Broadly reactive murine cytotoxic cells induced in vitro under syngeneic conditions

Mouse spleen cells became cytotoxic in short-term 51Cr-release assays for a wide variety of target cells after 5 days of culture in vitro with polyinosinic acid in a system that was otherwise entirely syngeneic. This study characterizes these effector cells with respect to target specificity, effect of donor age, time course of their appearance, mouse strain differences, and expression of differentiation antigens Thy-1, Lyt-1, Lyt-2, NK-1, and asialo GM1. The combination of properties of this cytotoxic cell response that make it unique are that a) the broadly reactive cytotoxic activity developed from unprimed spleen cells in the absence of either foreign cells or foreign serum; b) the response did not peak until 4 to 5 days of culture in vitro; c) the broad reactivity pattern included freshly dispersed primary syngeneic sarcoma cells and cultured syngeneic fibroblasts but did not include syngeneic lymphoblast target cells; d) the response was largely monoclonal as defined by target cell binding; and e) cytotoxic cell activity was sensitive in complement-mediated treatments to both anti-NK and anti-theta but not to anti-Lyt-2, anti-Lyt-1, or anti-asialo GM1. Both high- and low-responding mouse strains have been identified.     

Altered Expression Pattern of Molecular Factors Involved in Colonic Smooth Muscle Functions: An Immunohistochemical Study in Patients with Diverticular Disease

Background

The pathogenesis of diverticular disease (DD) is thought to result from complex interactions among dietary habits, genetic factors and coexistence of other bowel abnormalities. These conditions lead to alterations in colonic pressure and motility, facilitating the formation of diverticula. Although electrophysiological studies on smooth muscle cells (SMCs) have investigated colonic motor dysfunctions, scarce attention has been paid to their molecular abnormalities, and data on SMCs in DD are lacking. Accordingly, the main purpose of this study was to evaluate the expression patterns of molecular factors involved in the contractile functions of SMCs in the tunica muscularis of colonic specimens from patients with DD.

Methods and Findings

By means of immunohistochemistry and image analysis, we examined the expression of Cx26 and Cx43, which are prominent components of gap junctions in human colonic SMCs, as well as pS368-Cx43, PKCps, RhoA and αSMA, all known to regulate the functions of gap junctions and the contractile activity of SMCs.The immunohistochemical analysis revealed significant abnormalities in DD samples, concerning both the expression and distribution patterns of most of the investigated molecular factors.

Conclusion

This study demonstrates, for the first time, that an altered pattern of factors involved in SMC contractility is present at level of the tunica muscularis of DD patients. Moreover, considering that our analysis was conducted on colonic tissues not directly affected by diverticular lesions or inflammatory reactions, it is conceivable that these molecular alterations may precede and predispose to the formation of diverticula, rather than being mere consequences of the disease.     

The expression of Lamin A mutant R321X leads to endoplasmic reticulum stress with aberrant Ca2+ handling

Mutations in the Lamin A/C gene (LMNA), which encodes A‐type nuclear Lamins, represent the most frequent genetic cause of dilated cardiomyopathy (DCM). This study is focused on a LMNA nonsense mutation (R321X) identified in several members of an Italian family that produces a truncated protein isoform, which co‐segregates with a severe form of cardiomyopathy with poor prognosis. However, no molecular mechanisms other than nonsense mediated decay of the messenger and possible haploinsufficiency were proposed to explain DCM. Aim of this study was to gain more insights into the disease‐causing mechanisms induced by the expression of R321X at cellular level. We detected the expression of R321X by Western blotting from whole lysate of a mutation carrier heart biopsy. When expressed in HEK293 cells, GFP‐ (or mCherry)‐tagged R321X mislocalized in the endoplasmic reticulum (ER) inducing the PERK‐CHOP axis of the ER stress response. Of note, confocal microscopy showed phosphorylation of PERK in sections of the mutation carrier heart biopsy. ER mislocalization of mCherry‐R321X also induced impaired ER Ca²⁺ handling, reduced capacitative Ca²⁺ entry at the plasma membrane and abnormal nuclear Ca²⁺ dynamics. In addition, expression of R321X by itself increased the apoptosis rate. In conclusion, R321X is the first LMNA mutant identified to date, which mislocalizes into the ER affecting cellular homeostasis mechanisms not strictly related to nuclear functions.     

High‐Performance Liquid Chromatographic Resolution of Neutral and Cationic Hetero[6]Helicenes

Cationic hetero[6]helicenes 1⁺ , 2⁺ and 3⁺ have been recently disclosed. Herein we report on their enantiomeric separation using high‐performance liquid chromatography. Separation of the antipodes can be achieved in preparative scale on neutral adducts with Chiralcel OD‐I or Chiralpak ID CSP. Selectivity factors of 1.90, 1.67, and 1.96 were obtained for 1-H , 2-H , and 3-H , respectively. Separation can also be performed on the carbenium ions on regular Chiralpak IA CSP using water‐containing eluents, thus allowing for enantiomeric purity determinations in aqueous environments. Resolution of neutral and cationic helicenes is also achieved on more recently developed LARIHC columns. The versatility of the cyclofructan phases allows for baseline separations for both cases and their loading capabilities are demonstrated. Finally, the configurational stability of 1⁺ , 2⁺ , and 3⁺ was measured. For each replacement of an oxygen atom by an amino group, the racemization barrier increases significantly (ΔG^? = 29.8, 36.3 and >37 kcal mol^‐1 for 1⁺ , 2⁺ , and 3⁺ respectively). Chirality 28:282–289, 2016. © 2016 Wiley Periodicals, Inc.     

