Quaternary structure and functional properties of Penaeus monodon hemocyanin

The hemocyanin of the tiger shrimp, Penaeus monodon, was investigated with respect to stability and oxygen binding. While hexamers occur as a major component, dodecamers and traces of higher aggregates are also found. Both the hexamers and dodecamers were found to be extremely stable against dissociation at high pH, independently of the presence of calcium ions, in contrast to the known crustacean hemocyanins. This could be caused by only a few additional noncovalent interactions between amino acids located at the subunit-subunit interfaces. Based on X-ray structures and sequence alignments of related hemocyanins, the particular amino acids are identified. At all pH values, the p50 and Bohr coefficients of the hexamers are twice as high as those of dodecamers. While the oxygen binding of hexamers from crustaceans can normally be described by a simple two-state model, an additional conformational state is needed to describe the oxygen-binding behaviour of Penaeus monodon hemocyanin within the pH range of 7.0 to 8.5. The dodecamers bind oxygen according to the nested Monod-Whyman-Changeaux (MWC) model, as observed for the same aggregation states of other hemocyanins. The oxygen-binding properties of both the hexameric and dodecameric hemocyanins guarantee an efficient supply of the animal with oxygen, with respect to the ratio between their concentrations. It seems that under normoxic conditions, hexamers play the major role. Under hypoxic conditions, the hexamers are expected not to be completely loaded with oxygen. Here, the dodecamers are supposed to be responsible for the oxygen supply.     

CD8+ CD28- T regulatory lymphocytes inhibiting T cell proliferative and cytotoxic functions infiltrate human cancers

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+ CD28- T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+ CD28- T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+ CD25+ T regulatory lymphocytes associate with CD8+ CD28- T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.     

Adrenergic origin of very low-frequency blood pressure oscillations in the unanesthetized rat

Spectral analysis of cardiovascular signals has been extensively used to investigate circulatory homeostatic mechanisms. However, the nature of very low-frequency (VLF) fluctuations remains unclear. Because we previously observed enhanced VLF fluctuations in blood pressure (BP) in the sympathectomized rat (a model characterized by markedly increased plasma epinephrine levels), the aims of our study were to assess whether the genesis of VLF fluctuations in BP depends on circulating catecholamines and to determine which adrenergic receptor(s) and which membrane ion channel(s) are involved. We used continuous intra-arterial BP recordings from unanesthetized unrestrained rats to compute the power of VLF fluctuations in BP in the intact condition, during acute ganglionic blockade with hexamethonium, and after restoration of BP levels by infusion (in addition to hexamethonium) of adrenergic agonists (epinephrine, norepinephrine, and clonidine) or nonadrenergic vasoconstrictors (vasopressin). Effects of infusion of specific adrenergic receptor blockers (propranolol, prazosin, and yohimbine) with hexamethonium and catecholamines and infusion of various membrane ion channel blockers on VLF fluctuations in BP were also evaluated. Our results are as follows. 1) Ganglionic blockade drastically reduced BP levels and VLF fluctuations. 2) All vasoconstrictors restored BP levels, but only adrenergic vasoconstrictors generated striking VLF fluctuations in BP. 3) Catecholamine-induced fluctuations were abolished by alpha2-, but not alpha1- or beta-, adrenergic receptor blockade and by Ba2+-sensitive K+ channel or L-type Ca2+ channel, but not by other ion channel, blockers. We conclude that, in the conscious, unrestrained ganglion-blocked rat, catecholamine infusion generates VLF fluctuations in BP through stimulation of alpha2-receptors and activation of Ba2+-sensitive K+ channels. These fluctuations may have (patho)physiological relevance under conditions of disrupted circulatory homeostasis.     

