Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.

Gene targeting in embryonic stem (ES) cells has been used to mutate the murine DNA methyltransferase gene. ES cell lines homozygous for the mutation were generated by consecutive targeting of both wild-type alleles; the mutant cells were viable and showed no obvious abnormalities with respect to growth rate or morphology, and had only trace levels of DNA methyltransferase activity. A quantitative end-labeling assay showed that the level of m5C in the DNA of homozygous mutant cells was about one-third that of wild-type cells, and Southern blot analysis after cleavage of the DNA with a methylation-sensitive restriction endonuclease revealed substantial demethylation of endogenous retroviral DNA. The mutation was introduced into the germline of mice and found to cause a recessive lethal phenotype. Homozygous embryos were stunted, delayed in development, and did not survive past mid-gestation. The DNA of homozygous embryos showed a reduction of the level of m5C similar to that of homozygous ES cells. These results indicate that while a 3-fold reduction in levels of genomic m5C has no detectable effect on the viability or proliferation of ES cells in culture, a similar reduction of DNA methylation in embryos causes abnormal development and embryonic lethality.     

Intrinsic PEEP monitored in the ventilated ARDS patient with a mathematical method.

Under mechanical volume-controlled ventilation, the intensive care patient can develop intrinsic positive end-expiratory pressure (iPEEP); that is, the passive expiration is terminated by the following inspiration before the alveolar pressure comes to its physical equilibrium value. We present a mathematical method to estimate this alveolar dynamic iPEEP breath by breath, without the need of a maneuver. We tested it in paralyzed patients ventilated for adult respiratory distress syndrome after multiple trauma and/or sepsis, and we compared the results obtained with the new mathematical method with those from the occlusion method introduced by Pepe and Marini. The results agreed well (median difference of 0.8 mbar in 201 investigations in 12 patients). However, the mathematically determined values, representing dynamic iPEEP, are systematically slightly smaller than those measured by the occlusion maneuver. A variation of expiratory time suggests that this difference might be due to mechanical time-constant inhomogeneity, viscoelastic processes, or other mechanisms showing time dependence.     

Isolation of a specific granulopoiesis inhibitor from calf spleen and identification as glutathione

A small peptide was isolated from calf spleen, specifically inhibiting murine granulopoiesis in vitro. Purification involved ultrafiltration, anion-exchange chromatography with a FPLC system and reversed-phase chromatography on HPLC. Determination of the amino acid composition, following acid hydrolysis and phenyl isothiocyanate and dabsyl chloride derivatization, revealed the amino acids glutamic acid, cysteine and glycine. Although the N-terminal amino group was not blocked, peptide sequencing with common techniques was not possible. Comparison of the isolated peptide with the well-known tripeptide glutathione by HPLC and fast atom bombardment (FAB)/tandem mass spectrometry showed the identity of both substances. Moreover, glutathione was found to be a specific granulopoiesis inhibitor in vitro at 10-100 nM, a so far unknown property of this well-known peptide.     

Construction and properties of replication-competent murine retroviral vectors encoding methotrexate resistance.

A series of replication-competent Moloney murine leukemia virus vectors was constructed in which each vector contained a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat. Two of the resulting viruses, MLV (murine leukemia virus) DHFR*-5 and MLV DHFR*-7, were able to stably transfer methotrexate resistance to infected fibroblast cells upon multiple rounds of virus replication and in the absence of drug selection. Cell lines producing recombinant virus with high titers were established, which indicated that the insert did not grossly interfere with viral replication functions. These vectors should be useful for introducing and expressing foreign genes in vivo in tissues and whole animals in which virus spread is needed for efficient infection.     

Curcumin Intake Affects miRNA Signature in Murine Melanoma with mmu-miR-205-5p Most Significantly Altered

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in ‘o-glycan biosynthesis’, ‘endoplasmatic reticulum protein processing’ and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly.     

Proprotein Convertase Subtilisin/Kexin Type 3 Promotes Adipose Tissue-Driven Macrophage Chemotaxis and Is Increased in Obesity

Background

Matrix metalloproteinase (MMP)-dependent extracellular matrix (ECM) remodeling is a key feature in cardiometabolic syndrome-associated adipogenesis and atherosclerosis. Activation of membrane-tethered (MT) 1-MMP depends on furin (PCSK3). However, the regulation and function of the natural furin-inhibitor serpinB8 and thus furin/MT1-MMP-activity in obesity-related tissue inflammation/remodeling is unknown. Here we aimed to determine the role of serpinB8/furin in obesity-associated chronic inflammation.

Methods and Results

Monocyte → macrophage transformation was characterized by decreases in serpinB8 and increases in furin/MT1-MMP. Rescue of serpinB8 by protein overexpression inhibited furin-dependent pro-MT1-MMP activation in macrophages, supporting its role as a furin-inhibitor. Obese white adipose tissue-facilitated macrophage migration was inhibited by furin- and MMP-inhibition, stressing the importance of the furin-MMP axis in fat tissue inflammation/remodeling. Monocytes from obese patients (body mass index (BMI) >30kg/m²) had higher furin, MT1-MMP, and resistin gene expression compared to normal weight individuals (BMI<25kg/m²) with significant correlations of BMI/furin and furin/MT1-MMP. In vitro, the adipocytokine resistin induced furin and MT1-MMP in mononuclear cells (MNCs), while MCP-1 had no effect.

Conclusions

Acquisition of the inflammatory macrophage phenotype is characterized by an imbalance in serpinB8/furin, leading to MT1-MMP activation, thereby enhancing migration. Increases in MT1-MMP and furin are present in MNCs from obese patients. Dissecting the regulation of furin and its inhibitor serpinB8 should facilitate targeting inflammation/remodeling in cardiometabolic diseases.     

Importance of tree height and social position for drought-related stress on tree growth and mortality

Key message

A higher mortality of dominant trees under drought stress is explained by impacts of tree size, canopy- and root structure and the hydraulic transport system.

Abstract

Drought stress can trigger tree mortality but the impact depends on stress intensity (water demand and availability) and on the vulnerability of the individual. Therefore, most research focusses on the species-specific properties such as water use efficiency or hydraulic conductivity that determine vulnerability. At the ecosystem scale, however, tree properties that have been found important for drought sensitivity or resistance vary with individual size and resource availability within a forest—also within the same species. This is caused by different environmental conditions for each tree and hence different growth histories of individuals generating specific anatomical and physiological features. Individual drought stress sensitivity might thus be considerably different from stand scale sensitivity. Indeed, empirical evidence shows that drought stress impact depends on tree social position which can be defined in degrees of suppression but correlated to resource availability, stress sensitivity and stress exposure. In this review, we collect such evidence and discuss the role of microclimate and soil water distribution as well as anatomical and physiological adjustments, which might serve as foundation for better-adapted management strategies to mitigate drought stress impacts. Finally, we define model requirements aiming to capture stand-scale drought responses or management impacts related to drought stress mitigation.