Using MT(-/-) mice to study metallothionein and oxidative stress

Mice with null mutations for metallothionein genes MT-1 and MT-2 were used to study the role that metallothionein plays in protecting cellular targets in vivo from oxidative stress. Wild-type (MT(+/+)) and MT-null (MT(-/-)) mice were treated with either saline or zinc and exposed to two types of oxidative stress: gamma-irradiation or 2-nitropropane. There was no alteration in the antioxidant defense system (superoxide dismutase, catalase, or glutathione peroxidase and glutathione levels) to compensate for the lack of the metallothionein in the MT(-/-) mice. The amount of oxidative damage to liver DNA, lipids, and proteins were similar for the MT(-/-) and MT(+/+) mice even though the levels of metallothionein in the livers of the saline- or zinc-pretreated MT(+/+) mice were 5- to 100-fold greater than found in the MT(-/-) mice. To determine if metallothionein can protect mice from the lethal effects of ionizing radiation, the mean survivals of MT(-/-) and MT(+/+) mice exposed to whole body gamma-irradiation were measured and found to be similar. However, the mean survival increased significantly after zinc pretreatment for both the MT(-/-) and MT(+/+) mice. These results demonstrate that tissue levels of metallothionein do not protect mice in vivo against oxidative stress.     

An integrated assessment of histopathological changes of the enteric neuromuscular compartment in experimental colitis

Bowel inflammatory fibrosis has been largely investigated, but an integrated assessment of remodelling in inflamed colon is lacking. This study evaluated tissue and cellular changes occurring in colonic wall upon induction of colitis, with a focus on neuromuscular compartment. Colitis was elicited in rats by 2,4‐dinitrobenzenesulfonic acid (DNBS). After 6 and 21 days, the following parameters were assessed on paraffin sections from colonic samples: tissue injury and inflammatory infiltration by histology; collagen and elastic fibres by histochemistry; HuC/D, glial fibrillar acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), nestin, substance P (SP), von Willebrand factor, c‐Kit and transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) by immunohistochemistry. TMEM16A/ANO1 was also examined in isolated colonic smooth muscle cells (ICSMCs). On day 6, inflammatory alterations and fibrosis were present in DNBS‐treated rats; colonic wall thickening and fibrotic remodelling were evident on day 21. Colitis was associated with both an increase in collagen fibres and a decrease in elastic fibres. Moreover, the neuromuscular compartment of inflamed colon displayed a significant decrease in neuron density and increase in GFAP/PCNA‐positive glia of myenteric ganglia, enhanced expression of neural SP, blood vessel remodelling, reduced c‐Kit‐ and TMEM16A/ANO1‐positive interstitial cells of Cajal (ICCs), as well as an increase in TMEM16A/ANO1 expression in muscle tissues and ICSMCs. The present findings provide an integrated view of the inflammatory and fibrotic processes occurring in the colonic neuromuscular compartment of rats with DNBS‐induced colitis. These morphological alterations may represent a suitable basis for understanding early pathophysiological events related to bowel inflammatory fibrosis.     

Chlorophyta microalgae as dietary protein supplement: a comparative analysis of productivity related to photosynthesis

Journal of Applied Phycology - Microalgae are studied as innovative sources of a wide range of highly valuable products, including proteins for the food/feed sectors. However, protein content...     

Effect of different CO2 concentrations on biomass,pigment content,and lipid production of the marine diatom Thalassiosira pseudonana

The marine diatom Thalassiosira pseudonana grown under air (0.04% CO₂) and 1 and 5% CO₂ concentrations was evaluated to determine its potential for CO₂ mitigation coupled with biodiesel production. Results indicated that the diatom cultures grown at 1 and 5% CO₂ showed higher growth rates (1.14 and 1.29 div day⁻¹, respectively) and biomass productivities (44 and 48 mg_AFDWL⁻¹ day⁻¹) than air grown cultures (with 1.13 div day⁻¹ and 26 mg_AFDWL⁻¹ day⁻¹). The increase of CO₂ resulted in higher cell volume and pigment content per cell of T. pseudonana. Interestingly, lipid content doubled when air was enriched with 1–5% CO₂. Moreover, the analysis of the fatty acid composition of T. pseudonana revealed the predominance of monounsaturated acids (palmitoleic-16:1 and oleic-18:1) and a decrease of the saturated myristic acid-14:0 and polyunsaturated fatty acids under high CO₂ levels. These results suggested that T. pseudonana seems to be an ideal candidate for biodiesel production using flue gases.