Apoptotic pathways: involvement of lysosomal proteases

Apoptosis or programmed cell death is the major mechanism used by multicellular organisms to remove infected, excessive and potentially dangerous cells. Cysteine proteases from the caspase family play a crucial role in the process. However, there is increasing evidence that lysosomal proteases are also involved in apoptosis. In this review various lysosomal proteases and their potential contribution to propagation of apoptosis are discussed.     

Glycosylation enhances functional stability of the chemotactic cytokine CCL2

The human chemokine CCL2 gene was expressed in the yeast P.pastoris and gave rise to a mixture of differently glycosylated recombinant proteins. In comparison to non-glycosylated E.coli-derived CCL2, glycosylated yeast CCL2L was 4-20 times less active in a chemotactic assay in vitro. However, CCL2L could maintain full activity upon prolonged incubation at 37 degrees C, whereas the non-glycosylated chemokine readily lost activity. It could be hypothesized that glycosylation is a mechanism used by the organism to modulate CCL2 stability. The partial loss of specific activity due to glycosylation is balanced by the advantage of prolonging the effectiveness of chemokine. Thus, differential glycosylation allows one to obtain highly effective short-lived CCL2 or less-effective long-lived CCL2 and may thus represent a novel mechanism of adaptation to pathological versus physiological conditions.     

Prognostic significance of standardized AgNOR analysis and Ki-67 immunostaining in gastrointestinal stromal tumors

OBJECTIVE: To evaluate the prognostic utility of argyrophilic nucleolar organizer region (AgNOR) protein parameters and Ki-67-immunostained growth fraction (Ki-67 labelling index) and to correlate AgNORs with Ki-67 LI and the main clinicopathologic parameters in gastrointestinal stromal tumors (GISTs). STUDY DESIGN: On 55 patients with surgically excised GISTs, visualization and quantification of AgNORs were performed as specified in the guidelines of the Committee on AgNOR Quantification. RESULTS: AgNOR protein area (NORA) > or = 5.28 microns 2 was statistically associated with mitotic rate > or = 5 x 10 high-power fields (hpfs) (P < .001) and presence of necrosis (P < .001); Ki-67 LI > or = 9.69% was significantly associated with mitotic rate > or = 5 x 10 hpfs (P < .001), size > or = 5 cm (P = .033) and presence of necrosis (P < .001). Ki-67 LI and NORA strongly correlated. Preliminary Kaplan-Meier survival analysis showed that an increased value of NORA, Ki-67 LI, mitotic rate, tumor size and presence of necrosis had a negative influence on patient survival. Multivariate regression analysis showed that only NORA and Ki-67 LI were independent parameters in predicting the clinical outcome for patients with GISTs. Mitotic rate and necrosis remained as independent prognostic factors when NORA and Ki-67 LI were not allowed to enter in models. CONCLUSION: AgNOR protein quantity, as determined by image cytometry, and Ki-67 immunostaining seem to represent reliable predictive parameters in GISTs and are independent of mitotic rate, tumor dimension and necrosis.     

Synthesis,characterization and antitumor activity of platinum(II) complexes with diethyl and monoethyl 2-quinolylmethylphosphonates

A series of new platinum(II) complexes with diethyl (2-dqmp) and monoethyl (2-Hmqmp) 2-quinolylmethylphosphonates have been prepared and studied. Both organophosphorus ligands by reaction with [PtX(4)](2-) (X=Cl, Br) form either the molecular or ionic complexes depending on the acidity of the reaction solution. Dihalide adducts, trans-[PtL(2)X(2)] (L=2-dqmp, 2-Hmqmp), with N-bonded ligand through the quinoline nitrogen were obtained in the neutral medium, while under acidic conditions at pH<3 were isolated the ion-pair salt complexes, [LH](2)[PtX(4)], containing the protonated quinoline ligand as cation and tetrahaloplatinate complex as anion. In addition, 2-Hmqmp at pH approximately 3.5 forms quinolinium hexahalodiplatinum salt complexes, [2-H(2)mqmp](2)[Pt(2)X(6)], while the chelate complex, [Pt(2-mqmp)(2)].2H(2)O, with N,O-bonded ligand through the quinoline nitrogen and the deprotonated phosphonic acid oxygen was obtained at pH>6. The new complexes were characterized on the basis of elemental and thermogravimetric analyses, conductometric measurements, and by infrared and (1)H NMR spectral studies. As a preliminary assessment of their biological activity, complexes were evaluated for their in vitro cytostatic activity in an epidermoid human carcinoma (KB) and murine leukemia (L1210) cell lines. The results obtained were compared with those obtained for the corresponding Pd(II) complexes.     

