Mechanisms of apoptosis in chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by the accumulation of terminally differentiated, mature B cells that do not progress beyond the G1 stage of cell cycle, suggesting that these cells possess intrinsic defects in apoptosis. Treatment relies heavily on chemotherapy (primarily nucleoside analogs and glucocorticoids) that may initially be effective in patients, but ultimately give rise to refractory, untreatable disease. The purpose of this study was to determine whether key components of the apoptotic machinery were intact in CLL lymphocytes, especially in patients refractory to therapy. ^ Activation of proteases has been shown to be at the core of the apoptotic pathway and this work demonstrates that protease activation is required for glucocorticoid and nucleoside analog-induced apoptosis in CLL cells. Inhibitors of serine proteases as well as caspase inhibitors blocked induced DNA fragmentation, and a peptide inhibitor of the nuclear scaffold (NS) protease completely suppressed both induced and spontaneous apoptosis. However, the NS protease inhibitor actually promoted several pro-apoptotic events, such as caspase activation, exposure of surface phosphatidylserine, and loss of mitochondrial membrane potential. These results suggested that the NS protease may interact with the apoptotic program in CLL cells at two separate points. ^ In order to further investigate the role of the NS protease in CLL, patient isolates were treated with proteasome inhibitors because of previous results suggesting that the ISIS protease might be a β subunit of the proteasome. Proteasome inhibitors induced massive DNA fragmentation in every patient tested, even in those resistant to the effects of glucocorticoid and nucleoside analogs in vitro. Several other features of apoptosis were also promoted by the proteasome inhibitor, including mitochondrial alterations such as release of cytochrome c and drops in mitochondrial membrane potential. Proteasome inhibitor-induced apoptosis was associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in a number of systems. The NS protease inhibitor also caused a decrease in active NFκB, suggesting that the proapoptotic effects of this agent might be due to depletion of NFκB. ^ Given these findings, the role of NFκB, in conferring survival in CLL was investigated. Glucocorticoid hormone treatment was shown to cause decreases in the activity of the transcription factor, while phorbol dibutyrate, which blocks glucocorticoid-induced DNA fragmentation, was capable of upregulating NFκB. Compellingly, introduction of an undegradable form of the constitutive NFκB inhibitor, IκB, caused DNA fragmentation in several patient isolates, some of which were resistant to glucocorticoid in vitro. Transcription of anti-apoptotic proteins by NFκB was postulated to be responsible for its effects on survival, but Bcl-2 levels did not fluctuate with glucocorticoid or proteasome inhibitor treatment. ^ The in vitro values generated from these studies were organized into a database containing numbers for over 250 patients. Correlation of relevant clinical parameters revealed that levels of spontaneous apoptosis in vitro differ significantly between Rai stages. Importantly, in vitro resistance to nucleoside analogs or glucocorticoids predicted resistance to chemotherapy in vivo, and inability to achieve remission. ^

The Pneumococcal Polysaccharide Capsule and Pneumolysin Differentially Affect CXCL8 and IL-6 Release from Cells of the Upper and Lower Respiratory Tract

The polysaccharide capsule and pneumolysin toxin are major virulence factors of the human bacterial pathogen Streptococcus pneumoniae. Colonization of the nasopharynx is asymptomatic but invasion of the lungs can result in invasive pneumonia. Here we show that the capsule suppresses the release of the pro-inflammatory cytokines CXCL8 (IL-8) and IL-6 from the human pharyngeal epithelial cell line Detroit 562. Release of both cytokines was much less from human bronchial epithelial cells (iHBEC) but levels were also affected by capsule. Pneumolysin stimulates CXCL8 release from both cell lines. Suppression of CXCL8 homologue (CXCL2/MIP-2) release by the capsule was also observed in vivo during intranasal colonization of mice but was only discernable in the absence of pneumolysin. When pneumococci were administered intranasally to mice in a model of long term, stable nasopharyngeal carriage, encapsulated S. pneumoniae remained in the nasopharynx whereas the nonencapsulated pneumococci disseminated into the lungs. Pneumococcal capsule plays a role not only in protection from phagocytosis but also in modulation of the pro-inflammatory immune response in the respiratory tract.

Work values as predictors of entrepreneurial career intentions: A longitudinal analysis of gender effects.

