Calorie Restriction-Mediated Replicative Lifespan Extension in Yeast Is Non-Cell Autonomous

In laboratory yeast strains with Sir2 and Fob1 function, wild-type NAD⁺ salvage is required for calorie restriction (CR) to extend replicative lifespan. CR does not significantly alter steady state levels of intracellular NAD⁺ metabolites. However, levels of Sir2 and Pnc1, two enzymes that sequentially convert NAD⁺ to nicotinic acid (NA), are up-regulated during CR. To test whether factors such as NA might be exported by glucose-restricted mother cells to survive later generations, we developed a replicative longevity paradigm in which mother cells are moved after 15 generations on defined media. The experiment reveals that CR mother cells lose the longevity benefit of CR when evacuated from their local environment to fresh CR media. Addition of NA or nicotinamide riboside (NR) allows a moved mother to maintain replicative longevity despite the move. Moreover, conditioned medium from CR-treated cells transmits the longevity benefit of CR to moved mother cells. Evidence suggests the existence of a longevity factor that is dialyzable but is neither NA nor NR, and indicates that Sir2 is not required for the longevity factor to be produced or to act. Data indicate that the benefit of glucose-restriction is transmitted from cell to cell in budding yeast, suggesting that glucose restriction may benefit neighboring cells and not only an individual cell.     

THE FORMATION OF BASAL BODIES (CENTRIOLES) IN THE RHESUS MONKEY OVIDUCT

Basal body replication during estrogen-driven ciliogenesis in the rhesus monkey (Macaca mulatta) oviduct has been studied by stereomicroscopy, rotation photography, and serial section analysis. Two pathways for basal body production are described: acentriolar basal body formation (major pathway) where procentrioles are generated from a spherical aggregate of fibers; and centriolar basal body formation, where procentrioles are generated by the diplosomal centrioles. In both pathways, the first step in procentriole formation is the arrangement of a fibrous granule precursor into an annulus. A cartwheel structure, present within the lumen of the annulus, is composed of a central cylinder with a core, spoke components, and anchor filaments. Tubule formation consists of an initiation and a growth phase. The A tubule of each triplet set first forms within the wall material of the annulus in juxtaposition to a spoke of the cartwheel. After all nine A tubules are initiated, B and C tubules begin to form. The initiation of all three tubules occurs sequentially around the procentriole. Simultaneous with tubule initiation is a nonsequential growth of each tubule. The tubules lengthen and the procentriole is complete when it is about 200 mµ long. The procentriole increases in length and diameter during its maturation into a basal body. The addition of a basal foot, nine alar sheets, and a rootlet completes the maturation process. Fibrous granules are also closely associated with the formation of these basal body accessory structures.     

Early detection of colon cancer: new tests on the horizon

This year, the American Cancer Society reported that the rate of decline in both the incidence and mortality of colorectal cancer has increased over the last two decades. This success is felt to be attributable to the early detection and treatment of colonic adenomas and early-stage colorectal cancers. However, the current recommended 'menu of options' for screening is limited by poor patient acceptance, low sensitivity, and both high cost and poor accessibility for application to a large general screening population (colonoscopy). Computerized tomography and magnetic resonance colonography offer an alternative method for the identification of polyps and early lesions in certain patients, but have cost, access, and acceptance limitations that are similar to those of colonoscopy; thus, they present similar barriers to their use in broad population screening. These limitations provide a strong rationale for the development of early colorectal cancer detection biomarkers that are simple to use and are cost effective. A successful biomarker or biomarker panel, coupled with the colonoscopic follow-up of only those patients with positive results, would reduce the burden and morbidity associated with the screening of colonoscopy. This would most likely result in enhanced adherence to colorectal screening, as well as a dramatic reduction in the incidence and mortality rates of colorectal cancer. In this paper, we review recent advances in the discovery of potential colorectal cancer biomarkers. Their applicability to clinical population screening will require large prospective validation.     

Defining the risks of mesenchymal stromal cell therapy

We address the issue of the potential for malignant transformation of cultured mesenchymal stromal cells (MSC) commonly used in clinical cell-therapy protocols and describe the culture conditions under which tumorigenesis is likely to be an extremely uncommon event.     

Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage

Introduction

The repair capability of traumatized articular cartilage is highly limited so that joint injuries often lead to osteoarthritis. Migratory chondrogenic progenitor cells (CPC) might represent a target cell population for in situ regeneration. This study aims to clarify, whether 1) CPC are present in regions of macroscopically intact cartilage from human osteoarthritic joints, 2) CPC migration is stimulated by single growth factors and the cocktail of factors released from traumatized cartilage and 3) CPC migration is influenced by cytokines present in traumatized joints.

Methods

We characterized the cells growing out from macroscopically intact human osteoarthritic cartilage using a panel of positive and negative surface markers and analyzed their differentiation capacity. The migratory response to platelet-derived growth factor (PDGF)-BB, insulin-like growth factor 1 (IGF-1), supernatants obtained from in vitro traumatized cartilage and interleukin-1 beta (IL-1β) as well as tumor necrosis factor alpha (TNF-α) were tested with a modified Boyden chamber assay. The influence of IL-1β and TNF-α was additionally examined by scratch assays and outgrowth experiments.

Results

A comparison of 25 quadruplicate marker combinations in CPC and bone-marrow derived mesenchymal stromal cells showed a similar expression profile. CPC cultures had the potential for adipogenic, osteogenic and chondrogenic differentiation. PDGF-BB and IGF-1, such as the supernatant from traumatized cartilage, induced a significant site-directed migratory response. IL-1β and TNF-α significantly reduced basal cell migration and abrogated the stimulative effect of the growth factors and the trauma supernatant. Both cytokines also inhibited cell migration in the scratch assay and primary outgrowth of CPC from cartilage tissue. In contrast, the cytokine IL-6, which is present in trauma supernatant, did not affect growth factor induced migration of CPC.

Conclusion

These results indicate that traumatized cartilage releases chemoattractive factors for CPC but IL-1β and TNF-α inhibit their migratory activity which might contribute to the low regenerative potential of cartilage in vivo.     

Effect of epinephrine on the oxidative desaturation of fatty acids in the rat.

The effect of epinephrine on the oxidative desaturation of fatty acids by liver microsomal preparations of rats has been studied. Administration of epinephrine (1 mg/kg body weight) produced a significant decrease in desaturation of [l-14C]=linoleic acid to gamma-linolenic acid and of [L-14C]alpha-linolenic acid to actadeca-6,9,12,15-tetraenoic acid 12 hr after the infection. Lower doses produced a lesser effect on the delta6-desaturation activity. Epinephrine administration modified the V max of linoleic acid desaturation but not the K m. There was also a slight increase in palmityl desaturation activity. The effect of epinephrine on delta6-desaturation activity was postulated to be mediated through an enhancement of the intracellular cyclic AMP levels that lead to an increase of a glucose metabolite. This metabolite would inhibit delta6-desaturation activity.     

Prostatic Alpha-Linolenic Acid (ALA) Is Positively Associated with Aggressive Prostate Cancer: A Relationship Which May Depend on Genetic Variation in ALA Metabolism

Previous observational studies have reported associations between prostate cancer and alpha-linolenic acid (ALA). However, few investigations have been able to study this relationship prospectively and in well-controlled settings. Moreover, no studies have determined whether single nucleotide polymorphisms (SNPs) that influence ALA metabolism are associated with this common cancer. The purpose of this study was to explore associations between prostatic levels of ALA, SNPs and prostate cancer-specific biomarkers in samples collected from a previous randomized clinical trial conducted using a presurgical model and which tested the effects of flaxseed supplementation, a rich source of ALA, prior to prostatectomy (n = 134). Serum prostate-specific antigen (PSA) was determined and immunohistochemistry was used to assess tumor proliferation rate (Ki67). Prostatic ALA was determined with gas chromatography. Seven previously identified SNPs associated with delta-6 desaturase activity (rs99780, rs174537, rs174545, rs174572, rs498793, rs3834458 and rs968567) were tested for associations with prostatic ALA, PSA and Ki67. Despite consuming seven times more ALA per day, men in the flaxseed arm had similar amounts of prostatic ALA relative to men not consuming flaxseed. In unadjusted analysis, there were significant positive associations between prostatic ALA and PSA (ρ = 0.191, p = 0.028) and Ki67 (ρ = 0.186, p = 0.037). After adjusting for covariates (flaxseed, age, race, BMI and statin-use) the association between ALA and PSA remained (p = 0.004) but was slightly attenuated for Ki67 (p = 0.051). We did not observe associations between any of the SNPs studied and prostatic ALA; however, in models for PSA there was a significant interaction between rs498793 and ALA and for Ki67 there were significant interactions with ALA and rs99780 and rs174545. Independent and inverse associations were observed between rs174572 and Ki67. This study provides evidence that prostatic ALA, independent of the amount of ALA consumed, is positively associated with biomarkers of aggressive prostate cancer and that genetic variation may modify this relationship.     