Levels of atherogenic lipoproteins are unexpectedly reduced in interstitial fluid from type 2 diabetes patients

At a given level of serum cholesterol, patients with T2D have an increased risk of developing atherosclerosis compared with nondiabetic subjects. We hypothesized that T2D patients have an increased interstitial fluid (IF)-to-serum gradient ratio for LDL, due to leakage over the vascular wall. Therefore, lipoprotein profiles in serum and IF from 35 T2D patients and 35 healthy controls were assayed using fast performance liquid chromatography. The IF-to-serum gradients for VLDL and LDL cholesterol, as well as for apoB, were clearly reduced in T2D patients compared with healthy controls. No such differences were observed for HDL cholesterol. Contrary to our hypothesis, the atherogenic VLDL and LDL particles were not increased in IF from diabetic patients. Instead, they were relatively sparser than in healthy controls. The most probable explanation to our unexpected finding is that these lipoproteins are more susceptible to retainment in the extravascular space of these patients, reflecting a more active uptake by, or adhesion to, tissue cells, including macrophages in the vascular wall. Further studies are warranted to further characterize the mechanisms underlying these observations, which may be highly relevant for the understanding of why the propensity to develop atherosclerosis is increased in T2D.     

Synergistic inhibition of pancreatic adenocarcinoma cell growth by trichostatin A and gemcitabine

We investigated the ability of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to interact with gemcitabine (GEM) in inducing pancreatic cancer cell death. The combined treatment with TSA and GEM synergistically inhibited growth of four pancreatic adenocarcinoma cell lines and induced apoptosis. This effect was associated with the induction of reactive oxygen species (ROS) by GEM, increased expression of the pro-apoptotic BIM gene by both TSA and GEM and downregulation of the 5'-nucleotidase UMPH type II gene by TSA. The expression of other genes critical for GEM resistance (nucleoside transporters, deoxycytidine kinase, cytidine deaminase, and ribonucleotide reductase genes) was not affected by TSA. The functional role of ROS in cell growth inhibition by GEM was supported by (i) a significantly reduced GEM-associated growth inhibition by the free radical scavenger N-acetyl-L-cysteine, and (ii) a positive correlation between the basal level of ROS and sensitivity to GEM in 10 pancreatic cancer cell lines. The functional role of both Bim and 5'-nucleotidase UMPH type II in cell growth inhibition by TSA and GEM was assessed by RNA interference assays. In vivo studies on xenografts of pancreatic adenocarcinoma cells in nude mice showed that the association of TSA and GEM reduced to 50% the tumour mass and did not cause any apparent form of toxicity, while treatments with TSA or GEM alone were ineffective. In conclusion, the present study demonstrates a potent anti-tumour activity of TSA/GEM combination against pancreatic cancer cells in vitro and in vivo, strongly supporting the use of GEM in combination with an HDAC inhibitor for pancreatic cancer therapy.     

Immunohistochemical analysis of pRb2/p130, VEGF, EZH2, p53, p16(INK4A), p27(KIP1), p21(WAF1), Ki-67 expression patterns in gastric cancer

Although the considerable progress against gastric cancer, it remains a complex lethal disease defined by peculiar histological and molecular features. The purpose of the present study was to investigate pRb2/p130, VEGF, EZH2, p53, p16(INK4A), p27(KIP1), p21(WAF1), Ki-67 expressions, and analyze their possible correlations with clinicopathological factors. The expression patterns were examined by immunohistochemistry in 47 patients, 27 evaluated of intestinal-type, and 20 of diffuse-type, with a mean follow up of 56 months and by Western blot in AGS, N87, KATO-III, and YCC-2, -3, -16 gastric cell lines. Overall, stomach cancer showed EZH2 correlated with high levels of p53, Ki-67, and cytoplasmic pRb2/p130 (P < 0.05, and P < 0.01, respectively). Increased expression of EZH2 was found in the intestinal-type and correlated with the risk of distant metastasis (P < 0.05 and P < 0.01, respectively), demonstrating that this protein may have a prognostic value in this type of cancer. Interestingly, a strong inverse correlation was observed between p27(KIP1) expression levels and the risk of advanced disease and metastasis (P < 0.05), and a positive correlation between the expression levels of p21(WAF1) and low-grade (G1) gastric tumors (P < 0.05), confirming the traditionally accepted role for these tumor-suppressor genes in gastric cancer. Finally, a direct correlation was found between the expression levels of nuclear pRb2/p130 and low-grade (G1) gastric tumors that was statistically significant (P < 0.05). Altogether, these data may help shed some additional light on the pathogenetic mechanisms related to the two main gastric cancer histotypes and their invasive potentials.     

