Goran Poznanović

Ionizing radiation-induced expression of the genes associated with the acute response to injury in the rat

Total-body irradiation of rats with doses ranging from an LD10/30 to an LD100/30 induced a dose-dependent increase in the concentration of serum protein associated with the acute response to the irradiation. However, this increase was reached at a later time and was not as pronounced as described previously during the typical acute phase of the response found experimentally (A. Koj, in Structure and Function of Plasma Proteins, Vol. 1, pp. 73-131, Plenum Press, London, 1974). The greatest increase in the serum concentrations of acute-phase proteins was found from the third to the seventh days postirradiation. At these times, the serum concentrations of alpha 2-macroglobulin, haptoglobin, fibrinogen and cysteine protease inhibitor were raised from two- to fivefold, whereas alpha 1-acid glycoprotein was increased sixfold. Incorporation of [35S]methionine into total serum and acute-phase proteins indicated that the increase in the concentration of the acute-phase proteins was preceded by their de novo synthesis in the liver. The results that were obtained by dot-blot analysis showed that the basic course of change in the relative mRNA concentrations in the liver for the acute-phase proteins examined correlated with the changes in their protein concentrations in the serum; only the relative increase in the concentration of alpha 1-acid glycoprotein mRNA was significantly lower than the increase in proteins in the serum, suggesting that a fraction of the serum alpha 1-acid glycoprotein had an extrahepatic origin. On the basis of these results we concluded that total-body irradiation increased the expression of acute-phase protein genes.

Decreased O -GlcNAcylation of the key proteins in kinase and redox signalling pathways is a novel mechanism of the beneficial effect of α-lipoic acid in diabetic liver

The present study aimed to investigate the effects of the treatment with a-lipoic acid (LA), a naturally occurring compound possessing antioxidant activity, on liver oxidant stress in a rat model of streptozotocin (STZ)-induced diabetes by examining potential mechanistic points that influence changes in the expression of antioxidant enzymes such as catalase (CAT) and CuZn/Mn superoxide dismutase(s) (SOD). LA was administered for 4 weeks by daily intraperitoneal injections (10 mg/kg) to STZ-induced diabetic rats, starting from the last STZ treatment. LA administration practically normalised the activities of the indicators of hepatocellular injury, alanine and aspartate aminotransferases, and lowered oxidative stress, as observed by the thiobarbituric acid-reactive substance assay, restored the reduced glutathione:glutathione disulphide ratio and increased the protein sulfhydryl group content. The lower level of DNA damage detected by the comet assay revealed that LA reduced cytotoxic signalling, exerting a hepatoprotective effect. The LA-treated diabetic rats displayed restored specific enzymatic activities of CAT, CuZnSOD and MnSOD. Quantitative real-time PCR analysis showed that LA restored CAT gene expression to its physiological level and increased CuZnSOD gene expression, but the gene expression of MnSOD remained at the diabetic level. Although the amounts of CAT and CuZnSOD protein expression returned to the control levels, the protein expression of MnSOD was elevated. These results suggested that LA administration affected CAT and CuZnSOD expression mainly at the transcriptional level, and MnSOD expression at the post-transcriptional level. The observed LA-promoted decrease in the O-GlcNAcylation of extracellular signal-regulated kinase, protein 38 kinase, NF-kB, CCAAT/enhancer-binding protein and the antioxidative enzymes themselves in diabetic rats suggests that the regulatory mechanisms that supported the changes in antioxidative enzyme expression were also influenced by post-translational mechanisms.

STAT3/NFκB Interplay in the Regulation of α2‐Macroglobulin Gene Expression During Rat Liver Development and the Acute Phase Response

The synthesis of alpha‐2‐macroglobulin (α2M) is low in adult rat liver and elevated in fetal liver. During the acute‐phase (AP) response it becomes significantly increased in both adult and fetal liver. In this work, the cross talk of STAT3 and NF‐κB transcription factors during α2M gene expression was analysed. Using immunoblotting, their cellular compartmentalization was examined by comparing the cytoplasmic levels of STAT3 and NF‐κB with their active equivalents, the 86 and 91 kDa isoforms and p65‐subunit, respectively, in the nuclear extract and nuclear matrix. Different partitioning dynamics of the transcription factors were observed. At the level of protein‐DNA interactions, studied by α2M promoter affinity chromatography, it was established that different ratios of promoter‐binding STAT3 isoforms participated in elevated hepatic transcription in the basal state fetus and the AP‐adult, but only the 91 kDa isoform in the AP‐fetus. Unchanged levels of DNA‐bound p65 in the control and AP‐fetus suggest that it participated in constitutive transcription. The promoter‐binding of p65 observed in the AP‐adult suggests that it was involved in transcriptional stimulation of α2M expression. The selective enrichment of the AP‐adult nuclear matrix with promoter‐binding STAT3 disclosed the importance of this association in the induction of transcription. Protein‐protein interactions were examined by co‐immunoprecipitation. Interactions between the 86 kDa STAT3 isoform and p65 that were observed in the control and AP‐fetus and of both the 86 and 91 kDa STAT3 isoforms with p65 in the AP‐adult, suggest that protein‐protein interactions were functionally connected to increased transcription. We concluded that α2M gene expression is driven by developmental‐ and AP‐related mechanisms that rely on STAT3/NF‐κB interplay. IUBMB Life, 59: 170‐178, 2007

