Proteoglycan fragmentation and respiratory mechanics in mechanically ventilated healthy rats.

This research investigated whether stretching of lung tissue due to increased positive alveolar pressure swings during mechanical ventilation (MV) at various tidal volumes (V(T)) might affect the composition and/or structure of the glycosaminoglycan (GAG) components of pulmonary extracellular proteoglycans. Experiments were performed in 30 healthy rats: 1) anesthetized and immediately killed (controls, C-0); 2) anesthetized and spontaneously breathing for 4 h (C-4h); and 3) anesthetized, paralyzed, and mechanically ventilated for 4 h with air at 0-cmH(2)O end-expiratory pressure and V(T) of 8 ml/kg (MV-1), 16 ml/kg (MV-2), 24 ml/kg (MV-3), or 32 ml/kg (MV-4), adjusting respiratory rates at a minute ventilation of 270 ml/min. Compared with C-0 and C-4h, a significant reduction of dynamic and static compliance of the respiratory system and of the lung was observed only in MV-4, while extravascular lung water significantly increased in MV-3 and MV-4, but not in MV-1 and MV-2. However, even in MV-1, MV induced a significant fragmentation of pulmonary GAGs. Extraction of covalently bound GAGs and wash out of loosely bound or fragmented GAGs progressively increased with increasing V(T) and was associated with increased expression of local (matrix metalloproteinase-2) and systemic (matrix metalloproteinase-9) activated metalloproteases. We conclude that 1) MV, even at "physiological" low V(T), severely affects the pulmonary extracellular architecture, exposing the lung parenchyma to development of ventilator-induced lung injury; and 2) respiratory mechanics is not a reliable clinical tool for early detection of lung injury.     

ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first‐degree relatives of type 2 diabetics

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.     

Low- and high-affinity phorbol ester and diglyceride interactions with protein kinase C: 1-O-alkyl-2-acyl-sn-glycerol enhances phorbol ester- and diacylglycerol-induced activity but alone does not induce activity

Phorbol ester-induced conventional protein kinase C (PKCalpha, -betaIota/IotaIota, and -gamma) isozyme activities are potentiated by 1,2-diacyl-sn-glycerol. This has been attributed to a "cooperative" interaction of the two activators with two discrete sites termed the low- and high-affinity phorbol ester binding sites, respectively [Slater, S. J., Milano, S. K., Stagliano, B. A., Gergich, K. J., Ho, C., Mazurek, A., Taddeo, F. J., Kelly, M. B., Yeager, M. D., and Stubbs, C. D. (1999) Biochemistry 38, 3804-3815]. Here, we report that the 1-O-alkyl ether diglyceride, 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), like its 1,2-diacyl counterpart, 1-oleoyl-2-acetyl-sn-glycerol (OAG), also potentiated PKCalpha, -betaI/II, and -gamma activities induced by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). Similar to OAG, HAG was found to bind to the low-affinity phorbol ester binding site and to enhance high-affinity phorbol ester binding, and to decrease the level of Ca(2+) required for phorbol ester-induced activity, while being without effect on the Ca(2+) dependence of membrane association. Thus, similar to OAG, HAG may also potentiate phorbol ester-induced activity by interacting with the low-affinity phorbol ester binding site, leading to a reduced level of Ca(2+) required for the activating conformational change. However, HAG was found not to behave like a 1,2-diacyl-sn-glycerol in that alone it did not induce PKC activity, and also in that it enhanced OAG-induced activity. The results reveal HAG to be a member of a new class of "nonactivating" compounds that modulate PKC activity by interacting with the low-affinity phorbol ester binding site.     

Correlation between germ tube production, phospholipase activity and serotype distribution in Candida albicans

One-hundred and thirteen Candida albicans strains isolated from patients infected by the human immunodeficiency virus (HIV) and twenty five from HIV-negative individuals were studied. The C. albicans strains isolated from different sites of the body were tested for germ-tube (GT), phospholipase production and serotype. The results obtained indicate that the serotype A was predominant in all the groups except for the vaginal strains. No correlation was observed between phospholipase activity and serotype distribution. Germ tube (GT) production was higher among the serotype B strains. A positive correlation between GT induction and phospholipase activity was observed only for the isolates from the oral cavity. It is possible that the correlation between phospholipase activity and high GT production in C. albicans strains can facilitate the penetration through the mucosa.     

