Assessment of nutritional status of gastroenterology patients in Croatia

Malnutrition is a common feature of gastroenterological diseases. In this study, nutritional status of the patients admitted to Department of Gastroenterology at University Hospital Center Zagreb was assessed. Anthropometric, dietetic, biochemical methods and method of Subjective Global Assessment (SGA) was used. The study group included 284 patients admitted to the Hospital. Malnutrition, as defined by SGA, was found in 61.1% of the patients, of whom 75% were moderately and 25% severely malnourished. Those patients classified as moderately and extremely malnourished by SGA were found to have statistically lower values of BMI, albumin, total proteins, calcium, iron, triglycerides, cholesterol, vitamin A and lymphocytes as compared to those who were adequately nourished. The prevalence of malnutrition in hospitalized patients treated at the Department of Gastroenterology is high. The use of nutritional screening with multiple measures would be important in the early identification and treatment of these patients and would help decrease this high prevalence.     

Simian Virus 40 Activates ATR-Δp53 Signaling To Override Cell Cycle and DNA Replication Control

During infection, simian virus 40 (SV40) attempts to take hold of the cell, while the host responds with various defense systems, including the ataxia-telangiectasia mutated/ATM-Rad3 related (ATM/ATR)-mediated DNA damage response pathways. Here we show that upon viral infection, ATR directly activates the p53 isoform Δp53, leading to upregulation of the Cdk inhibitor p21 and downregulation of cyclin A-Cdk2/1 (AK) activity, which force the host to stay in the replicative S phase. Moreover, downregulation of AK activity is a prerequisite for the generation of hypophosphorylated, origin-competent DNA polymerase α-primase (hypo-Polα), which is, unlike AK-phosphorylated Polα (P-Polα), recruited by SV40 large T antigen (T-Ag) to initiate viral DNA replication. Prevention of the downregulation of AK activity by inactivation of ATR-Δp53-p21 signaling significantly reduced the T-Ag-interacting hypo-Polα population and, accordingly, SV40 replication efficiency. Moreover, the ATR-Δp53 pathway facilitates the proteasomal degradation of the 180-kDa catalytic subunit of the non-T-Ag-interacting P-Polα, giving rise to T-Ag-interacting hypo-Polα. Thus, the purpose of activating the ATR-Δp53-p21-mediated intra-S checkpoint is to maintain the host in S phase, an optimal environment for SV40 replication, and to modulate the host DNA replicase, which is indispensable for viral amplification.Infection of quiescent CV-1 cells with the primate polyomavirus simian virus 40 (SV40) induces cell cycle progression and stimulates host cell DNA replication, which is mandatory for viral amplification. SV40 uses only a single viral protein, T antigen (T-Ag), for its own replication; all other components have to be provided by the host. Initially, a specifically phosphorylated subclass of T-Ag binds to a palindromic sequence in the SV40 origin (43), and in the presence of ATP, T-Ag forms a double-hexamer nucleoprotein complex leading to structural distortion and unwinding of origin DNA sequences (5). In concert with the cellular single-strand DNA binding protein RPA and topoisomerase I, the DNA helicase activity of T-Ag promotes more-extensive origin unwinding, forming a preinitiation complex (pre-RC), resulting in an initiation complex (53). Once the initiation complex forms, the primase activity of the heterotetrameric DNA polymerase α-primase (Polα) complex, consisting of the p180 catalytic subunit, the p70 regulatory subunit, and the p48/58 primase subunits, synthesizes a short RNA primer on each template strand, which is extended by the DNA polymerase activity of Polα (6, 17). Immediately after the first nascent RNA/DNA primer is synthesized, the complete replication machinery is assembled, and elongation at both forks by the processive DNA polymerase δ ensues (62). Thus, during the initiation of SV40 replication, T-Ag performs many of the functions attributed to the eukaryotic pre-RC complex proteins, including Orc, Cdc6, Cdt1, and kinase-independent cyclin E, which facilitates loading of the putative replication helicase Mcm2-7 onto the eukaryotic origin (4, 18). Biochemical evidence shows that initiation of SV40 and eukaryotic DNA replication occurs by the physical interaction of Polα with the appropriate pre-RC in the immediate vicinity of the origin. In SV40, Polα is loaded onto the origin by direct physical contact between the helicase T-Ag and its p180 N-terminal domain C (14, 15, 16). In eukaryotes, Cdc45, Mcm10, and And-1 cooperate to recruit Polα to the origin-initiation complex, thereby tethering the replicase to the origin-loaded Mcm2-7 helicase (34, 61).Although SV40 and chromosomal DNA replication share the same essential replication factors that are recruited to the appropriate pre-RC, there are noticeable differences between the SV40 and eukaryotic replication systems. The viral system allows unregulated multiple firing of the origin, whereas in the eukaryotic system, origin-dependent initiation of replication is regulated and restricted to firing only once per cell cycle. Reinitiation at origins within a cell cycle is prevented by the inactivation of pre-RC components in S and G₂. The cyclin-dependent kinases (Cdks) play a central role in establishing a block to rereplication through phosphorylation of each of the components. At present, several proteins of the mammalian pre-RC, such as Orc1, Cdt1, Cdc6, and the Mcm complex are phosphorylated by cyclin A (cycA)-Cdk2/1 (AK) and, as a result, are degraded or inactivated (1, 26, 30, 33, 40). Nevertheless, not all of the pre-RC components mentioned above are utilized by SV40, and accordingly, not all are involved in viral initiation control. However, in both replication systems, DNA synthesis is initiated by Polα and its initiation activity is regulated by Cdks (55). Moreover, AK-phosphorylated Polα is not recruited to mammalian origins in vivo (13) and is unable to initiate SV40 replication in vitro (47, 57, 58). Considering that cellular mechanisms blocking the rereplication of DNA act by AK phosphorylation of the replication factors mentioned above, especially Polα, it is feasible to suggest that downregulation or even inhibition of this kinase activity promotes dysregulation of replication control in SV40-infected cells.One pathway that leads to downregulation of AK activity in response to cellular stress is the intra-S checkpoint, which employs the novel p53 isoform Δp53 (45). In UV-damaged S-phase cells, ATR (ataxia-telangiectasia mutated [ATM]-Rad3 related)-activated Δp53 upregulates the Cdk inhibitor p21, resulting in a transient reduction in AK activity and decelerated S-phase progression (45). Here we demonstrate that SV40 lytic infection triggers the ATR signaling cascade, leading to the activation of Δp53 and accordingly a p21-mediated drop in AK activity to prevent AK-catalyzed inactivation of the hypophosphorylated, T-Ag-interacting Polα (hypo-Polα) subclass, which is essential for the initiation of viral DNA replication.     