Poly(ADP‐ribose) polymerase‐1: Association with nuclear lamins in rodent liver cells

The distribution of poly(ADP‐ribose) polymerase‐1 (PARP‐1) over different nuclear compartments was studied by nuclear fractionation procedures and Western analysis revealing a prominent role of the nuclear matrix. This structure is operationally defined by the solubility properties of the A‐ and B‐type lamins under defined experimental conditions. We consistently observed that most of the nuclear matrix‐associated PARP‐1 partitioned, in an active form, with the insoluble, lamin‐enriched protein fractions that were prepared by a variety of established biochemical procedures. These PARP‐1–protein interactions resisted salt extraction, disulfide reduction, RNase and DNase digestion. An inherent ability of PARP‐1 to reassemble with the lamins became evident after a cycle of solubilization/dialysis using either urea or Triton X‐100 and disulfide reduction, indicating that these interactions were dominated by hydrophobic forces. Together with in vivo crosslinking and co‐immunoprecipitation experiments our results show that the lamins are prominent PARP‐1‐binding partners which could contribute to the functional sequestration of the enzyme on the nuclear matrix.

The protein composition of the hepatocyte nuclear matrix is differentiation-stage specific.

The protein composition of hepatocyte nuclear matrices was examined in rats from the 16th day of gestation to 75 days after birth (adult). An overall increase in size of the nuclear matrix was accompanied by quantitative and qualitative changes in its protein content. Quantitative changes of the major proteins of the peripheral lamina surrounding the isolated nuclear matrix were detected. By Western analysis we established that in pre- and postnatal nuclear matrices the relative concentrations of lamin C were greater than lamin A. After birth, the relative concentrations of both lamins progressively increased. In the adult nuclear matrix, the concentration of lamin A was greater than lamin C. In contrast, the relative concentrations of lamin B remained unchanged throughout development and growth. The relative concentrations of two nuclear matrix-associated regulatory proteins studied changed with development and growth: transcription factor C/EBPalpha isoforms, which were detected during the gestation period, increased notably after the first postnatal day, attaining a maximum at the adult stage; the high concentrations of the proliferating cell nuclear antigen (PCNA) perceptibly decreased after the 21st prenatal day. Changes in the composition of the nuclear matrix protein suggest that this structure coordinates nuclear functioning during cell differentiation.

Pharmacological interaction between oxcarbazepine and two COX inhibitors in a rat model of inflammatory hyperalgesia.

Oxcarbazepine, ibuprofen and etodolac have efficacy in inflammatory pain. The combination of different drugs activates both central and peripheral pain inhibitory pathways to induce additive or synergistic antinociception, and this interaction may allow lower doses of each drug combined and improve the safety profile, with lower side-effects. This study aimed to examine the effects of oxcarbazepine-ibuprofen and oxcarbazepine-etodolac combinations, in a rat model of inflammatory hyperalgesia, and determine the type of interaction between drugs. Rats were intraplantarly injected with carrageenan (0.1 ml, 1%) and the hyperalgesia was assessed by modified paw pressure test. The anti-hyperalgesic effects of oxcarbazepine, ibuprofen and etodolac and oxcarbazepine-ibuprofen and oxcarbazepine-etodolac combinations were examined. Drugs were co-administered in a fixed-dose fractions of the ED₅₀ and the type of interaction was determined by isobolographic analysis. Oxcarbazepine (40-160 mg/kg; p.o.), ibuprofen (10-120 mg/kg; p.o.) and etodolac (5-20 mg/kg; p.o.) produced a significant, dose-dependent anti-hyperalgesia in carrageenan-injected rats. ED₅₀ values (mean±SEM) for oxcarbazepine, ibuprofen and etodolac were 88.17±3.65, 47.07±10.27 and 13.05±1.42 mg/kg, respectively. Oxcarbazepine-ibuprofen and oxcarbazepine-etodolac combinations induced significant and dose-dependent anti-hyperalgesia. Isobolographic analysis revealed that oxcarbazepine exerts a synergistic interaction with ibuprofen, with almost 4-fold reduction of doses of both drugs in combination. In contrast, there was an additive interaction with etodolac. Synergistic interaction of oxcarbazepine with ibuprofen and its additive interaction with etodolac provide new information about the combination pain treatment and could be explored further in patients with inflammatory pain. Adverse effect analysis of the combinations is necessary to verify possible clinical use of the mixtures.

