in the aASC phenotype was demonstrated as ASCs from subcutaneous fat displayed reduced ROS accumulation, long-term culture propagation, improved fat differentiation and reduced cytokine expression compared with aASCs.     

Phosphoenolpyruvate carboxykinase overexpression selectively attenuates insulin signaling and hepatic insulin sensitivity in transgenic mice

The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steady-state levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.     

The four-celled female gametophyte of Illicium (Illiciaceae; Austrobaileyales): implications for understanding the origin and early evolution of monocots, eumagnoliids,and eudicots

The recent consensus that Amborellaceae, Nymphaeales, and Austrobaileyales form the three earliest-diverging lineages of angiosperms has led comparative biologists to reconsider the origin and early developmental evolution of the angiosperm seven-celled/eight-nucleate (Polygonum-type) female gametophyte. Illicium mexicanum (Illiciaceae; Austrobaileyales) develops a four-celled/four-nucleate female gametophyte. The ontogenetic sequence of the Illicium female gametophyte is consistent with that of all other Austrobaileyales and also with all Nymphaeales and is likely a plesiomorphy of angiosperms. A character analysis based on more than 250 embryological studies indicates that a transition from an ancestrally four-celled/four-nucleate Illicium-like female gametophyte to a seven-celled/eight-nucleate female gametophyte occurred in the common ancestor of the sister group to Austrobaileyales (a clade that includes monocots, eumagnoliids, and eudicots). Comparative analysis of reconstructed ancestral female gametophyte ontogenies identifies specific early stages of ontogeny that were modified during this transition. These modifications generated two important angiosperm novelties-a set of three persistent antipodal cells and a binucleate central cell, which upon fertilization yields a triploid endosperm. Early angiosperms are anatomically quite diverse in these two features, although triploid endosperm, composed of one paternal genome and two maternal genomes, is a conserved feature of the overwhelming majority of angiosperms.     

Interferon-induced revertants of ras-transformed cells: resistance to transformation by specific oncogenes and retransformation by 5-azacytidine.

Prolonged alpha/beta interferon (IFN-alpha/beta) treatment of NIH 3T3 cells transformed by a long terminal repeat-activated Ha-ras proto-oncogene resulted in revertants that maintained a nontransformed phenotype long after IFN treatment had been discontinued. Cloned persistent revertants (PRs) produced large amounts of the ras-encoded p21 and were refractile to transformation by EJras DNA and by transforming retroviruses which carried the v-Ha-ras, v-Ki-ras, v-abl, or v-fes oncogene. Transient treatment either in vitro or in vivo with cytidine analogs that alter gene expression by inhibiting DNA methylation resulted in transformation of PR, but not of NIH 3T3, cells. The PR retransformants reverted again with IFN, suggesting that DNA methylation is involved in IFN-induced persistent reversion.     

Dietary self-selection by Heliothis zea larvae: roles of metabolic feedback and chemosensory stimuli

The effects on dietary self-selection of phagostimulation and metabolic feedback from nutrients were seperated by using two unusual carbohydrates alone or in combination: mannitol, which is nutritive but not phagostimulatory for H. zea larvae; and sorbose, which is weakly phagostimulatory but not nutritive. Last instar H. zea larvae recognized and responded positively to mannitol by metabolic feedback when it was included in a diet with casein. When offered a choice of two diets, one containing casein but lacking a utilizable carbohydrate and another lacking casein but including either mannitol or sorbose, they did not self-select for either mannitol or sorbose. However, in a similar experiment, in which the carbohydrate diet contained a mixture of mannitol and sorbose, there was a positive response, demonstrating that both metabolic feedback and phagostimulation are required to elicit self-selection.

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Résumé Les chenilles d'H. zea peuvent choisir un mélange nutritif optimal parmi deux régimes séparés, répondant à des besoins alimentaires différents. Cette aptitude à l'autosélection pose la question des voies de sélection d'un régime optimal par l'insecte. L'autosélection est probablement réalisée par un feedback métabolique des aliments; la stimulation sensorielle par les aliments pourrait jouer elle aussi un rôle. Notre objectif est de séparer ces deux facteurs et d'en déterminer l'importance relative.Des L 5 venant de muer ont pu s'alimenter sur 2 régimes nutritionnellement complets, mais dont l'un contenait des carbohydrates (sucrose) mais non des protéines, tandis que l'autre contenait des protéines (caséine) mais non des carbohydrates. L'auto-sélection caséine/sucrose a été 81/19; elle correspond à la portion idéale pour le dernier stade des chenilles H. zea établie lors d'expériences antérieures (Waldbauer et al., 1984a). Pour comparer l'importance relative du feedback métabolique et de la stimulation sensorielle, 2 carbohydrates ont été substitués au sucrose, qui est à la fois nutritif et phagostimulant: le mannitol, nutritif mais ne stimulant pas de prise de nourriture, et le sorbose, faiblement stimulant mais non nutritif. Quand elles ont le choix, les chenilles ne sélectionnent ni mannitol, ni sorbose, mais consomment presque exclusivement le régime alternatif à la caséine. Quand sorbose et mannitol sont mélangés et proposés ensemble, la réponse est positive, montrant que le feedback métabolique et la stimulation sensorielle sont impliqués tous deux dans l'autosélection alimentaire.Des expériences en deux étapes d'autosélection ont montré que le feedback métabolique est de première importance. Dans la première, les chenilles avaient le choix entre caséine seule et caséine et mannitol. Les chenilles ont consommé significativement plus d'aliment avec mannitol que sans carbohydrate, bien que les goûts des deux régimes aient du être identiques, puisque le mannitol n'a pas de goût pour H. zea. La caséine a été vraisemblablement assez phagostimulante pour maintenir suffisamment les chenilles sur l'aliment avec mannitol et ainsi permettre au feedback métabolique du mannitol d'influer sur leur maintien sur cet aliment sans en rechercher un autre.Dans d'autres expériences, les chenilles ont eu le choix entre un régime alimentaire classique avec de la caséinne et d'autres n'ayant plus leur caséine mais avec des quantités décroissantes de sucrose. Elles n'ont pas réagi en consommant plus d'aliments avec du sucrose dilué, mais en consommant plus de caséine. Cependant, si du mannitol, pourtant non phagostimulant, est ajouté à des aliments avec du sucrose dilué pour remplacer le sucrose manquant, les chenilles ne compensent plus en consommant plus d'aliments avec de la caséine, mais de la même façon qu'elles le feraient avec un aliment contenant la concentration adéquate de sucrose, montrant qu'elles perçoivent ainsi le feedback métabolique du mannitol, qui est pourtant sans goût.

     

Human and porcine early kidney precursors as a new source for transplantation

Kidney transplantation has been one of the major medical advances of the past 30 years. However, tissue availability remains a major obstacle. This can potentially be overcome by the use of undifferentiated or partially developed kidney precursor cells derived from early embryos and fetal tissue. Here, transplantation in mice reveals the earliest gestational time point at which kidney precursor cells, of both human and pig origin, differentiate into functional nephrons and not into other, non-renal professional cell types. Moreover, successful organogenesis is achieved when using the early kidney precursors, but not later-gestation kidneys. The formed, miniature kidneys are functional as evidenced by the dilute urine they produce. In addition, decreased immunogenicity of the transplants of early human and pig kidney precursors compared with adult kidney transplants is demonstrated in vivo. Our data pinpoint a window of human and pig kidney organogenesis that may be optimal for transplantation in humans.