Structural Basis of the Regulation of the CbpA Co-chaperone by its Specific Modulator CbpM

CbpA, one of the Escherichia coli DnaJ homologues, acts as a co-chaperone in the DnaK chaperone system. Despite its extensive similarity in domain structure and function to DnaJ, CbpA has a unique and specific regulatory mechanism mediated through the small protein CbpM. Both CbpA and CbpM are highly conserved in bacteria. Earlier studies showed that CbpM interacts with the N-terminal J-domain of CbpA inhibiting its co-chaperone activity but the structural basis of this interaction is not known. Here, we have combined NMR spectroscopy, site-directed mutagenesis and surface plasmon resonance to characterize the CbpA/CbpM interaction at the molecular level. We have determined the solution structure of the CbpA J-domain and mapped the residues that are perturbed upon CbpM binding. The NMR data defined a broad region on helices α2 and α3 as involved in the interactions. Site-directed mutagenesis has been used to further delineate the CbpA J-domain/CbpM interface. We show that the binding sites of CbpM and DnaK on CbpA J-domain overlap, which suggests a competition between DnaK and CbpM for binding to CbpA as a mechanism for CbpA regulation. This study also provides the explanation for the specificity of CbpM for CbpA versus DnaJ, by identifying the key residues for differential binding.     