Purpose – Work values are an important characteristic to understand gender differences in career intentions, but how gender affects the relationship between values and career intentions is not well established. The purpose of this paper is to investigate whether gender moderates the effects of work values on level and change of entrepreneurial intentions (EI). Design/methodology/approach – In total, 218 German university students were sampled regarding work values and with EI assessed three times over the course of 12 months. Data were analysed with latent growth modelling. Findings – Self‐enhancement and openness to change values predicted higher levels and conservation values lower levels of EI. Gender moderated the effects of enhancement and conservation values on change in EI. Research limitations/implications – The authors relied on self‐reported measures and the sample was restricted to university students. Future research needs to verify to what extent these results generalize to other samples and different career fields, such as science or nursing. Practical implications – The results imply that men and women are interested in an entrepreneurial career based on the same work values but that values have different effects for men and women regarding individual changes in EI. The results suggest that the prototypical work values of a career domain seem important regarding increasing the career intent for the gender that is underrepresented in that domain. Originality/value – The results enhance understanding of how gender affects the relation of work values and a specific career intention, such as entrepreneurship.

Modeling episodic fluid-release events in the ductile carbonates of the Glarus thrust

The exposed Glarus thrust displays midcrustal deformation with tens of kilometers of displacement on an ultrathin layer, the principal slip zone (PSZ). Geological observations indicate that this structure resulted from repeated stick-slip events in the presence of highly overpressured fluids. Here we show that the major characteristics of the Glarus thrust movement (localization, periodicity, and evidence of pressurized fluids) can be reconciled by the coupling of two processes, namely, shear heating and fluid release by carbonate decomposition. During this coupling, slow ductile creep deformation raises the temperature through shear heating and ultimately activates the chemical decomposition of carbonates. The subsequent release of highly overpressurized fluids forms and lubricates the PSZ, allowing a ductile fault to move tens of kilometers on millimeter-thick bands in episodic stick-slip events. This model identifies carbonate decomposition as a key process for motion on the Glarus thrust and explains the source of overpressured fluids accessing the PSZ.

The effect of extracellular nucleotides on cytokine production and phagocytosis of macrophages

Bacterial sepsis is a severe clinical condition, leading to severe sepsis, septic shock, and death. The complex pathophysiology of sepsis is not yet fully understood. Cytokines, released by immune cells such as macrophages, play an important role in the pathophysiology of sepsis. Kupffer cells are the largest population of macrophages in the body. Purinergic signaling, mediated by different nucleosides and nucleotides, and purinergic receptors, has been shown to have various effects on cytokine release, inflammatory processes and the immune system. In our work with in vitro experiments we studied the effect of extracellular nucleotides on the release of TNFα by primary murine Kupffer cells, and the effect of extracellular nucleotides on the phagocytosis of murine RAW 264.7 and human U-937 cell culture macrophages. Secretion of TNFα was measured using ELISA, phagocytosis of bio particles was measured using a plate reader phagocytosis assay and flow cytometry. Our experiments show, that extracellular LPS stimulate release of TNFα in murine Kupffer cells and that extracellular nucleotides inhibit this effect in a dose dependent matter. Our other experiments show phagocytosis of fluorescence labeled bio particles by both macrophage cell lines RAW 264.7 and U-937 in a dose dependent manner. The experiments could not show an effect of extracellular nucleotides on phagocytosis of cell culture macrophages.

Dynamics of Sodium‐22 and Cesium‐134 in Young Wheat Plants

The dynamics of the two alkali metals sodium and cesium in crop plants are relevant in an ecological context. Redistribution processes for these elements in young wheat plants were investigated in the work reported here. Two days old wheat plants (Triticum aestivum L. cv. Arina) were fed for 24 h with sodium‐22 (22Na) and cesium‐134 (134Cs) via the main root and incubated afterwards in a culture room. Cesium‐134 accumulated in newly formed parts of the main root and in the expanding leaves during the first 20 days after labeling, while 22Na accumulated transiently in these plant parts, reached a peak and declined after a few days. A high percentage of 22Na was released from the roots to the medium. Total Na in leaves also accumulated transiently, but its highest accumulation appeared later than the peak of 22Na. Therefore, the distribution and retranslocation processes differ considerably for sodium and cesium in wheat plants. Such differences must be considered for the evaluation of environmental effects (e.g., release of pollutants into the environment) on the quality of harvested cereal products.

