Lessons from development: A role for asymmetric stem cell division in cancer

Asymmetric stem cell division has emerged as a major regulatory mechanism for physiologic control of stem cell numbers. Reinvigoration of the cancer stem cell theory suggests that tumorigenesis may be regulated by maintaining the balance between asymmetric and symmetric cell division. Therefore, mutations affecting this balance could result in aberrant expansion of stem cells. Although a number of molecules have been implicated in regulation of asymmetric stem cell division, here, we highlight known tumor suppressors with established roles in this process. While a subset of these tumor suppressors were originally defined in developmental contexts, recent investigations reveal they are also lost or mutated in human cancers. Mutations in tumor suppressors involved in asymmetric stem cell division provide mechanisms by which cancer stem cells can hyperproliferate and offer an intriguing new focus for understanding cancer biology. Our discussion of this emerging research area derives insight from a frontier area of basic science and links these discoveries to human tumorigenesis. This highlights an important new focus for understanding the mechanism underlying expansion of cancer stem cells in driving tumorigenesis.     

Biochemical and physiological characterization of the efrotomycin fermentation

Summary An efrotomycin fermentation was characterized through physical, chemical and biochemical studies. Growth of the actinomycete,Nocardia lactamdurans occurred during the first 50 h of the fermentation cycle at the expense of glucose, protein, and triglycerides. The initiation of efrotomycin biosynthesis was observed when glucose dropped to a low concentration. Upon glucose depletion, cell growth ceased and a switch in the respiratory quotient occurred. Efrotomycin biosynthesis was supported by the utilization of soybean oil and starch. Analysis of triglyceride metabolism showed that no diglycerides or monoglycerides accumulated during the fermentation. The activity of extracellular enzymes (lipase, protease, and amylase) increase during the cell growth phase and decreased significantly after 150 h. The concentrations of DNA, tetrahydro-vitamin K₂ (a membrane component), and free amino acids in the supernatant increased dramatically late in the fermentation cycle (225 h), indicating massive cell lysis. During this same time period, a reduction in cellular respiratory activity and efrotomycin biosynthesis were observed.     

Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.     

Genetic variation within a fragmented population of swietenia humilis zucc

With large tracts of once continuous forest now modified by human use to leave reduced and highly fragmented stands of trees, the determination of the genetic consequences of forest fragmentation is a priority for ascertaining the conservation value of resultant stands, and in formulating sustainable management strategies. The levels and distribution of genetic diversity over 10 microsatellite loci were investigated within a fragmented population of the neotropical tree Swietenia humilis Zucc. High levels of genetic variation, typical of a highly outcrossing species, were found in all fragments at all loci (mean HE = 0.548). The majority of the variation was within rather than between fragments (RST = 0.032), giving high indirect estimates of gene flow (Nm = 8.9), probably reflecting the genetic structure of the trees present under more continuous forest. A high proportion of loci also showed significant departures from Hardy-Weinberg equilibrium with associated significant levels of FIS. The initial effects of fragmentation were, however, seen in the fragments through the loss of low-frequency alleles present in the continuous 'control' stand. The percentage of this allelic loss increased with a decrease in fragment size.     

Regulation of 5-hydroxyeicosanoid dehydrogenase activity in monocytic cells

The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric Gbetagamma (GTP-binding protein betagamma subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCeta (protein kinase Ceta) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PKCeta, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by Gbetagamma, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that betagamma-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLCbeta3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLCbeta3, which is necessary to activate PKCeta and PKD in that Golgi compartment, via DAG production.     

Fatty acid esterification in the yolk sac membrane of the avian embryo

The transfer of lipid from the yolk to the avian embryo is mediated by the yolk sac membrane (YSM). Some, but not all, of the published morphological evidence supports the view that the lipid undergoes a cycle of hydrolysis and re-esterification during translocation across the YSM. The present study aims to test this view by investigating the capacity of the YSM to perform esterification of free fatty acids to form acyl-lipids. YSM pieces (area vasculosa), obtained from the chicken embryo at day 10 of development, were incubated in vitro in medium containing [¹⁴C]-palmitic acid. Radioactivity was rapidly incorporated into the tissue lipid indicating a high capacity for esterification. The incorporation was linear with time during the 1-h incubation. Approximately 84% of the incorporated label was recovered in triacylglycerol, 12% was incorporated into phospholipid and less than 1% was detected in cholesteryl ester. [¹⁴C]-palmitic acid was incorporated primarily at the sn-1/3 positions in the triacylglycerol molecule and at the sn-1 position of phospholipid. The incorporation of label into tissue pieces obtained from the non-vascularized peripheral region of the YSM (area vitellina) was much more limited than that observed for the area vasculosa. The results support the hypothesis that yolk lipid is hydrolyzed and re-esterified during transfer across the YSM.Abbreviations YSM yolk sac membrane - VLDL very-low density lipoprotein Communicated by G. Heldmaier     

