Intracellular dynamics of Smad-mediated TGFbeta signaling

The transforming growth factor-beta (TGFbeta) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGFbeta on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFbeta action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGFbeta. In the current study, elements of the Smad component of the TGFbeta intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFbeta1, TGFbeta2, and TGFbeta3 was demonstrated. TGFbeta-initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGFbeta-inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad-mediated TGFbeta signaling system in cells of the developing murine palate.     

Microbial population dynamics in laboratory-scale solid waste bioreactors in the presence or absence of biosolids

Aims:  Decomposition of solid waste is microbially mediated, yet little is known about the associated structure and temporal changes in prokaryotic communities. Bioreactors were used to simulate landfill conditions and archaeal and bacterial community development in leachate was examined over 8 months.
Methods and Results:  Municipal solid waste (MSW) was deposited in laboratory bioreactors with or without biosolids and combustion residues (ash). The near-neutral pH fell about half a log by day 25, but recovered to ∼7·0 by day 50. Cell concentrations in bioreactors containing only MSW were significantly higher than those from co-disposal bioreactors. Archaeal and bacterial community structure was analysed by denaturing gradient gel electrophoresis targeting 16S rRNA genes, showing temporal population shifts for both domains. mcrA sequences retrieved from a co-disposal bioreactor were predominantly affiliated with the orders Methanosarcinales and Methanomicrobiales .
Conclusion:  Regardless of waste composition, microbial communities in bioreactor leachates exhibited high diversity and distinct temporal trends. The solid waste filled bioreactors allowed simulation of solid waste decomposition in landfills while also reducing the variables.
Significance and Impact of the Study:  This study advances the basic understanding of changes in microbial community structure during solid waste decomposition, which may ultimately improve the efficiency of solid waste management.     

Follicular dynamics in water buffalo superovulated in presence or absence of a dominant follicle

The ovaries of 12 buffalo were examined daily by ultrasound beginning at Day 3 of the estrous cycle, followed by superovulation between Days 10 and 13 of the cycle. The buffalo were divided into 2 groups on the basis of the presence (dominant, n = 7) or absence (nondominant, n = 5) of a dominant follicle at the start of superovulation. Daily ultrasonographic observations of the ovaries were recorded on a videotape and were used to assess the progression of both the large (dominant) follicle and the next-to-the-large (subdominant) follicle as well as the numbers of follicles in the small (4 to 6 mm), medium (7 to 10 mm), and large (>10 mm) size categories, before and during the superovulation treatment. A greater number of small size (P < 0.05) follicles was available before the start of the superovulatory treatment in the buffalo superovulated in the absence of a dominant follicle. The turnover of follicles from medium to large size classes also occurred sooner (P < 0.01), and was of higher magnitude (P < 0.01) during treatment in buffalo of the nondominant follicle group. The number of corpora lutea at palpation per rectum was higher (P < 0.05) in buffalo of the nondominant than the dominant group (4.6 +/- 0.6 vs 2.7 +/- 0.5). However, there was no significant difference among the groups in the means of serum progesterone concentration (3.6 +/- 1.3 vs 2.2 +/- 0.6 ng/ml), total number of embryos (2.0 +/- 0.6 vs 1.1 +/- 0.7), transferable embryos (1.6 +/- 0.5 vs 1.0 +/- 0.6) and unfertilized ova recovered (0.4 +/- 0.2 vs 0) on Day 6. It is concluded that in buffalo, the superovulatory response could possibly be improved by ultrasongraphic observation of the status of follicular dominance prior to treatment.     

Intracellular redox changes during apoptosis

In the current paradigm for apoptotic cell death, the activity of a family of proteases related to interleukin 1-beta converting enzyme (ICE) orchestrates the multiple downstream events (such as cell shrinkage and chromatin degradation) that comprise apoptosis. A variety of stimuli can induce this type of cell death. One of the most reproducible inducers is mild oxidative stress, although it is unclear how an oxidative stimulus activates ICE-like proteases. Oxidative modification of proteins and lipids have also been observed in cells undergoing apoptosis in response to non-oxidative stimuli, suggesting that intracellular oxidation may be a general feature of the effector phase of apoptosis. However, attempts to consistently detect a requirement for reactive oxygen species in apoptosis have been inconclusive. Recent experiments revealing that apoptosis is typically accompanied by a depletion of intracellular reduced glutathione (GSH) are also discussed. In JURKATT lymphocytes treated with antibodies to the Fas/APO-1 surface receptor, this depletion results from an accelerated efflux of the reduced thiol rather than any intracellular oxidation. As GSH is the most abundant cytosolic reductant, we propose that its efflux may provide a non-oxidative mechanism by which the reducing environment of apoptotic cells is lost. An increase in oxidative damage to proteins and lipids would then result even in the absence of an increase in the production of oxidants. This may explain the seemingly contradictory findings that increased oxidative stress is not required for apoptosis even though antioxidants often inhibit the process and peroxidised products accumulate in apoptotic cells.     