Influence of root canal disinfectants on growth factor release from dentin

INTRODUCTION During dentinogenesis, growth factors become entrapped in the dentin matrix that can later be released by demineralization. Their effect on pulpal stem cell migration, proliferation, and differentiation could be beneficial for regenerative endodontic therapies. However, precondition for success, as for conventional root canal treatment, will be sufficient disinfection of the root canal system. Various irrigation solutions and intracanal dressings are available for clinical use. The aim of this study was 2-fold: to identify a demineralizing solution suitable for growth factor release directly from dentin and to evaluate whether commonly used disinfectants for endodontic treatment will compromise this effect. METHODS Dentin disks were prepared from extracted human teeth and treated with EDTA or citric acid at different concentrations or pH for different exposure periods. The amount of transforming growth factor-β1 (TGF-β1), fibroblast growth factor 2, and vascular endothelial growth factor were quantified via enzyme-linked immunosorbent assay and visualized by gold labeling. Subsequently, different irrigation solutions (5.25% sodium hypochloride, 0.12% chlorhexidine digluconate) and intracanal dressings (corticoid-antibiotic paste, calcium hydroxide: water-based and oil-based, triple antibiotic paste, chlorhexidine gel) were tested, and the release of TGF-β1 was measured after a subsequent conditioning step with EDTA. RESULTS Conditioning with 10% EDTA at pH 7 rendered the highest amounts of TGF-β1 among all test solutions. Fibroblast growth factor 2 and vascular endothelial growth factor were detected after EDTA conditioning at minute concentrations. Irrigation with chlorhexidine before EDTA conditioning increased TGF-β1 release; sodium hypochloride had the opposite effect. All tested intracanal dressings interfered with TGF-β1 release except water-based calcium hydroxide. CONCLUSIONS Growth factors can be released directly from dentin via EDTA conditioning. The use of disinfecting solutions or medicaments can amplify or attenuate this effect.

Influence of sample matrix on the alkaline extraction of Cr(VI) in soils and industrial materials

An accurate and efficient determination of the highly toxic Cr(VI) in solid materials is important to determine the total Cr(VI) inventory of contaminated sites and the Cr(VI) release potential from such sites into the environment. Most commonly, total Cr(VI) is extracted from solid materials following a hot alkaline extraction procedure (US EPA method 3060A) where a complete release of water-extractable and sparingly soluble Cr(VI) phase is achieved. This work presents an evaluation of matrix effects that may occur during the hot alkaline extraction and in the determination of the total Cr(VI) inventory of variably composed contaminated soils and industrial materials (cement, fly ash) and is compared to water-extractable Cr(VI) results. Method validation including multiple extractions and matrix spiking along with chemical and mineralogical characterization showed satisfying results for total Cr(VI) contents for most of the tested materials. However, unreliable results were obtained by applying method 3060A to anoxic soils due to the degradation of organic material and/or reactions with Fe2+-bearing mineral phases. In addition, in certain samples discrepant spike recoveries have to be also attributed to sample heterogeneity. Separation of possible extracted Cr(III) by applying cation-exchange cartridges prior to solution analysis further shows that under the hot alkaline extraction conditions only Cr(VI) is present in solution in measurable amounts, whereas Cr(III) gets precipitated as amorphous Cr(OH)3(am). It is concluded that prior to routine application of method 3060A to a new material type, spiking tests are recommended for the identification of matrix effects. In addition, the mass of extracted solid material should to be well adjusted to the heterogeneity of the Cr(VI) distribution in the material in question.

Isolation and Characterization of a Microorganism from Groundwater that Reduces Arsenate

This is an investigation into the microbially mediated processes involved in the transformation of arsenic. With the recent change in the Federal Maximum Contaminant Level for arsenic in drinking water, an increasing amount of resources are being devoted to understanding the mechanisms involved in the movement of arsenic. Arsenic in drinking water typically comes from natural sources, but the triggers that result in increased release of arsenic from parent material are poorly understood. Knowledge of these processes is necessary in order to make sound engineering decisions regarding drinking water management practices. Recent years have brought forth the idea that bacteria play a significant role in arsenic cycling. Groundwater is a major source of potable water in this and many other countries. To date, no reports have been made indicating the presence and activity of arsenate reducing bacteria in groundwater settings, which may increase dissolved arsenic concentrations. This research was designed to address this question and has shown that these bacteria are present in Maine groundwater. Two Maine wells were sampled in order to culture resident bacteria that are capable of dissimilatory arsenate reduction. Samples were collected using anaerobic techniques fiom wells in Northport and Green Lake. These samples were amended with specific compounds to enrich the resident population of arsenate utilizing bacteria. These cultures were monitored over time to establish rates of arsenate reduction. Cultures fiom both sites exhibited arsenate reduction in initial enrichment cultures. Isolates obtained fiom the Green Lake enrichments, however, did not reduce arsenate. This indicates either that a symbiotic relationship was required for the observed arsenate reduction or that fast-growing fermentative organisms that could survive in high arsenate media were picked in the isolation procedure. The Northport cultures exhibited continued arsenate reduction after isolation and successive transfers into fiesh media. The cultured bacteria reduced the majority of 1 a arsenate solutions in less than one week, accompanied by a corresponding oxidation of lactate. The 16s rRNA fiom the isolate was arnplifled and sequenced. The results of the DNA sequence analysis indicate that the rRNA sequence of the bacteria isolated at the Northport site is unique. This means that this strain of bacteria has not been reported before. It is in the same taxonomic subgroup as two previously described arsenate respirers. The implications of this study are significant. The fact that resident bacteria are capable of reducing arsenate has implications for water management practices. Reduction of arsenate to arsenite increases the mobility of the compound, as well as the toxicity. An understanding of the activity of these types of organisms is necessary in order to understand the contribution they are making to arsenic concentrations in drinking water. The next step in this work would be to quantitj the actual loading of dissolved arsenic present in aquifers because of these organisms.

