A complicated pregnancy in a patient with lipodystrophic diabetes attributable to a peroxisome proliferator-activated receptor gamma (PPARG) mutation

Diabet. Med. 29, e398-e401 (2012) ABSTRACT: Background We describe an unplanned pregnancy in a 19-year-old with lipodystrophic diabetes caused by a mutation in the peroxisome proliferator-activated receptor gamma (PPARG) gene. The pregnancy was complicated by poor compliance with treatment, severe hypertriglyceridaemia and pancreatitis. Case report The patient presented at 6?weeks' gestation with an HbA(1c) of 140?mmol/mol (15%), cholesterol 8.1?mmol/l and triglycerides 20.1?mmol/l. She wished to continue the pregnancy so lipid-lowering therapy was discontinued. She was severely insulin resistant and poorly compliant with diet and medication. A continuous subcutaneous insulin infusion was required for efficient delivery of large doses of basal insulin, alongside injected mealtime boluses, (up to 300?units insulin per day). At 17?weeks' gestation she developed acute pancreatitis secondary to hypertriglyceridaemia (triglycerides >?100?mmol/l) and required plasmapheresis. Lipid-lowering therapy was reinstated in the third trimester and plasmapheresis was required repeatedly to maintain triglycerides 相似文献   

Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, independently of PPARγ in human glioma cells

Peroxisome proliferator-activated receptor γ (PPARγ) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPARγ in CGZ-induced cell death was examined. At concentrations of greater than 30 μM, CGZ, a synthetic PPARγ agonist, activated caspase-3 and induced apoptosis in T98G cells. Treatment of T98G cells with less than 30 μM CGZ effectively induced cell death after pretreatment with 30 μM of the PPARγ antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPARγ was down-regulated cells by siRNA, lower concentrations of CGZ (<30 μM) were sufficient to induce cell death, although higher concentrations of CGZ (≥30 μM) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPARγ. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPARγ in glioma cells, by down-regulating Akt activity and inducing MMP collapse.     