Changes in the ciliate assemblage along a fluvial system related to physical, chemical and geomorphological characteristics

Samples were collected monthly from the water-sediment interface at six stations along the Mincio River (northern Italy) during a 1-year study of the ciliated protozoan communities. Four stations were located upstream of the Mantua lakes in the hyporhithron fluvial zone and two stations were located in the potamon fluvial zone between the Mantua lakes and the confluence with the Po River. A total of 133 species of active trophic ciliates belonging to 76 genera were found. Community structures revealed in this data were analysed using some statistical methods (similarity index, and categorical principal component analysis (CATPCA)) and this allowed the determination of differences between stations and between ciliate communities characteristic of stations. Species typical of the ecotypes located in both rhithron and potamon fluvial zones were defined. The saprobic index and valency analysis methods were used to quantify organic input and to follow changes in saprobicity along the river. A change in the ciliate communities was observed between stations located upstream and stations located downstream of the town of Mantua. The former were composed mainly of beta-mesosaprobic species, typical of the hill zone of running waters, while in the latter increased numbers of alpha-mesosaprobic species are associated with the higher anthropogenic pressures. Our results reiterate the high sensitivity shown by ciliated protozoa as indicators of organic load in watercourses.     

AMPA receptor expression in mouse testis and spermatogonial GC-1 cells: A study on its regulation by excitatory amino acids

Excitatory amino acids (EAAs) are found present in the nervous and reproductive systems of animals. Numerous studies have demonstrated a regulatory role for Glutamate (Glu), d -aspartate ( d -Asp) and N-methyl- d -aspartate (NMDA) in the control of spermatogenesis. EAAs are able to stimulate the Glutamate receptors, including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Here in, we assess expression of the main AMPAR subunits, GluA1 and GluA2/3, in the mouse testis and in spermatogonial GC-1 cells. The results showed that both GluA1 and GluA2/3 were localized in mouse testis prevalently in spermatogonia. The subunit GluA2/3 was more highly expressed compared with GluA1 in both the testis and the GC-1 cells. Subsequently, GC-1 cells were incubated with medium containing l -Glu, d -Glu, d -Asp or NMDA to determine GluA1 and GluA2/3 expressions. At 30 minutes and 2 hours of incubation, EAA-treated GC-1 cells showed significantly higher expression levels of both GluA1 and GluA2/3. Furthermore, p-extracellular signal-regulated kinase (ERK), p-Akt, proliferating cell nuclear antigen (PCNA), and Aurora B expressions were assayed in l -Glu-, d -Glu-, and NMDA-treated GC-1 cells. At 30 minutes and 2 hours of incubation, treated GC-1 cells showed significantly higher expression levels of p-ERK and p-Akt. A consequent increase of PCNA and Aurora B expressions was induced by l -Glu and NMDA, but not by d -Glu. Our study demonstrates a direct effect of the EAAs on spermatogonial activity. In addition, the increased protein expression levels of GluA1 and GluA2/3 in EAA-treated GC-1 cells suggest that EAAs could activate ERK and Akt pathways through the AMPAR. Finally, the increased PCNA and Aurora B levels may imply an enhanced proliferative activity.     

A specific inhibitor of lactate dehydrogenase overcame the resistance toward gemcitabine in hypoxic mesothelioma cells,and modulated the expression of the human equilibrative transporter-1

ABSTRACT

Malignant pleural mesothelioma (MPM) is a very hypoxic malignancy, and hypoxia has been associated with resistance towards gemcitabine. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis. Therefore we investigated the combination of a new LDH-A inhibitor (NHI-1) with gemcitabine in MPM cell lines. Under hypoxia (O₂ tension of 1%) the cell growth inhibitory effects of gemcitabine, were reduced, as demonstrated by a 5- to 10-fold increase in IC₅₀s. However, the simultaneous addition of NHI-1 was synergistic (combination index < 1). Flow cytometry demonstrated that hypoxia caused a G1 arrest, whereas the combination of NHI-1 significantly increased gemcitabine-induced cell death. Finally, the mRNA expression levels of the human equilibrative transporter-1 (hENT1) were significantly down-regulated under hypoxia, but treatment with NHI-1 was associated with a recovery of hENT1 expression. In conclusion, our data show that hypoxia increased MPM resistance to gemcitabine. However, cell death induction and modulation of the key transporter in gemcitabine uptake may contribute to the synergistic interaction of gemcitabine with the LDH-A inhibitor NHI-1 and support further studies for the rational development of this combination.