Hymenolepis diminuta: Effect of infection on ion transport in colon and blood picture of rats

The aim of this study was to examine the effect of an infection with Hymenolepis diminuta on ion transport in an isolated colon and blood picture of rats. Fifty rats were orally infected with five cysticercoids of H. diminuta. The experimental groups of rats were assigned to four groups: group I - 8 days post-infection (dpi), group II - 16 dpi, group III - 40 dpi and group IV- 60 dpi. The control group comprised non-infected rats. The experiments consisted of measuring the transepithelial electrical potential difference (PD) and the transepithelial electrical resistance (R) of the rat colon under controlled conditions as well as during mechanical stimulation (MS) using a modified Ussing chamber. Ion transport was modified using inhibitors of the epithelial sodium channel (amiloride - AMI) and the epithelial chloride channel (bumetanide - BUME), and also using capsaicin (CAPSA), a substance which activates C-fibres. The experimental data presented in this study indicates that experimental hymenolepidosis inhibits sodium and chloride ion transport in the epithelium of the rat colon, with preserved tight junction continuity (except at 40 dpi) and a decreased mechanical sensitivity. The effect of capsaicin on ion transport in the rat colon was varied. In control rats it increased ionic current, and in H. diminuta-infected rats it did not cause any changes in PD.Blood picture in this study showed a statistically significantly lower red blood cells (RBC) count and haemoglobin (HGB) concentration in infected rats in comparison to non-infected. Red cell distribution width (RDW) values and platelet (PLT) count were negatively correlated with the duration of infection, whereas mean corpuscular volume (MCV) value was positively correlated. We did not observe leukocytosis during infection, and amongst the differential leukocyte counts eosinophils and basophils showed statistically significant lower values in infected rats in comparison to non-infected.Our results indicate that hymenolepidosis is associated with the activation of inflammatory mediators and stimulation of nervous fibres, which significantly affects the function of ion channels in the epithelium of the colon in the host. At the same time, a significant decrease in eosinophil count during infection suggests that such an infection did not trigger a strong immunological reaction in rats.     

Fuzzy-oil-drop hydrophobic force field--a model to represent late-stage folding (in silico) of lysozyme

A model of hydrophobic collapse (in silico), which is generally considered to be the driving force for protein folding, is presented in this work. The model introduces the external field in the form of a fuzzy-oil-drop assumed to represent the environment. The drop is expressed in the form of a three-dimensional Gauss function. The usual probability value is assumed to represent the hydrophobicity distribution in the three-dimensional space of the virtual environment. The differences between this idealized hydrophobicity distribution and the one represented by the folded polypeptide chain is the parameter to be minimized in the structure optimization procedure. The size of fuzzy-oil-drop is critical for the folding process. A strong correlation between protein length and the dimension of the native and early-stage molecular form was found on the basis of single-domain proteins analysis. A previously presented early-stage folding (in silico) model was used to create the starting structure for the procedure of late-stage folding of lysozyme. The results of simulation were found to be promising, although additional improvements for the formation of beta-structure and disulfide bonds as well as the participation of natural ligand in folding process seem to be necessary.     

Immuno-proteasome subunit LMP7 is up-regulated in the ischemic kidney in an experimental model of renovascular hypertension

Immuno-proteasome is thought to be responsible for the processing of intracellular antigens and is induced when cells are treated with the inflammatory cytokines promoting cellular immunity. We tested the possibility that immuno-proteasome can be up-regulated in renal cells exposed to a long-lasting ischemia and inflammation in an experimental model of two-kidney, one-clip renovascular hypertension in the rat. Western blotting showed that immuno-proteasome subunit, LMP7, was up-regulated in the clipped ischemic kidney that was atrophic, but not in the contralateral unclipped kidney that underwent compensatory hypertrophy. Immunohistochemical analysis revealed that LMP7 was highly expressed in cortical epithelial and endothelial cells of the ischemic kidney. Surprisingly, the second immuno-subunit, LMP2, was almost undetectable, indicating that renal ischemia may induce exclusively the LMP7 subunit. We also found that renal ischemia neither reduced the SDS-stimulated proteasomal activity nor affected a high level of the PA28 activator. Thus, the results provide evidence that LMP7 immuno-subunit is induced in renal cells exposed to a long-lasting renal ischemia and inflammation, and that there is a direct link between LMP induction and renal atrophy. This opens an opportunity to study a role for LMP-containing proteasomes in the kidneys and other organs undergoing reduction in mass in diseases accompanied by a long-lasting ischemia and inflammatory responses.     