The Physical Foundation of Vasoocclusion in Sickle Cell Disease

The pathology of sickle cell disease arises from the occlusion of small blood vessels because of polymerization of the sickle hemoglobin within the red cells. We present measurements using a microfluidic method we have developed to determine the pressure required to eject individual red cells from a capillary-sized channel after the cell has sickled. We find that the maximum pressure is only ∼100 Pa, much smaller than typically found in the microcirculation. This explains why experiments using animal models have not observed occlusion beginning in capillaries. The magnitude of the pressure and its dependence on intracellular concentration are both well described as consequences of sickle hemoglobin polymerization acting as a Brownian ratchet. Given the recently determined stiffness of sickle hemoglobin gels, the observed obstruction seen in sickle cell disease as mediated by adherent cells can now be rationalized, and surprisingly suggests a window of maximum vulnerability during circulation of sickle cells.Human capillaries are narrower than the erythrocytes they convey. In sickle cell disease, red cells can become rigid in those capillaries, because the hemoglobin inside the red cell will aggregate into stiff polymers. This happens once the molecules deliver their oxygen, and led to the long-held view that capillary occlusion was central to the pathophysiology of the disease (1,2). This was challenged when microscopic study of animal model tissues perfused with sickle blood revealed blockages that began further downstream, in the somewhat larger venules (3–5), at the site of adherent red or white cells which diminished the vessel lumen without fully obstructing the flow. Yet no rationale has been presented for the failure of the prior assumption of capillary blockage. Microfluidic methods (6) are ideally suited to discover why cells don’t get stuck in the capillaries, yet occlude subsequent vessels, and we have constructed a system to address this question. Our measurements show that the pressure differences across capillaries in vivo can easily dislodge a cell sickled within a capillary, giving an experimental answer to the question of why sickled cells don’t stick in capillaries. It turns out that the pressure a cell can withstand is quantitatively explained by the Brownian ratchet behavior of sickle hemoglobin polymerization.We constructed single-cell channels in transparent polydimethylsiloxane, with a cross section (1.5 μm × 4 μm) that is smaller than the resting diameter of red cells (Fig. 1). These channels are much narrower than those that have been employed in other recent studies of the sickling process (7,8), and they resemble human capillaries in permitting only one cell at a time to pass through them. We used a laser photolysis method to create ligand free (deoxygenated) cells, and this requires that the hemoglobin bind CO, which can then be readily removed by strong illumination, in contrast to bound O₂ which is released with far lower efficiency than CO. The microfluidic chips were enclosed in a gas-tight chamber flushed with CO to avoid introduction of oxygen and keep the cells fully ligated before photolysis. The profiles of the channels were confirmed by microscopic observation. To confirm that liquid did not pass around the cells when they were trapped in the channels, fluorescent beads were introduced into some cell solutions. The beads did not pass the cells, nor did they approach the cell when it was occluded, verifying that no significant flow occurred around the cell when it was stuck.Open in a separate windowFigure 1An erythrocyte enters a channel (moving left to right) and is positioned in the center, where it will be photolyzed. The channel cross section is 1.5 μm × 4 μm, smaller than a resting red cell diameter.Optical measurements were carried out on a microspectrophotometer constructed on an optical table. The system employed ×32 LWD objectives (Leitz, Wetzlar, Germany), which were autofocused during collection of absorption spectra to minimize aberrations. Spectra were obtained using a series 300 camera (Photometrics, Tucson, AZ); video imaging was done with a high-speed camera (Photron, San Diego, CA). Photolysis was provided by a 2020 Argon Ion laser (Spectra Physics, Houston, TX). Sickle cells were obtained from patients at the Marian Anderson Sickle Cell Center at St. Christopher''s Hospital for Children, Philadelphia, PA by phlebotomy into EDTA-containing tubes. The blood was centrifuged at 5°C at 1200g for 4 min, and then the pellet was washed 4× with 15 volumes of buffer (120 mM NaCl, 2 mM KCl, 10 mM dibasic Na Phosphate, 7 mM monobasic Na Phosphate, 3.4 mM Na Bicarbonate, and 6 mM Dextrose) by repeated suspension and centrifugation at 30g for 4 min. This minimizes fibrinogen and platelets in the final suspension, to insure that these studies are controlled by the mechanical properties of the cells themselves.Our experiment began by parking a cell in the center of a channel (Fig. 1). The cell, its hemoglobin, and the microchannel environment all were saturated with CO. Because the thickness of the channel is known, we were able to determine the hemoglobin concentration inside the cell from its absorption spectrum (Fig. 2 A). Steady-state laser illumination then removed the CO, allowing the hemoglobin to polymerize, in which condition it remained while the laser was kept on. Removal of CO was confirmed by observing the spectral difference between COHb and deoxyHb. Photolysis of COHb generates negligible heating (9–11). During illumination, hydrostatic pressure was applied until the cell broke