Synthesis of 4-amino-6-(hetero)arylalkylamino-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives as potent A(2A) adenosine receptor antagonists

In previous papers (Colotta, V. et al. Arch. Pharm. Pharm. Med. Chem. 1999, 332, 39. Colotta, V. et al. J. Med. Chem. 2000, 43, 1158) we reported the synthesis and binding affinity at bovine (b) A₁ and A_2A and human (h) A₃ adenosine receptors (ARs) of the 4-amino-6-benzylamino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (compound A) which resulted in a potent and selective A_2A AR antagonist. Compound A provided the lead compound of a series of 6- or 8-(hetero)arylalkylamino-4-amino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives (compounds 1–20) which are the object of this paper. Most of the newly synthesized compounds are inactive at hA₃ ARs while they possess both nanomolar bA_2A affinities and different degrees of bA_2A versus bA₁ selectivity. The binding data show that hydrophilic substituents on the benzyl moiety are the most profitable for bA_2A receptor affinity. Furthermore, their steric hindrance seems to play an important role for the bA_2A AR interaction, thus suggesting that the 6-aralkylamino moiety of these ligands interacts with a size-limited binding pocket of this AR subtype. Thus, the SAR studies provided us some new insights about the structural requirements of the bA_2A AR recognition site.     

The CD45 locus in cattle: allelic polymorphism and evidence for exceptional positive natural selection

Cattle in Africa are a genetically diverse population that has resulted from successive introduction of Asian Bos indicus and European B. taurus cattle. However, analysis of mitochondrial genetic diversity in African cattle identified three lineages, one associated with Asian B. indicus, one with European B. taurus, and a third ascribed to an indigenous African sub-species of cattle. Due to their extended coevolution, indigenous African herbivores are generally tolerant to endemic African pathogens. We are interested in identifying alleles derived from the indigenous African cattle that may be associated with tolerance to African pathogens. An analysis of the locus which encodes the abundant plasma membrane-associated tyrosine phosphatase, CD45, identified three highly divergent allelic families in Kenya Boran cattle. Analysis of allelic distribution in a diverse range of cattle populations suggests a European B. taurus, an Asian B. indicus, and an African origin. This demonstrates not only significant allelic polymorphism at the CD45 locus in cattle but also convincing autosomal evidence for a distinct African sub-species of cattle. Furthermore, maximum-likelihood analysis of selection pressures revealed that the CD45 locus is subject to exceptionally strong natural selection which we suggest may be pathogen driven.     

Impact of glycosylation on the effect of cytokines. A special focus on oncology

The clinical use of cytokines is largely confined to the field of cancer treatment. Industrial procedures for the preparation of cytokines may or may not affect the glycosylation status of several cytokines. The glycosylation of cytokines may be of importance for the accomplishment of their biological functions, including their biological stability, their interaction with specific receptors and their pharmacokinetic behavior. The purpose of this review is thus to describe and discuss the role of cytokine glycosylation, based on experimental data, and with the clinical perspective on oncology. There is no general rule governing the effects induced by the presence of glycosylation on cytokine activity. Among different cytokines concerned by glycosylation status, data concerning G-CSF are sufficient to translate experimental findings into clinical evidence. For this cytokine, glycosylation confers advantages for both stability and biological activity. Although at weight equivalence, the glycosylated form confers greater biological activity than the non-glycosylated form, clinical studies have demonstrated that efficacy remains the same when glycosylated and non-glycosylated G-CSF are prescribed at bioequivalent doses.     

Unorthodox view of the functioning of a GABAA synapse

1. Studies about the permeation of labelled chloride and GABA across single plasma membranes microdissected from vestibular Deiters' neurons have yielded two unexpected results: (a) intracellular GABA stimulates chloride permeation in an asymmetric fashion (efflux being favoured); (b) under certain conditions GABA permeates by a diffusion mechanism in the out in direction across these plasma membranes.2. These two main results have been obtained over many years together with a host of other indications about the fine mechanism of these events. Thus, a picture has emerged of their physiological meaning within the context of the functioning of the GABA_A synapses between the Purkinje cells and the Deiters' neurons.3. In short, it is proposed that at these synapses GABA accumulates into the postsynaptic neuron after its release and activation of the postsynaptic receptors. GABA accumulated in the Deiters' neurons is involved in the process of chloride extrusion to build an inward directed electrochemical gradient for chloride.