Population dynamics in cladoceran zooplankton in the presence and absence of fishes

The pattern of annual changes in zooplankton composition andabundance was investigated in Lake Mikolajskie and in a small,artificial pond. Changes in the cladoceran abundance in thelake do not accord with the competitive exclusion principle,which appears to occur in the pond. In the lake, though notin the pond, fish predation on zooplankton seems to weaken interspecificcompetition sufficiently to allow related cladocerans to achievetheir highest levels of abundance simultaneously.     

Intracellular calcium dynamics during photolysis.

The objective of this investigation was to gain a deeper understanding of the intracellular events that precede photolysis of cells. A model system, consisting of malignant melanoma cells pretreated with the calcium sensitive fluorescent dye, Fluo-3, was used to examine the intracellular calcium dynamics in single-cell photolysis experiments. Exposure of the cells to 632 nm laser light in the presence of photosensitizer, tin chlorin e6, resulted in a rise in intracellular calcium. The increase in intracellular calcium was blocked using a variety of calcium channel blocking agents, including verapamil, nifedipine, and nickel. Treatment with the channel blockers was also effective in either decreasing or eliminating cell death despite the presence of lethal doses of photosensitizer and irradiation. These results show that intracellular calcium rises prior to plasma membrane lysis, and that this early rise in intracellular calcium is necessary for membrane rupture.     

Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak

The serine/threonine kinase Akt inhibits mitochondrial cytochrome c release and apoptosis induced by a variety of proapoptotic stimuli. The antiapoptotic activity of Akt is coupled, at least in part, to its effects on cellular metabolism. Here, we provide genetic evidence that Akt is required to maintain hexokinase association with mitochondria. Targeted disruption of this association impairs the ability of growth factors and Akt to inhibit cytochrome c release and apoptosis. Targeted disruption of mitochondria-hexokinase (HK) interaction or exposure to proapoptotic stimuli that promote rapid dissociation of hexokinase from mitochondria potently induce cytochrome c release and apoptosis, even in the absence of Bax and Bak. These effects are inhibited by activated Akt, but not by Bcl-2, implying that changes in outer mitochondrial membrane (OMM) permeability leading to apoptosis can occur in the absence of Bax and Bak and that Akt inhibits these changes through maintenance of hexokinase association with mitochondria.     

Sex differences in brain developing in the presence or absence of gonads

Brain sexual differentiation results from the interaction of genetic and hormonal influences. This study used a unique agonadal mouse model to determine relative contributions of genetic and gonadal hormone influences in the differentiation of selected brain regions. SF-1 knockout (SF-1 KO) mice are born without gonads and adrenal glands and are not exposed to endogenous sex steroids during fetal/neonatal development. Consequently, male and female SF-1 KO mice are born with female external genitalia and if left on their own, die shortly after birth due to adrenal insufficiency. In this study, SF-1 KO mice were rescued by neonatal adrenal transplantation to examine their brain morphology in adult life. To determine potential brain loci that might mediate functional sex differences, we examined the area and distribution of immunoreactive calbindin and neuronal nitric oxide synthase in the preoptic area (POA) and ventromedial nucleus of the hypothalamus, two areas previously reported to be sexually dimorphic in the mammalian brain. A sex difference in the positioning of cells containing immunoreactive calbindin in a group within the POA was clearly gonad dependent based on the elimination of the sex difference in SF-1 KO mice. Several other differences in the area of ventromedial hypothalamus and in POA were maintained in male and female SF-1 KO mice, suggesting gonad-independent genetic influences on sexually dimorphic brain development.     