In vitro pluripotency of epiblasts derived from bovine blastocysts

Two experiments were conducted to compare the utility of in vitro- and in vivo-derived bovine blastocysts for the isolation of pluripotent epiblasts. In experiment 1, the inner cell masses (ICMs) of in vivo-collected blastocysts yielded a higher proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts (P = .0157). In experiment 2, ICMs of in vivo-collected blastocysts that hatched on day 8 yielded a greater proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts that hatched on day 8. The difference was reversed but smaller for blastocysts that hatched on day 9 (Interaction, P = .0125). Epiblasts from blastocysts that hatched on day 8 regardless of their source generated more differentiated cell lines in extended culture than did blastocysts that hatched on day 9. Extended epiblast culture yielded cells identifiable as products of the three embryonic germ layers that included epithelial cells, fibroblasts, neuronal cells, hepatocyte-like cells, and macrophage-like cells. Alkaline phosphatase activity combined with cell morphology identified the bovine epiblast cells and distinguished them from trophectoderm and endoderm that frequently contaminated epiblast cell cultures. In vivo-derived blastocysts, especially from early-hatching blastocysts, were a superior source of pluripotent epiblasts. Epiblast cells in this study all differentiated or senesced indicating that standard conditions for mouse embryonic stem cell culture do not maintain bovine epiblast cells in an undifferentiated state. © 1995 wiley-Liss, Inc.

1 This artilce is a US Government work and, as such, is in the public domain in the United States of America.

     

Structural chemistry and In vitro antitubercular activity of acetylpyridine benzoyl hydrazone and its copper complex against Mycobacterium smegmatis

Acetylpyridine benzoyl hydrazone (APBH) 1 and its copper complex [[(APBH)CuCl](2)].(EtOH) 2 were structurally characterized by elemental analysis, magnetic measurements, spectroscopy, electrochemistry and single crystal X-ray diffraction studies. The ligand assumes Z-isomeric form and planar geometry in solid state, coordinating through pyridyl nitrogen, azomethine nitrogen and the carbonyl oxygen of the benzoyl group. The copper complex is dimeric and has a distorted octahedral geometry in which the two copper atoms are bridged by two chloride atoms. Antimycobacterial screening of ligand and its copper compound against Mycobacterium smegmatis shows clear enhancement in the antitubercular activity upon copper complexation.     

Energy,Water and Fish: Biodiversity Impacts of Energy-Sector Water Demand in the United States Depend on Efficiency and Policy Measures

Rising energy consumption in coming decades, combined with a changing energy mix, have the potential to increase the impact of energy sector water use on freshwater biodiversity. We forecast changes in future water use based on various energy scenarios and examine implications for freshwater ecosystems. Annual water withdrawn/manipulated would increase by 18–24%, going from 1,993,000–2,628,000 Mm³ in 2010 to 2,359,000–3,271,000 Mm³ in 2035 under the Reference Case of the Energy Information Administration (EIA). Water consumption would more rapidly increase by 26% due to increased biofuel production, going from 16,700–46,400 Mm³ consumption in 2010 to 21,000–58,400 Mm³ consumption in 2035. Regionally, water use in the Southwest and Southeast may increase, with anticipated decreases in water use in some areas of the Midwest and Northeast. Policies that promote energy efficiency or conservation in the electric sector would reduce water withdrawn/manipulated by 27–36 m³GJ⁻¹ (0.1–0.5 m³GJ⁻¹ consumption), while such policies in the liquid fuel sector would reduce withdrawal/manipulation by 0.4–0.7 m³GJ⁻¹ (0.2–0.3 m³GJ⁻¹ consumption). The greatest energy sector withdrawal/manipulation are for hydropower and thermoelectric cooling, although potential new EPA rules that would require recirculating cooling for thermoelectric plants would reduce withdrawal/manipulation by 441,000 Mm³ (20,300 Mm³ consumption). The greatest consumptive energy sector use is evaporation from hydroelectric reservoirs, followed by irrigation water for biofuel feedstocks and water used for electricity generation from coal. Historical water use by the energy sector is related to patterns of fish species endangerment, where water resource regions with a greater fraction of available surface water withdrawn by hydropower or consumed by the energy sector correlated with higher probabilities of imperilment. Since future increases in energy-sector surface water use will occur in areas of high fish endemism (e.g., Southeast), additional management and policy actions will be needed to minimize further species imperilment.