Co‐localization of PARP‐1 and lamin B in the nuclear architecture: A halo‐fluorescence‐ and confocal‐microscopy study

A functional interaction between poly(ADP‐ribose) polymerase‐1 (PARP‐1) and lamin B has recently been proposed by nuclear fractionation, crosslinking, and immunoprecipitation experiments. Here we use fluorescence microscopy to verify and extend these findings. We analyze nuclear halo preparations by fluorescence in situ immuno staining (FISIS), which shares attributes with traditional nuclear fractionation techniques, and by confocal laser scanning microscopy (CLSM). The results agree in that a major part of the enzyme co‐localizes with lamin B under physiological conditions, where PARP‐1 only has basal activity. After DNA damage and the associated activation of PARP‐1, and during the subsequent entry into apoptosis, dramatic changes occur: a gradual release of the enzyme from the lamina, accompanied by its accumulation in nucleoli. Our observations are in line with biochemical evidence for lamin B‐PARP‐1 interactions under physiological conditions and suggest ways by which these interactions are modified to support PARP‐functions in damage and its fate in apoptosis.

The acute phase response of rats to soman intoxication

The capacity of an organophosphate to elicit the acute phase response (APR) was assessed by studying the effects of acute soman intoxication on two major processes which characterize inflammation, cytokine production in macrophages and the expression of acute phase protein (APP) genes in the liver. It was established that the concentration of lymphostimulatory substances secreted by the macrophages of soman-intoxicated rats was increased to a level characteristic of the primary inflammatory reaction. Macrophage activation was followed by increased transcription rates of APP genes and the corresponding mRNA and protein synthesis in the liver. The pattern of the DNA-protein complexes obtained with nuclear extracts and the cis-element of the rat haptoglobin gene in the gel-retardation assay suggested that the molecular events which underlie the expression of APP genes of intoxicated rats are similar to those that occur during the APR. From these findings we concluded that soman intoxication was a metabolic injury which elicited the typical APR.

Association of the glucocorticoid receptor with STAT3, C/EBPβ, and the hormone-responsive element within the rat haptoglobin gene promoter during the acute phase response

Upregulation of haptoglobin (Hp) expression in the rat during the acute phase (AP) response is the result of synergistic effects of IL‐6–, IL‐1β–, and corticosterone‐activated signaling pathways. IL‐6 signaling terminates in cis–trans interactions of the Hp gene hormone‐responsive element (HRE) with transcription factors STAT3 and C/EBPβ. The aim of this study was to examine the unresolved molecular mechanism of glucocorticoid action. A 3‐fold rise in serum corticosterone at 2 and 4 h of the AP response induced by turpentine administration preceded a 2.3‐fold increase in the rate of Hp gene transcription at 12 h that was accompanied by a 4.8‐fold increase in glucocorticoid receptor (GR), the appearance of an 86‐kDa STAT3 isoform and 3.9‐, 1.9‐, and 1.7‐fold increased amounts of 91‐kDa STAT3, 35‐ and 42‐kDa C/EBPβ isoforms in the nucleus. These events resulted in 4.6‐ and 2.5‐fold increased Hp levels in the liver and serum at 24 h. HRE affinity chromatography and immunoblot analysis revealed that maximal occupancy of the HRE with GR, STAT3, and C/EBPβ at 12 h correlated with increased transcriptional activity of the Hp gene. Coimmunoprecipitation experiments showed that activated GR established de novo interaction with STAT3 isoforms while GR–C/EBPβ interactions observed during basal transcription increased during the AP response. Computer analysis of the HRE disclosed two potential GR‐binding sites: one overlapping STAT3, another adjacent to a C/EBPβ‐binding site. This finding and the experimental results suggest that activated GR through direct interactions with STAT3 and C/EBPβ, participates in Hp gene upregulation as a transcriptional coactivator.

Proteolytic events in cryonecrotic cell death: Proteolytic activation of endonuclease P23 ☆

Although cryosurgery is attaining increasing clinical acceptance, our understanding of the mechanisms of cryogenic cell destruction remains incomplete. While it is generally accepted that cryoinjured cells die by necrosis, the involvement of apoptosis was recently shown. Our studies of liver cell death by cryogenic temperature revealed the activation of endonuclease p23 and its de novo association with the nuclear matrix. This finding is strongly suggestive of a programmed-type of cell death process. The presumed order underlying cryonecrotic cell death is addressed here by examining the mechanism of p23 activation. To that end, nuclear proteins that were prepared from fresh liver, which is devoid of p23 activity, were incubated with protein fractions isolated from liver exposed to freezing/thawing that possessed a presumed p23 activation factor. We observed that the activation of p23 was the result of a proteolytic event in which cathepsin D played a major role. Different patterns of proteolytic cleavage of nuclear proteins after in vitro incubation of nuclei and in samples isolated from frozen/thawed liver were observed. Although both processes induced p23 activation, the incubation experiments generated proteolytic hallmarks of apoptosis, while freezing/thawing of whole liver resulted in typical necrotic PARP-1 cleavage products and intact lamin B. As an explanation we offer a hypothesis that after freezing, cells possess the potential to die through necrotic as well as apoptotic mechanisms, based on our finding that the cytosol of cells exposed to cryogenic temperatures contains both necrotic and apoptotic executors of cell death.