Mitochondrial dynamics during apoptosis

Mitochondria exist as dynamic networks that often change shape and subcellular distribution. The morphology of mitochondria within a cell is controlled by precisely regulated rates of organelle fusion and fission. Several reports have described dramatic alterations in mitochondrial morphology during the early stages of apoptosis: a fragmentation of the network and the cristae remodeling. However, whether this mitochondrial fragmentation is a required step for apoptosis is highly debated. In this review the recent progress in understanding the mechanisms governing mitochondrial morphology during apoptosis and the latest advances connecting the regulation of mitochondrial morphology with apoptosis are discussed.     

Characteristics of interhemispheric relationships in the absence or presence of a skill

A comparative analysis of the time and amplitude characteristics of the negative N200 and positive P300 components of visual evoked potentials recorded at symmetric points of the frontal, parietal, temporal, and occipital areas of the right and left hemispheres of the cerebral cortex has been performed in subjects with or without the skill of operating a computer. Subjects inexperienced in an operator’s work exhibited an interhemispheric difference in the time and amplitude characteristics of the studied components. In subjects that had the skill of operating a computer, the interhemispheric difference was little, which suggests that the cortex plays only a small role in the cerebral control of this activity.     

Censored linear regression in the presence or absence of auxiliary survival information

There has been a rising interest in better exploiting auxiliary summary information from large databases in the analysis of smaller-scale studies that collect more comprehensive patient-level information. The purpose of this paper is twofold: first, we propose a novel approach to synthesize information from both the aggregate summary statistics and the individual-level data in censored linear regression. We show that the auxiliary information amounts to a system of nonsmooth estimating equations and thus can be combined with the conventional weighted log-rank estimating equations by using the generalized method of moments (GMM) approach. The proposed methodology can be further extended to account for the potential inconsistency in information from different sources. Second, in the absence of auxiliary information, we propose to improve estimation efficiency by combining the overidentified weighted log-rank estimating equations with different weight functions via the GMM framework. To deal with the nonsmooth GMM-type objective functions, we develop an asymptotics-guided algorithm for parameter and variance estimation. We establish the asymptotic normality of the proposed GMM-type estimators. Simulation studies show that the proposed estimators can yield substantial efficiency gain over the conventional weighted log-rank estimators. The proposed methods are applied to a pancreatic cancer study for illustration.     

A structural analysis of cytoskeleton components during the execution phase of apoptosis

Summary During the execution phase of apoptosis, the cell undergoes a set of morphological changes which reveal the activation of a complex machinery leading the cell to its disruption into small, spherical, membrane-bounded fragments called apoptotic bodies. In the present study, we have focused on the implications of the micro-filament network in the early stages of the active phase of apoptosis. By using confocal microscopy, we have analysed the location of the actin microfilaments and two actin-binding proteins, -actinin and myosin, in F9 embryonal carcinoma cells undergoing apoptosis during the stages previous to their fragmentation. Our results show that these proteins locate in the centre of the disrupting cell and form a three-dimensional structure which suggests the existence of a fully functional contractile system involved in the fragmentation of the cell and the formation of apoptotic bodies.Abbreviations CI-II calpain inhibitor II - CD cytochalasin D - CSLM confocal scanning laser microscopy - EC embryonal carcinoma - FALS forward angle side scattered - FCS fetal calf serum - FITC fluorescein isothyocyanate - ISS integrated side scattered - PBS phosphate buffered saline: PI propidium iodide - RA retinoic acid - TEM transmission electron microscopy     

Double base lesions in DNA X-irradiated in the presence or absence of oxygen

Previously, double lesions in which two adjacent bases are modified were identified in DNA oligomers exposed in solution to ionizing radiation. However, the formation of such lesions in polymer DNA had not been demonstrated. Using reference oligomer containing a specific double lesion and employing liquid chromatography-mass spectrometry (LC-MS), it was possible to show directly that double lesions are formed in irradiated calf thymus DNA. The double lesion in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized to 8-oxoguanine was detected in DNA X-irradiated in oxygenated aqueous solution. The double lesion in which the methyl carbon atom of a thymine base is covalently linked to carbon at the 8-position of an adjacent guanine base was detected in DNA irradiated in a deoxygenated environment.     