The role of drug use in welfare to work: Evidence from Houston, Texas

The purpose of this study is to examine the prevalence of drug abuse among welfare recipients in Houston, TX and compare the work activities and employment barriers of drug abusers in order to better understand the potential effects of welfare reform for this population. Four hypotheses were tested comparing the work activities and employment barriers of drug abusers to others on welfare and the relative importance of drug abuse and employment barriers in predicting work activity. ^ This cross-sectional study examined the characteristics and work activities of 447 welfare recipients (81 drug abusers and 366 non-abusers) who were surveyed between October 1998 and April 1999 in Houston, TX. Subjects were introduced and recruited to participate in the study through a flyer, door to door visits, and peer driven recruitment/referral. ^ About 18% were found to be drug abusers, which is consistent with the national average (10–33%) among welfare recipients. Compared to others on welfare, drug abusers were less involved in work activities, and had more employment barriers. Employment barriers were found to be more predictive of welfare to work activities than drug abuse. The results suggest that alleviating employment barriers should be stressed in programs aimed at welfare recipients with drug abuse problems. ^

The impact of school year work on adolescent *development

In recent decades, work has become an increasingly common feature of adolescent life in the United States. Once assumed to be an inherently positive experience for youth, school year work has recently been associated with several adverse effects, especially as the number of hours of weekly work increases. The purpose of this dissertation was to describe the impact of school year work on adolescent development in a sample of high school students from rural South Texas, an area where economically-disadvantaged and Hispanic students are heavily represented.^ The first study described the prevalence and work circumstances of 3,565 10$\rm\sp{th}$ and 12$\rm\sp{th}$ grade students who responded to anonymous surveys conducted in regular classrooms. The overall prevalence of current work was 53%. Prevalence differed by grade, college-noncollege-bound status, and parent education. Fifty percent of employed students worked to support consumer spending.^ The second study examined the effects of four levels of work intensity on the academic, behavioral, social, mental and physical health of students. The following negative effects of intense work were reported: (1) decreased engagement in school, satisfaction with leisure time, and hours of weeknight and weekend sleep, and (2) increased health risk behaviors and psychological stress. The negative effects of intense work differed by gender, grade, ethnicity, but not by parent education.^ The third study described the prevalence of injury in the study population. A dose response effect was observed where increasing hours of weekly work were significantly related to work-related injury. The likelihood of being injured while employed in restaurant, farm/ranch, and construction work was greater than the probability of being injured while working in factory/office/skilled, yard, or retail work when compared to babysitting. Cuts, shocks/burns and sprains were the most common injuries in working teens.^ Students, parents, educators, health professionals and policymakers should continue to monitor the number of weekly hours that students work during the school year. ^

Helium isotope ratios and sedimentation rate models of ODP holes

A continuous age model for the brief climate excursion at the Paleocene-Eocene boundary has been constructed by assuming a constant flux of extraterrestrial 3He (3He[ET]) to the seafloor. 3He[ET] measurements from ODP Site 690 provide quantitative evidence for the rapid onset (of global warming and of the associated disturbance to the Earth's surficial carbon budget at this time. These observations support astronomically calibrated age models indicating extremely rapid release of isotopically light carbon, possibly from seafloor methane hydrate, as the proximal cause of the event. However, the 3He[ET] technique indicates a previously unrecognized and extreme increase in sedimentation rate coincident with the return of climate proxies to pre-event values. The 3He[ET]-based age model thus suggests a far more rapid recovery from the climatic perturbation than previously proposed or predicted on the basis of the modern carbon cycle, and so may indicate additional or accelerated mechanisms of carbon removal from the ocean-atmosphere system during this period. 3He[ET] was also measured at ODP Site 1051 to test the validity of the Site 690 chronology. Comparison of these data sets seems to require removal of several tens of kyr of sediment within the climatic excursion at Site 1051, an observation consistent with sediment structures and previous age modeling efforts. The Site 1051 age model shows a ~30 kyr period in which climate proxies return toward pre-event values, after which they remain invariant for ~80 kyr. If this rise represents the recovery interval identified at Site 690, then the 3HeET-based age models of the two sites are in good agreement. However, alternative interpretations are possible, and work on less disrupted sites is required to evaluate the reliability of the proposed new chronology of the climate excursion. Regardless of these details, this work shows that the 3HeET technique can provide useful independent evidence for the development and testing of astronomically calibrated age models.

