Molecular and histological traits of reduced lysosomal acid lipase activity in the fatty liver

Recent studies demonstrated reduced blood lysosomal acid lipase (LAL) activity in patients with nonalcoholic fatty liver disease (NAFLD). We aimed to verify hepatic LAL protein content and activity in in vitro and in vivo models of fat overload and in NAFLD patients. LAL protein content and activity were firstly evaluated in Huh7 cells exposed to high-glucose/high-lipid (HGHL) medium and in the liver of C57BL/6 mice fed with high-fat diet (HFD) for 4 and 8 months. LAL protein was also evaluated by immunohistochemistry in liver biopsies from 87 NAFLD patients and 10 controls, and correlated with hepatic histology. Huh7 cells treated with HGHL medium showed a significant reduction of LAL activity, which was consistent with reduced LAL protein levels by western blotting using an antibody towards the N-term of the enzyme. Conversely, antibodies towards the C-term of the enzyme evidenced LAL accumulation, suggesting a post-translational modification that masks the LAL N-term epitope and affects enzymatic activity. Indeed, we found a high rate of ubiquitination and extra-lysosomal localization of LAL protein in cells treated with HGHL medium. Consistent with these findings, inhibition of proteasome triggered dysfunctional LAL accumulation and affected LAL activity. Accumulation of ubiquitinated/dysfunctional LAL was also found in the liver of HFD fed mice. In NAFLD patients, hepatic levels of non-ubiquitinated/functional LAL were lower than in controls and inversely correlated with disease activity and some of the hallmarks of reduced LAL. Fat overload leads to LAL ubiquitination and impairs its function, possibly reducing hepatic fat disposal and promoting NAFLD activity

CATALYTIC OXIDATION REACTIONS OF AROMATIC DIAMINES BY TRANSITION METAL COMPLEXES.

The catalytic reactions of M(TPP)Cl (M = Fe (1), Mn (2)), Fe(TMP)Cl (3), and Fe(TDCPP)Cl (4) (H2TPP = 5,10,15,20-tetraphenylporphyrin; H(2)TMP = 5,10,15,20-tetramesitylporphyrin; H2TDCPP = 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin) with 2,4-diaminotoluene (5) and p-phenylenediamine (6) were carried out using t-BuOOH (3 M isooctane solution) as oxidant. With the former substrate, the nitro derivatives 2-amino-4-nitrotoluene (5a) and 2,4-dinitrotoluene (5b) (total yield of 5a + 5b ranges between 27-55%) were obtained with low yields and their amounts depend on the catalyst used. An other oxidation product of 5 was 3-amino-4(tert-butyldioxy)-4-methyl-2,5-cyclohexadien-1-mine hydrochloride (5c) (20%). In the oxidation reaction of 6,4-nitroaniline (6a) (11%) was obtained only with catalyst 1, being an unidentified black solid (6b) the major oxidation product. 6b was always the main product with all the studied catalysts (1-3), while the yields and nature of the recovered azobenzenes depend on the metalloporphyrin used: the highest yield of 4,4-dinitroazobenzene (6c) (37%) was obtained with 3 in the presence of acetic acid, 4,4'-diaminoazobenzene (6d) (19%) was obtained with 4, and 4-amino,4'-nitroazobenzene (6e) (17%) with 3. The catalytic reactions of Mo(O)(O-2)(2)(H2O)(HMPA) (7) (HMPA = hexamethylphosphotriamide), with 5, 6, and 4-aminodiphenylamine (8), in the presence of H2O2 (35% w/w water solution) as oxidant, were studied. The corresponding mononitro derivatives, 5a, 6a, and 4-nitrodiphenylamine (8a) were obtained with fair selectivity (35, 70, and 60%, respectively)

Single cell analysis of the localization of the hematopoietic stem cells within the bone marrow architecture identifies niche-specific proliferation dynamics

IntroductionHematopoietic stem cells (HSC) reside in the bone marrow (BM) in specialized niches which provide support for their self-replication and differentiation into the blood cells. Recently, numerous studies using sophisticated molecular and microscopic technology have provided snap-shots information on the identity of the BM niches in mice. In adults, HSC are localized around arterioles and sinusoids/venules whereas in juvenile mice they are in close to the osteoblasts. However, although it is well recognized that in mice the nature of the hematopoietic niche change with age or after exposure to inflammatory insults, much work remains to be done to identify changes occurring under these conditions. The dynamic changes occurring in niche/HSC interactions as HSC enter into cycle are also poorly defined. MethodsWe exploit mice harboring the hCD34tTA/Tet-O-H2BGFP transgene to establish the feasibility to assess interactions of the HSC with their niche as they cycle. In this model, H2BGFP expression is driven by the TET trans-activator under the control of the human CD34 promoter which in mice is active only in the HSC. Since Doxycycline inhibits TET, HSC exposed to this drug no longer express H2BGFP and loose half of their label every division allowing establishing the dynamics of their first 1-3 divisions. To this aim, we first validated user-friendly confocal microscopy methods to determine HSC divisions by hemi-decrement changes in levels of GFP expression. We then tracked the interaction occurring in old mice between the HSC and their niche during the first HSC divisions. ResultsWe determined that in old mice, most of the HSC are located around vessels, both arterioles which sustain quiescence and self-replication, and venules/sinusoids, which sustain differentiation. After just 1 week of exposure to Doxycycline, great numbers of the HSC around the venules lost most of their GFP label, indicating that they had cycled. By contrast, the few HSC surrounding the arterioles retained maximal levels of GFP expression, indicating that they are either dormant or cycle at very low rates. ConclusionThese results reveal that in old mice, HSC cycle very dynamically and are biased toward interactions with the niche that instructs them to differentiate