Harmonic dynamics of a DNA hexamer in the absence and presence of the intercalator ethidium

Vibrational normal mode calculations are presented for a DNA hexanucleoside pentaphosphate, d(CpGpCpGpCpG)2, and for its complex with the cationic intercalator ethidium. Two intercalation sites are modeled that differ in DNA backbone torsion angles. Normal mode frequencies for the DNA fragment itself are significantly lower than those reported earlier using different force fields, but an analysis of "effective" frequencies suggests that somewhat higher frequencies are more appropriate. Intercalation leads to significant lowering of mobility for the base pairs adjacent to the drug; in this sequence, the ethidium binding affects the guanosine atoms more strongly than the cytosine atoms. Motions of the bases and the intercalator are analyzed in terms of "twist" about the local helix axis and a "tilt" angle relative to this axis, and the results are compared to fluorescence studies of ethidium-DNA complexes.     

Open-field responses of domestic chicks in the presence or absence of familiar cues

Male domestic chicks were reared in groups with distinctive cues (red crosses) placed on the walls of the home boxes from 2 to 7 d of age and their subsequent behaviour in an open field in the presence or absence of the familiar rearing cues was recorded.Ambulation, peeping, pecking, jumping and standing were all significantly higher and the latency to the first step, duration of freezing, sitting time, lying time and time spent with the eyes closed were significantly lower when the familiar rearing cues were present in the test situation. These observations are consistent with the suggestion that the birds are less fearful in the presence of familiar stimuli. The presence of the familiar cues also increased the time spent feeding and drinking. The preference effect was a specific one, for birds exposed to red crosses in early life preferred such stimuli to black circles in a subsequent two-choice situation, whereas chicks reared with black circles preferred them to red crosses. There was no generalisation of attachment from one stimulus to the other. As the chicks spent more time feeding and drinking in the presence of the familiar cues, it is assumed that fear was reduced. Thus, the results tend to confirm that the frequency of certain behaviour patterns is indicative of certain levels of fear.     

Preparation of gel-entrapped mycorrhizal inoculum in the presence or absence of Yarowia lipolytica

Two arbuscular mycorrhizal fungi (Glomus deserticola and Glomus fasciculatum) were entrapped in calcium alginate, alone or in combination with a phosphate-solubilizing yeast (Yarowia lipolytica) and, after storage for 60 days, were inoculated into soil microcosms with tomato as the test plant. The average extent of root colonization by gel-entrapped G. deserticola and G. fasciculatum were 32 ± 5.6 and 24 ± 12.1%, respectively. Improved infective potential and colonization efficiency were observed when Y. lipolytica was co-entrapped with the mycorrhizal fungi. The best value, 49%, of mycorrhizal colonization was in roots of plants inoculated with G. deserticola co-entrapped with Y. lipolytica.     

Effects of dexmedetomidine on glioma cells in the presence or absence of cisplatin

With the extensive use of dexmedetomidine (Dex) in the surgical resection of tumours for its potent sedative and analgesic properties, its effects on various properties of tumours have received increased attention. The study described herein aimed to investigate the effects of Dex on glioma cells in the presence or absence of cisplatin (DDP). Glioma U251 and U87MG cells were treated with different doses (1-50 nM) of Dex for 12 hours, then recultured in a Dex-free medium. In addition, Dex was added to U251 and U87MG cells 12 hours before or simultaneously with a 12-hour DDP treatment. Treatment with Dex increased the viability of both cell lines; this effect continued for at least 24 hours after Dex was removed. A cell invasion assay indicated that Dex inhibited cell invasion at 50 nM, but not at 10 nM. Western blot analysis showed that Dex increased the expression of phosphorylated extracellular-signal-regulated kinase 1/2, phosphoitide 3-kinase and p-AKT, but decreased ROCK protein levels at a dose of 50 nM. Intracellular Ca ²⁺ concentration was decreased by Dex in a dose-dependent manner. DDP toxicity was attenuated by 10 nM Dex added either before or with DDP treatment. However, pretreatment with 50 nM Dex instead enhanced the toxicity of DDP. Single-dose treatment with Dex did not significantly change glioma volume in nude mice, but changed the expression of Ki67 and matrix metalloproteinase-3 in the tumour. In conclusion, this study provides evidence of the regulatory effects of Dex on proliferation, invasion and chemosensitivity of glioma cells, and outlines potential mechanisms for these effects.