A system for studying epithelial-stromal interactions reveals distinct inductive abilities of stromal cells from benign prostatic hyperplasia and prostate cancer

The development of normal and abnormal glandular structures in the prostate is controlled at the endocrine and paracrine levels by reciprocal interactions between epithelium and stroma. To study these processes it is useful to have an efficient method of tissue acquisition for reproducible isolation of cells from defined histologies. Here we assessed the utility of a standardized system for acquisition and growth of prostatic cells from different regions of the prostate with different pathologies, and we compared the abilities of stromal cells from normal peripheral zone (PZ-S), benign prostatic hyperplasia (BPH-S), and cancer (CA-S) to induce the growth of a human prostatic epithelial cell line (BPH-1) in vivo. Using the tissue recombination method, we showed that grafting stromal cells (from any histology) alone, or BPH-1 epithelial cells alone produced no visible grafts. Recombining PZ-S with BPH-1 cells also produced no visible grafts (n = 15). Recombining BPH-S with BPH-1 cells generated small, well-organized and sharply demarcated grafts approximately 3-4 mm in diameter (n = 9), demonstrating a moderate inductive ability of BPH-S. Recombining CA-S with BPH-1 cells generated highly disorganized grafts that completely surrounded the host kidney and invaded into adjacent renal tissue, demonstrating induction of an aggressive phenotype. We conclude that acquisition of tissue from toluidine blue dye stained specimens is an efficient method to generate high quality epithelial and/or stromal cultures. Stromal cells derived by this method from areas of BPH and cancer induce epithelial cell growth in vivo which mimics the natural history of these diseases.

Support for global climate reorganization during the "Medieval Climate Anomaly"

Widely distributed proxy records indicate that the Medieval Climate Anomaly (MCA; *900–1350 AD) was characterized by coherent shifts in large-scale Northern Hemisphere atmospheric circulation patterns. Although cooler sea surface temperatures in the central and eastern equatorial Pacific can explain some aspects of medieval circulation changes, they are not sufficient to account for other notable features, including widespread aridity through the Eurasian sub-tropics, stronger winter westerlies across the North Atlantic and Western Europe, and shifts in monsoon rainfall patterns across Africa and South Asia. We present results from a full-physics coupled climate model showing that a slight warming of the tropical Indian and western Pacific Oceans relative to the other tropical ocean basins can induce a broad range of the medieval circulation and climate changes indicated by proxy data, including many of those not explained by a cooler tropical Pacific alone. Important aspects of the results resemble those from previous simulations examining the climatic response to the rapid Indian Ocean warming during the late twentieth century, and to results from climate warming simulations—especially in indicating an expansion of the Northern Hemisphere Hadley circulation. Notably, the pattern of tropical Indo-Pacific sea surface temperature (SST) change responsible for producing the proxy-model similarity in our results agrees well with MCA-LIA SST differences obtained in a recent proxy-based climate field reconstruction. Though much remains unclear, our results indicate that the MCA was characterized by an enhanced zonal Indo-Pacific SST gradient with resulting changes in Northern Hemisphere tropical and extra-tropical circulation patterns and hydroclimate regimes, linkages that may explain the coherent regional climate shifts indicated by proxy records from across the planet. The findings provide new perspectives on the nature and possible causes of the MCA—a remarkable, yet incompletely understood episode of Late Holocene climatic change.

Evidence for global climate reorganization during medieval times

A synthesis of global climate model results and inferences from proxy records suggests an increased sea surface temperature gradient between the tropical Indian and Pacific Oceans during medieval times.

The effects of retinoic acid on human corneal stromal keratocytes cultured in vitro under serum-free conditions

Purpose: Retinoic acid (RA) is a metabolite of vitamin A that plays a fundamental role in the development and function of the human eye. The purpose of this study was to investigate the effects of RA on the phenotype of corneal stromal keratocytes maintained in vitro for extended periods under serum-free conditions. Methods: Keratocytes isolated from human corneas were cultured up to 21 days in serum-free media supplemented with RA or DMSO vehicle. The effects of RA and of its removal after treatment on cell proliferation and morphology were evaluated. In addition, the expression of keratocyte markers was quantified at the transcriptional and protein levels by quantitative PCR and immunoblotting or ELISA, respectively. Furthermore, the effects of RA on keratocyte migration were tested using scratch assays. Results: Keratocytes cultured with RA up to 10×10-6 M showed enhanced proliferation and stratification, and reduced mobility. RA also promoted the expression of keratocyte-characteristic proteoglycans such as keratocan, lumican, and decorin, and increased the amounts of collagen type-I in culture while significantly reducing the expression of matrix metalloproteases 1, 3, and 9. RA effects were reversible, and cell phenotype reverted to that of control after removal of RA from media. Conclusions: RA was shown to control the phenotype of human corneal keratocytes cultured in vitro by regulating cell behaviour and extracellular matrix composition. These findings contribute to our understanding of corneal stromal biology in health and disease, and may prove useful in optimizing keratocyte cultures for applications in tissue engineering, cell biology, and medicine.

Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward higher-order oligomers

We report on the assembly of tumor necrosis factor receptor 1 (TNF-R1) prior to ligand activation and its ligand-induced reorganization at the cell membrane. We apply single-molecule localization microscopy to obtain quantitative information on receptor cluster sizes and copy numbers. Our data suggest a dimeric pre-assembly of TNF-R1, as well as receptor reorganization toward higher oligomeric states with stable populations comprising three to six TNF-R1. Our experimental results directly serve as input parameters for computational modeling of the ligand-receptor interaction. Simulations corroborate the experimental finding of higher-order oligomeric states. This work is a first demonstration how quantitative, super-resolution and advanced microscopy can be used for systems biology approaches at the single-molecule and single-cell level.

Paracrine effects of TLR4-polarised mesenchymal stromal cells are mediated by extracellular vesicles

Mesenchymal stromal cells (MSCs) are adult stem cells able to give rise to bone, cartilage and fat cells. In addition, they possess immunomodulatory and immunosuppressive properties that are mainly mediated through secretion of extracellular vesicles (EVs). In a previous issue of Journal of Translational Medicine, Ti and colleagues demonstrated that preconditioning of MSCs with bacterial lipopolysaccharides results in secretion of EVs that can polarise mac‑ rophages towards anti-inflammatory M2 phenotype. Moreover, the authors suggest that EVs of lipopolysaccharide (LPS)-treated MSCs are superior to EVs of untreated MSCs concerning their ability to support wound healing. Our commentary critically discusses parallel efforts of other laboratories to generate conditioned media from stem cells for therapeutic applications, and highlights impact and significance of the study of Ti et al. Finally, we summarise its limitations and spotlight areas that need to be addressed to better define the underlying molecular mechanisms.

A critical assessment of unbalanced surface stresses: Some complementary considerations

The wide-ranging survey of twisted growth in polymers by Lotz and Cheng cites extensive evidence consistent with the relief of surface stress being the underlying cause. This complementary note contributes to the discussion by making three main points. First, it is necessary to go further and explain the key issue of how a consistent twist is maintained when, as commonly, this habit has a lower symmetry than the crystallographic lattice. Detailed study has shown that, in polyethylene, this occurs by reorganization of the initial fold surfaces. Second, the suggested explanation by Keith and Padden that. in polyethylene, the asymmetric habit derives from molecules adding to lamellae with inclined fold surfaces is invalid being doubly inconsistent with observation. Third, twisting has now been linked to faster growth by study of row structures in polyethylene. This produces inherently rough fold surfaces in Regime II whose internal stresses drive reorganization and twisting. For slower (Regime I) growth, fold surfaces form with and maintain ordered packing so providing no basis for twisting. These new insights radically alter the context of twisted growth and provide a firm factual basis for further work. (c) 2005 Elsevier Ltd. All rights reserved.

Ultrastructural alterations in mesophyll and bundle sheath chloroplasts of two maize cultivars in response to chilling at high irradiance

Maize (Zea mays L.) seedlings of two cultivars (cv. Bastion adapted to W. Europe, and cv. Batan 8686 adapted to the highlands of Mexico), raised in a glasshouse (19-25 degrees C), were transferred to 4.5 or 9 degrees C at photon flux density (PPFD) of 950 mu mol m(-2) s(-1) with 10-h photoperiod for 58 h and then allowed to recover at 22 degrees C for 16 h (14 h dark and 2 h at PPFD of 180 mu mol m(-2) s(-1)). The ultrastructural responses after 4 h or 26 h at 4.5 degrees C were the disappearance of starch grains in the bundle sheath chloroplasts and the contraction of intrathylakoid spaces in stromal thylakoids of the mesophyll chloroplasts. At this time, bundle sheath chloroplasts of cv. Batan 8686 formed peripheral reticulum. Prolonged stress at 4.5 degrees C (50 h) caused plastid swelling and the dilation of intrathylakoid spaces, mainly in mesophyll chloroplasts. Bundle sheath chloroplasts of cv. Batan 8686 seedlings appeared well preserved in shape and structure. Batan 8686 had also higher net photosynthetic rates during chilling and recovery than Bastion. Extended leaf photobleaching developed during the recovery period after chilling at 4.5 degrees C. This was associated with collapsed chloroplast envelopes, disintegrated chloroplasts and very poor staining.

Evolution of microbial virulence: the benefits of stress

Although genome sequencing of microbial pathogens has shed light on the evolution of virulence, the drivers of the gain and loss of genes and of pathogenicity islands (gene clusters), which contribute to the emergence of new disease outbreaks, are unclear. Recent experiments with the bean pathogen Pseudomonas syringae pv. phaseolicola illustrate how exposure to resistance mechanisms acts as the driving force for genome reorganization. Here we argue that the antimicrobial conditions generated by host defences can accelerate the generation of genome rearrangements that provide selective advantages to the invading microbe. Similar exposure to environmental stress outside the host could also drive the horizontal gene transfer that has led to the evolution of pathogenicity towards both animals and plants.