Yersinia YopP-induced apoptotic cell death in murine dendritic cells is partially independent from action of caspases and exhibits necrosis-like features

Yersinia outer protein P (YopP) is a virulence factor of Yersinia enterocolitica that is injected into the cytosol of host cells where it targets MAP kinase kinases (MKKs) and inhibitor of κB kinase (IKK)-β resulting in inhibition of cytokine production as well as induction of apoptosis in murine macrophages and dendritic cells (DC). Here we show that DC death was only partially prevented by the broad spectrum caspase inhibitor zVAD-fmk, indicating simultaneous caspase-dependent and caspase-independent mechanisms of cell death induction by YopP. Microscopic analyses and measurement of cell size demonstrated necrosis-like morphology of caspase-independent cell death. Application of zVAD-fmk prevented cleavage of procaspases and Bid, decrease of the inner transmembrane mitochondrial potential ΔΨ_m and mitochondrial release of cytochrome c. From these data we conclude that YopP-induced activation of the mitochondrial death pathway is mediated upstream via caspases. In conclusion, our results suggest that YopP simultaneously induces caspase-dependent apoptotic and caspase-independent necrosis-like death in DC. However, it has to be resolved if necrosis-like DC death occurs independently from apoptotic events or as an apoptotic epiphenomenon.     

A mutation in the herbicide target site acetohydroxyacid synthase produces morphological and structural alterations and reduces fitness in Amaranthus powellii

We investigated the effect of a herbicide resistance-conferring mutation on fitness in Amaranthus powellii. Morphological and histological observations were made. Growth and leaf appearance were recorded for six resistant and six susceptible populations. The competitiveness of a susceptible population was compared with that of a resistant population using a replacement series experiment. Leaves of the resistant plants were distorted and much smaller than those of susceptible plants. Additionally, they exhibited an abnormal morphological and structural pattern consisting of a mosaic of heterogeneous areas in the same leaf blade. The roots and stems had similar structures in susceptible and resistant plants, but the former were up to four times more developed. The resistant plants were slower to develop and produced 67% less biomass and 58% lower leaf area than susceptible plants. Under competitive conditions, one susceptible population outperformed one resistant population by 7-15 times. The Trp(574)Leu acetohydroxyacid synthase (AHAS) mutation appears to have considerable pleiotropic effects on the early growth and development of the plants which, in competitive conditions, greatly reduce fitness.     

Role of bradykinin B1 and B2 receptors in normal blood pressure regulation

With inhibition or absence of the bradykinin B2 receptor (B2R), B1R is upregulated and assumes some of the hemodynamic properties of B2R, indicating that both participate in the maintenance of normal vasoregulation or to development of hypertension. Herein we further evaluate the role of bradykinin in normal blood pressure (BP) regulation and its relationship with other vasoactive factors by selectively blocking its receptors. Six groups of Wistar rats were treated for 3 wk: one control group with vehicle alone, one with concurrent administration of B1R antagonist R-954 (70 microg x kg(-1) x day(-1)) and B2R antagonist HOE-140 (500 microg x kg(-1) x day(-1)), one with R-954 alone, one with HOE 140 alone, one with concurrent administration of both R-954 and HOE-140 plus the angiotensin antagonist losartan (5 mg x kg(-1) x day(-1)), and one with only losartan. BP was measured continuously by radiotelemetry. Only combined administration of B1R and B2R antagonists produced a significant BP increase from a baseline of 107-119 mmHg at end point, which could be partly prevented by losartan and was not associated with change in catecholamines, suggesting no involvement of the sympathoadrenal system. The impact of blockade of bradykinin on other vasoregulating systems was assessed by evaluating gene expression of different vasoactive factors. There was upregulation of the eNOS, AT1 receptor, PGE2 receptor, and tissue kallikrein genes in cardiac and renal tissues, more pronounced when both bradykinin receptors were blocked; significant downregulation of AT2 receptor gene in renal tissues only; and no consistent changes in B1R and B2R genes in either tissue. The results indicate that both B1R and B2R contribute to the maintenance of normal BP, but one can compensate for inhibition of the other, and the chronic inhibition of both leads to significant upregulation in the genes of related vasoactive systems.