Left ventricular end-diastolic pressure-volume relationship in septic rats with open thorax

To estimate changes in compliance, we evaluated the effects of sepsis on the end-diastolic pressure-volume relationship (EDPVR) in the left ventricle of rats that had undergone an open thorax procedure. Sepsis was induced in male Wistar Hannover rats (n = 7; 240 to 270 g) by intraperitoneal administration of a slurry of cecal contents; control rats (n = 7) were given 5% dextrose only. On the third day after induction of sepsis, left ventricular (LV) pressure and LV dimensions were recorded simultaneously in animals of both groups. Using a micromanometer and ultrasonic crystals, measurements were obtained at baseline and during the increase of afterload. Blood samples were taken for determination of complete blood count, white blood cell differential count, and lactate concentration, and for bacteriologic examination. Septic rats lost weight, and developed changes in body temperature, ascites, and abscesses in the abdominal and thoracic cavities, gram-negative bacteremia, and increase in heart rate. On the third day after induction of sepsis, LV EDPVR decreased, compared with that in the control rats (regression coefficients: control group, 8.41 to 23.95; sepsis group, 3.94 to 7.92). Myocardial compliance in the left ventricle increased on the third day of sepsis in the open-thorax rat model, as evidenced by the downward shift of LV EDPVR in rats with sepsis, compared with controls.     

The plant invertase inhibitor shares structural properties and disulfide bridges arrangement with the pectin methylesterase inhibitor

Attempts to purify the inhibitor of pectin methylesterase (PMEI) from the soluble extract of ripe apricot (Prunus armeniaca) fruit led to isolation of a protein (Pa-INH) similar to PMEI, but having invertase inhibitory activity against vacuolar invertase from tomato. The molecular charge, the native and SDS-PAGE molecular weights were similar to those of PMEI. Partial amino acid sequence indicated a high level of identity with invertase inhibitors and a significant identity with PMEI. Circular dichroism analysis showed a mainly -helix secondary structure for both the inhibitors and a higher thermostability of Pa-INH. Four Cys residues forming disulfide bridges in PMEI were conserved in Pa-INH. Similarly to PMEI, these residues were linked by disulfide bridges (first to second and third to fourth). The free Cys139 of PMEI is substituted by Ala in Pa-INH. The results reported in this study suggest a common structural arrangement of the two inhibitors.     

Effects of hypoxic preconditioning on the hypoxicreoxygenated atria from fed and fasted rats

Hypoxic preconditioning (PC) was studied using rat atria set up isometrically in 10 mM dextrose medium and paced at 1 Hz, applying three different protocols wherein fed and 24-h fasted rats were used in protocols 1 and 2 and only the fed in protocol 3. In protocol 1, PC was achieved applying a 5 min hypoxia followed by 10 min of reoxygenation before the onset of a 60 min hypoxia and 60 min reoxygenation. In protocol 2 the 5 min and a posterior 45 min hypoxia were applied in the absence of dextrose whereas in the 10 min and 60 min reoxygenation periods dextrose was present. In protocol 3, two cycles of 5 min dextrose-free hypoxic periods were applied before the sustained hypoxia (dextrose-free) and reoxygenation periods (10 min and final 45 min, both in the presence of dextrose). In the control groups of all protocols, the equilibration periods were prolonged to compensate the duration of PC. In the control groups of protocols 1 and 2, the sustained hypoxia evoked greater disturbances of contractility and a smaller post-hypoxic recovery in the fasted than in the fed rat atria. In protocol 1, PC markedly reduced the rise in resting tension and improved the post-hypoxic recovery in the fasted rat atria whereas in the fed rat atria protective effects were small and brief. In protocol 2, PC evoked a small reduction of contracture only in the atria from fasted rats and in protocol 3, PC exacerbated the hypoxic disturbances. These data suggest that PC effects depend both on the severity of the PC stress and the sustained hypoxia; and that PC does not require coronary flow.     

Procedures to characterize and study P2Z/P2X7 purinoceptor: flow cytometry as a promising practical, reliable tool

The expression of P2Z/P2X7 purinoceptor in different cell types is well established. This receptor is a member of the ionotropic P2X receptor family, which is composed by seven cloned receptor subtypes (P2X1 - P2X7). Interestingly, the P2Z/P2X7 has a unique feature of being linked to a non-selective pore which allows the passage of molecules up to 900 Da depending on the cell type. Early studies of P2Z/P2X7 purinoceptor were exclusively based on classical pharmacological studies but the recent tools of molecular biology have enriched the analysis of the receptor expression. The majority of assays and techniques chosen so far to study the expression of P2Z/P2X7 receptor explore directly or indirectly the effects of the opening of P2Z/P2X7 linked pore. In this review we describe the main techniques used to study the expression and functionality of P2Z/P2X7 receptor. Additionally, the increasing need and importance of a multifunctional analysis of P2Z/P2X7 expression based on flow cytometry technology is discussed, as well as the adoption of a more complete analysis of P2Z/P2X7 expression involving different techniques.     

Automated technique to evaluate thymocyte adhesion to thymic epithelial cells

Thymocyte adhesion to thymic epithelial cells is a relevant issue during intrathymic T-cell differentiation, and directly intervenes in the generation and expansion of the T-cell repertoire. In view of these data, it was apparent the usefulness of an automated strategy to evaluate the degree of thymic epithelial cell-thymocyte adhesion. This prompted us to develop an ELISA procedure (using an anti-Thy1 reagent) to determine the degree of thymocyte adhesion onto cultured thymic epithelial cells. The procedure described herein is simple, non-radioactive and reproducible. Additionally, it can potentially be applied to quantitate the degree of thymocyte adhesion to any cellular or non-cellular substrate (for example, extracellular matrix). Moreover, it detected fluctuations of thymocyte adhesion secondary to glucocorticoid treatment of epithelial cells. Thus, it can be regarded as a further tool to analyze intrathymic interactions.