TGF-beta superfamily members and ovarian follicle development

In recent years, exciting progress has been made towards unravelling the complex intraovarian control mechanisms that, in concert with systemic signals, coordinate the recruitment, selection and growth of follicles from the primordial stage through to ovulation and corpus luteum formation. A plethora of growth factors, many belonging to the transforming growth factor-beta (TGF-beta) superfamily, are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as intraovarian regulators of folliculogenesis. Two such factors, bone morphogenetic proteins, RMP-4 and BMP-7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition. In contrast, evidence indicates a negative role for anti-Mullerian hormone (AMH, also known as Mullerian-inhibiting substance) of pre-granulosa/granulosa cell origin in this key event and subsequent progression to the antral stage. Two other TGF-beta superfamily members, growth and differentiation factor-9 (GDF-9) and BMP-15 (also known as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play key roles in promoting follicle growth beyond the primary stage; mice with null mutations in the gdf-9 gene or ewes with inactivating mutations in gdf-9 or bmp-15 genes are infertile with follicle development arrested at the primary stage. Studies on later stages of follicle development indicate positive roles for granulosa cell-derived activin, BMP-2, -5 and -6, theca cell-derived BMP-2, -4 and -7 and oocyte-derived BMP-6 in promoting granulosa cell proliferation, follicle survival and prevention of premature luteinization and/or atresia. Concomitantly, activin, TGF-beta and several BMPs may exert paracrine actions on theca cells to attenuate LH-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection in monovular species may depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Changes in intrafollicular activins, GDF-9, AMH and several BMPs may contribute to this selection process by modulating both FSH- and IGF-dependent signalling pathways in granulosa cells. Activin may also play a positive role in oocyte maturation and acquisition of developmental competence. in addition to its endocrine role to suppress FSH secretion, increased output of inhibin by the selected dominant follicle(s) may upregulate LH-induced androgen secretion that is required to sustain a high level of oestradiol secretion during the pre-ovulatory phase. Advances in our understanding of intraovarian regulatory mechanisms should facilitate the development of new approaches for monitoring and manipulating ovarian function and improving fertility in domesticated livestock, endangered species and man.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine ( DCVC) resulted in a > 1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Oxidation-responsiveness of nanomaterials for targeting inflammatory reactions

Oxidation is an almost ubiquitous feature of inflammatory reactions. We discuss the development of nanocarriers that respond to the presence of oxidants with profound physical reorganization, which could in perspective allow their use for delivering anti-inflammatory principles in an inflammation-responsive fashion. We also present a study demonstrating that the response of polysulfide nanoparticles has a bulk character, i.e., the odixation reactions happen homogeneously throughout the nanoparticles, and not interfacially.

Coordination-driven self-assembly of a novel carbonato-bridged heteromolecular neutral nickel(II) triangle by atmospheric CO2 fixation

Formation of a quasi-symmetrical mu(3)-carbonato-bridged self-assembled heteromolecular triangle of Ni(II), [(mu(3)-CO3){Ni-2(salmeNH)(2)(NCS)(2)}[Ni(salmeNH(2))(2)]center dot Et2O center dot H2O (HsalmeNH = 2-[(3-methylamino-propylimino)-methyl]-phenol) involves atmospheric CO2 uptake in a neutral medium, by spontaneous self-reorganization of the starting mononuclear Ni(II)-Schiff-base complex, [Ni(salmeNH)(2)]. The environment around Ni(II) in two of the subunits is different from the third one. The starting complex, (Ni(salmeNH)(2)], and one of the possible intermediate species, [Ni(salmeNH(2))(2)(NCS)(2)], which has a very similar coordination environment to that in the third Ni(II) center, have been characterized structurally. A plausible mechanism for the formation of such a triangle has also been proposed. The compound shows a very strong antiferromagnetic coupling. Fit as a regular triangular arrangement gave J = -53.1, g = 2.24, and R = 1.5 x 10(-4).

PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E-3-ligase MIR

PDGF is a potent chemotactic mitogen and a strong inductor of fibroblast motility. In Swiss 3T3 fibroblasts, exposure to PDGF but not EGF or IGF-1 causes a rapid loss of actin stress fibers (SFs) and focal adhesions (FAs), which is followed by the development of retractile dendritic protrusions and induction of motility. The PDGF-specific actin reorganization was blocked by inhibition of Src-kinase and the 26S proteasome. PDGF induced Src-dependent association between the multifunctional transcription/translation regulator hnRNP-K and the mRNA-encoding myosin regulatory light-chain (MRLC)-interacting protein (MIR), a E3-ubiquitin ligase that is MRLC specific. This in turn rapidly increased MIR expression, and led to ubiquitination and proteasome-mediated degradation of MRLC. Downregulation of MIR by RNA muting prevented the reorganization of actin structures and severely reduced the migratory and wound-healing potential of PDGF-treated cells. The results show that activation of MIR and the resulting removal of diphosphorylated MRLC are essential for PDGF to instigate and maintain control over the actin-myosin-based contractile system in Swiss 3T3 fibroblasts. The PDGF induced protein destabilization through the regulation of hnRNP-K controlled ubiquitin-ligase translation identifies a novel pathway by which external stimuli can regulate phenotypic development through rapid, organelle-specific changes in the activity and stability of cytoskeletal regulators.