free.Open in a separate windowFigure 2(A) Absorption of the cell (points), fit to a standard spectrum (9). (B) Pressure to dislodge a cell sickled in the microchannel, as a function of intracellular concentration. Note that typical intracellular concentrations are ∼32 g/dL. (Line) Brownian-ratchet theory described in the text. The coefficient of friction (0.036) is within the observed range, and is the only parameter varied.The magnitude of the dislodging pressure, measured by simple height difference between input and output cell reservoirs, is shown in Fig. 2 B. The pressure needed to dislodge the cell increased with increasing intracellular Hb concentration, implying that an increased mass of polymerized hemoglobin is more difficult to dislodge. A clear concentration threshold for capture is apparent. While there is a well-known solubility below which polymers cannot form (18.5 g/dL for the 22°C of this experiment (12)), the threshold here is significantly higher.Central to explaining these observations is a Brownian ratchet mechanism (13) which derives from the metastable nature of this polymerization process. Unless disrupted, as by centrifugation, polymerization in sickle hemoglobin terminates before the thermodynamic limit of monomer solubility is reached (14,15). This arises from the fact that polymers only grow at their ends, which are easily occluded in the dense mass of polymers that form. This end obstruction leaves the system in a metastable state and fluctuations accordingly provide polymers with space into which they can incrementally grow. This Brownian ratchet has been shown to lead to dramatic fiber buckling when individual fibers are isolated in sickle cells (16). The force can be simply expressed as f = (kT/δ) ln S(c), where k is Boltzmann’s constant, T the absolute temperature, δ the net spatial elongation from addition of a single monomer, and S is the supersaturation of the solution when the metastable limit is reached, at monomer concentration c. In this calculation, c is taken as the terminal concentration, computed from our empirical finding (15) that in this metastable system the amount of polymerized hemoglobin Δ is Δ(∞) = 2/3 (c_o-c_s), rather than the expected thermodynamic limit c_o-c_s, where c_o is the initial concentration and c_s is the solubility.For determining the net force, the total number of fibers must be known, and can be calculated based on the double nucleation mechanism (17) which has been quantitatively successful in describing polymerization. The concentration of polymers [p(t)] initially grows exponentially, described by[p(t)]=(AB2J)exp(Bt),where A and B are parameters related to nucleation, and J is the polymer elongation rate, as described in Ferrone et al. (17). Because A and B are both extremely concentration-dependent (9), they will drop dramatically once monomers begin to add to polymers in any significant numbers, and thereby diminish the remaining monomer pool. Thanks to the extreme concentration dependence of the reaction, this rapidly shuts off further polymerization. This happens at approximately the 10th time (the time when the reaction has reached 1/10 of its maximum). Thus, the [p(t_1/10)] ≈ [p(∞)]. Moreover, at one-tenth of the reaction,Δ(t1/10)=12Aexp(Bt1/10)=Δ(∞)10,and thus[p(∞)]=(BJ)(Δ(∞)10)=(BJ)((co−cs)15).For computing the number of fibers, the volume of the cell was taken as 90 μm³. This calculation shows, as expected, that the number of polymers in the cell is highly concentration-dependent, and very few fibers are produced at concentrations just above solubility, but the number grows sharply as concentration rises. This is the main contribution to the threshold in holding force shown by the data.With the force per fiber, and the total number of fibers, the net force against the wall is known. With a coefficient of friction, this reveals the force that a trapped cell can withstand. If the force is divided by the cross-sectional area across which the force is applied, we get a prediction of the dislodging pressure, which can be compared to the data. For a quantitative comparison with the results, two further corrections, of order unity, were applied. Because only normal force will contribute to friction, the calculated force was determined by integrating cos θ. This integration is not over all angles (π) because of the possibility that large incidence angles of the fibers against the wall will lead to fiber runaway (18). Therefore, the integration described is taken to the runaway threshold, here ∼1 rad. Finally, it is necessary to assign a coefficient of friction. Known values span the range of 0.03–0.06 (19). We therefore selected a value within the range, 0.036, as the best match for the data. The predicted pressures match the measurements well, as the line in Fig. 2 B shows.Because the flow resistance is comparable for red cells traversing glass channels and endothelial-lined capillaries (20), we conclude that in vivo the pressures a sickled cell inside a capillary can withstand are no more than hundreds of Pa. This is significantly smaller than typical arteriovenous pressure differentials that have been measured, which range from 0.7 kPa (in hamster skin (21)) to 7.9 kPa (in rat mesentery (22)).Our measurements coupled with recent determination of the stiffness of sickle hemoglobin gels (23) provide the missing physical basis for the processes of vasoocclusion seen in ex vivo tissue and animal models of sickle cell disease, arguing that these observations indeed represent fundamental behavior of sickle cell disease. We now understand this behavior in terms of three possible outcomes, all intimately connected with kinetics:

• 1.Certain escape: A cell that does not polymerize until after passing the obstruction can reach the lungs where it reoxygenates and resets its polymerization clock.
• 2.Possible escape: A cell that polymerizes within the capillary will assume an elongated sausage shape. The forces that it can exert against the wall cannot hold it there, and it will emerge into the postcapillary venule. There it has some chance of passing a subsequent obstruction, though it might also obstruct flow were it to rotate before reaching the adherent cell, so as to present its long dimension to the reduced space it must traverse.
• 3.Certain occlusion: A cell that does not polymerize in the capillary reassumes a larger diameter as soon as it escapes. If the cell then polymerizes before it encounters a cell attached to the venule wall, this rigidified cell will not be able to squeeze past the adherent cell, because that kind of deformation takes MPa (23). This would precipitate the type of blockage that is observed. This suggests that there is a window of greatest vulnerability, toward which therapies might be addressed.

     

Disaggregating Tropical Disease Prevalence by Climatic and Vegetative Zones within Tropical West Africa

Tropical infectious disease prevalence is dependent on many socio-cultural determinants. However, rainfall and temperature frequently underlie overall prevalence, particularly for vector-borne diseases. As a result these diseases have increased prevalence in tropical as compared to temperate regions. Specific to tropical Africa, the tendency to incorrectly infer that tropical diseases are uniformly prevalent has been partially overcome with solid epidemiologic data. This finer resolution data is important in multiple contexts, including understanding risk, predictive value in disease diagnosis, and population immunity. We hypothesized that within the context of a tropical climate, vector-borne pathogen prevalence would significantly differ according to zonal differences in rainfall, temperature, relative humidity and vegetation condition. We then determined if these environmental data were predictive of pathogen prevalence. First we determined the prevalence of three major pathogens of cattle, Anaplasma marginale, Babesia bigemina and Theileria spp, in the three vegetation zones where cattle are predominantly raised in Ghana: Guinea savannah, semi-deciduous forest, and coastal savannah. The prevalence of A. marginale was 63%, 26% for Theileria spp and 2% for B. bigemina. A. marginale and Theileria spp. were significantly more prevalent in the coastal savannah as compared to either the Guinea savanna or the semi-deciduous forest, supporting acceptance of the first hypothesis. To test the predictive power of environmental variables, the data over a three year period were considered in best subsets multiple linear regression models predicting prevalence of each pathogen. Corrected Akaike Information Criteria (AICc) were assigned to the alternative models to compare their utility. Competitive models for each response were averaged using AICc weights. Rainfall was most predictive of pathogen prevalence, and EVI also contributed to A. marginale and B. bigemina prevalence. These findings support the utility of environmental data for understanding vector-borne disease epidemiology on a regional level within a tropical environment.     

Mercury-tolerant Transgenic Poplars Expressing Two Bacterial Mercury-metabolizing Genes

Mercury is one of the most toxic metals to various organisms, including humans. Genes involved in mercury metabolism have been cloned fromStaphylococcus aureus, and were modified here to be expressed in plants. Transgenic poplars containing both chimeric genes (p35S-merA andp35S-merB) were developed via two rounds of transformation usingnos-nptll andnos-hpt genes as selectable markers. Although expression levels varied among transgenic lines, tolerance to either ionic mercury or organic mercury matched well with the degree of expression revealed by northern hybridization. In culture, these trees were tolerant to 50 μM HgCl₂ and 2 μM CH₃HgCI. Variations in mercury tolerance among the transgenic lines indicates that vigorous selection is required to select the best clones for use in phytoremediation.     

Plants measure the time

All eukaryotes, including plants, and most prokaryotes have developed elaborate mechanisms to anticipate external environmental changes associated with the Earth’s rotation. These mechanisms are mediated by a circadian clock, which regulates several physiological and biochemical processes. Microarray experiments using Affymetrix chips that included about 8000 of the 27000 Arabidopsis genes have demonstrated that as much as 6% of that genome may be under the control of this clock. While our understanding of such mechanisms is lagging, molecular genetics studies of Arabidopsis have allowed us to make great progress toward identifying and characterizing components of the plant circadian clock since its first component was isolated in 1995. The generation of 24-h rhythms by this clock appears to rely on mechanisms similar to those found in other organisms. However, an entirely different set of molecular components are recruited to perform these functions in Arabidopsis. In this review, we introduce useful and powerful approaches for identifying clock-associated genes and determining how they can act together in the interlocking feedback loops that comprise this particular clock.     

Arabidopsis homologs of components of the SWR1 complex regulate flowering and plant development

The SWR1 complex (SWR1C) in yeast catalyzes the replacement of nucleosomal H2A with the H2AZ variant, which ensures full activation of underlying genes. We compared the phenotype of mutants in the homologs of SWR1C components in Arabidopsis thaliana. Mutations in Arabidopsis SWC6 (AtSWC6), SUPPRESSOR OF FRIGIDA 3 (SUF3) and PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 (PIE1), homologs of SWC6, ARP6 and SWR1, respectively, caused similar developmental defects, including leaf serration, weak apical dominance, flowers with extra petals and early flowering by reduction in expression of FLOWERING LOCUS C (FLC), a strong floral repressor. Chromatin immunoprecipitation assays showed that AtSWC6 and SUF3 bind to the proximal region of the FLC promoter, and protoplast transfection assays showed that AtSWC6 colocalizes with SUF3. Protein interaction analyses suggested the formation of a complex between PIE1, SUF3, AtSWC6 and AtSWC2. In addition, H2AZ, a substrate of SWR1C, interacts with both PIE1 and AtSWC2. Finally, knockdown of the H2AZ genes by RNA interference or artificial microRNA caused a phenotype similar to that of atswc6 or suf3. Our results strongly support the presence of an SWR1C-like complex in Arabidopsis that ensures proper development, including floral repression through full activation of FLC.