Expression of human T cell receptor-gamma delta structural forms

The human TCR-gamma delta occurs in three biochemically distinct forms (forms 1, 2bc, and 2abc). A 40-kDa TCR gamma-chain is disulfide-linked to the TCR delta-chain in form 1, whereas 40-kDa or 55-kDa TCR-gamma polypeptides are noncovalently associated with the TCR delta-chain in forms 2bc and 2abc, respectively. Sequence analysis of TCR-gamma cDNA clones indicates that form 1 utilizes the C gamma 1 gene segment, whereas forms 2bc and 2abc appear to use allelic C gamma 2 gene segments containing either two copies (b and c) or three copies (a, b, and c) of the CII exon, respectively. We transfected TCR-gamma cDNA encoding form 1 or form 2abc into the MOLT-13 cell line that expresses form 2bc. The transfected TCR gamma-chains associate with the resident MOLT-13 TCR-delta, normally part of form 2bc, to yield CD3-associated TCR-gamma delta heterodimers identical to those seen on the donor cell lines (form 1 or 2abc). These transfection experiments show directly that, 1) when a single TCR-delta subunit is available, the presence or absence of disulfide linkage between TCR gamma- and TCR delta-chains is controlled by the TCR gamma-chain, and 2) the difference in the amount of N-linked carbohydrate attached to the transfected TCR-gamma proteins of form 2bc vs form 2abc is influenced by the presence or absence of CII exon copy "a" which appears to alter the secondary and/or tertiary structure of these TCR gamma-chain constant regions, thereby affecting the attachment of N-linked glycans. In contrast to the similar structure and usage of C beta 1 and C beta 2, TCR-gamma delta forms show striking differences in structure and are not equally represented in peripheral blood. Although the role of each form is unknown, it is possible that variable or joining-gene segment selection events or functional differences account for their unequal usage.     

DNA damage response in peripheral mouse blood leukocytes in vivo after variable,low-dose rate exposure

Environmental contamination and ingestion of the radionuclide Cesium-137 (137Cs) is a large concern in fallout from a nuclear reactor accident or improvised nuclear device, and highlights the need to develop biological assays for low-dose rate, internal emitter radiation. To mimic low-dose rates attributable to fallout, we have developed a VAriable Dose-rate External 137Cs irradiatoR (VADER), which can provide arbitrarily varying and progressive low-dose rate irradiations in the range of 0.1–1.2 Gy/day, while circumventing the complexities of dealing with radioactively contaminated biomaterials. We investigated the kinetics of mouse peripheral leukocytes DNA damage response in vivo after variable, low-dose rate 137Cs exposure. C57BL/6 mice were placed in the VADER over 7 days with total accumulated dose up to 2.7 Gy. Peripheral blood response including the leukocyte depletion, apoptosis as well as its signal protein p53 and DNA repair biomarker γ-H2AX was measured. The results illustrated that blood leukocyte numbers had significantly dropped by day 7. P53 levels peaked at day 2 (total dose = 0.91 Gy) and then declined; whereas, γ-H2AX fluorescence intensity (MFI) and foci number generally increased with accumulated dose and peaked at day 5 (total dose = 2.08 Gy). ROC curve analysis for γ-H2AX provided a good discrimination of accumulated dose < 2 Gy and ≥ 2 Gy, highlighting the potential of γ-H2AX MFI as a biomarker for dosimetry in a protracted, environmental exposure scenario.