Normal hepatic glucose production in the absence of GLUT2 reveals an alternative pathway for glucose release from hepatocytes

Glucose production by liver is a major physiological function, which is required to prevent development of hypoglycemia in the postprandial and fasted states. The mechanism of glucose release from hepatocytes has not been studied in detail but was assumed instead to depend on facilitated diffusion through the glucose transporter GLUT2. Here, we demonstrate that in the absence of GLUT2 no other transporter isoforms were overexpressed in liver and only marginally significant facilitated diffusion across the hepatocyte plasma membrane was detectable. However, the rate of hepatic glucose output was normal. This was evidenced by (i) the hyperglycemic response to i.p. glucagon injection; (ii) the in vivo measurement of glucose turnover rate; and (iii) the rate of release of neosynthesized glucose from isolated hepatocytes. These observations therefore indicated the existence of an alternative pathway for hepatic glucose output. Using a [14C]-pyruvate pulse-labeling protocol to quantitate neosynthesis and release of [14C]glucose, we demonstrated that this pathway was sensitive to low temperature (12°C). It was not inhibited by cytochalasin B nor by the intracellular traffic inhibitors brefeldin A and monensin but was blocked by progesterone, an inhibitor of cholesterol and caveolae traffic from the endoplasmic reticulum to the plasma membrane. Our observations thus demonstrate that hepatic glucose release does not require the presence of GLUT2 nor of any plasma membrane glucose facilitative diffusion mechanism. This implies the existence of an as yet unsuspected pathway for glucose release that may be based on a membrane traffic mechanism.

Omega images at the active zone may be endocytotic rather than exocytotic: Implications for the vesicle hypothesis of transmitter release

A Ca2+-dependent synaptic vesicle-recycling pathway emanating from the plasma membrane adjacent to the dense body at the active zone has been demonstrated by blocking pinch-off of recycling membrane by using the Drosophila mutant, shibire. Exposure of wild-type Drosophila synapses to low Ca2+/high Mg2+ saline is shown here to block this active zone recycling pathway at the stage in which invaginations of the plasma membrane develop adjacent to the dense body. These observations, in combination with our previous demonstration that exposure to high Ca2+ causes “docked” vesicles to accumulate in the identical location where active zone endocytosis occurs, suggest the possibility that a vesicle-recycling pathway emanating from the active zone may exist that is stimulated by exposure to elevated Ca2+, thereby causing an increase in vesicle recycling, and is suppressed by exposure to low Ca2+ saline, thereby blocking newly forming vesicles at the invagination stage. The presence of a Ca2+-dependent endocytotic pathway at the active zone opens up the following possibilities: (i) electron microscopic omega-shaped images (and their equivalent, freeze fracture dimples) observed at the active zone adjacent to the dense body could represent endocytotic images (newly forming vesicles) rather than exocytotic images; (ii) vesicles observed attached to the plasma membrane adjacent to the dense body could represent newly formed vesicles rather than vesicles “docked” for release of transmitter.

The role of the synthetic enzyme GAD65 in the control of neuronal γ-aminobutyric acid release

We have studied GABAergic synaptic transmission in retinal ganglion cells and hippocampal pyramidal cells to determine, at a cellular level, what is the effect of the targeted disruption of the gene encoding the synthetic enzyme GAD65 on the synaptic release of γ-aminobutyric acid (GABA). Neither the size nor the frequency of GABA-mediated spontaneous inhibitory postsynaptic currents (IPSCs) were reduced in retina or hippocampus in GAD65−/− mice. However, the release of GABA during sustained synaptic activation was substantially reduced. In the retina both electrical- and K+-induced increases in IPSC frequency were depressed without a change in IPSC amplitude. In the hippocampus the transient increase in the probability of inhibitory transmitter release associated with posttetanic potentiation was absent in the GAD65−/− mice. These results indicate that during and immediately after sustained stimulation the increase in the probability of transmitter release is not maintained in GAD65−/− mice. Such a finding suggests a decrease in the size or refilling kinetics of the releasable pool of vesicles, and various mechanisms are discussed that could account for such a defect.