Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

Sequence of deltaF508 CFTR allele identified at present is lacking in medieval specimens from Central Poland. Preliminary results

deltaF508 is the most common (70%) among over 1000 mutations of the gene encoding ATP-regulated chloride channel, namely CFTR--cystic fibrosis transmembrane regulator. The time which passed from the calculated mutation event was anticipated on the basis of the frequency of contemporary haplotypes, but not on its direct identification. The presence of three base pairs deletion in the ancient DNA (aDNA) isolated from skeletal remains of the Middle Ages origin was investigated. Teeth excavated in the area of three sites located in Central Poland were processed for a DNA. 6 out of 82 samples did not produce amplificable fragments of DNA. Although the number of specimens analyzed was sufficient to confirm the presence of the rare mutation, the deltaF508 CFTR sequence was not found in the remains of individuals living back 35 - 45 generations. The absence of the mutated allele in the particular geographic region cannot state for the status of mutated allele throughout the country, especially at times when migrations were limited and movements of people were more area restricted than at present days.     

Carbonic anhydrase inhibitors. Selective inhibition of human tumor-associated isozymes IX and XII and cytosolic isozymes I and II with some substituted-2-mercapto-benzenesulfonamides

A series of 2-mercapto-substituted-benzenesulfonamides has been prepared by a unique two-step procedure starting from the corresponding 2-chloro-substituted benzenesulfonamides. Compounds bearing an unsubstituted mercapto group and the corresponding S-benzoyl derivatives were investigated as inhibitors of four isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), i.e., the cytosolic, ubiquitous isozymes CA I and II, as well as the transmembrane, tumor associated isozymes CA IX and XII. These derivatives were medium potency hCA I inhibitors (K(I)s in the range of 1.5-5.7 microM), two derivatives were strong hCA II inhibitors (K(I)s in the range of 15-16 nM), whereas the others showed weak activity. These compounds inhibited hCA IX with inhibition constants in the range 160-1950 nM and hCA XII with inhibition constants in the range 1.2-413 nM. Some of these derivatives showed a certain degree of selectivity for inhibition of the tumor-associated over the cytosolic isoforms, being thus interesting leads for the development of potentially novel applications in the management of hypoxic tumors which overexpress CA IX and XII.     

Decrease of serum chemerin concentration in patients with end stage renal disease after successful kidney transplantation

Chemerin is an adipokine associated with metabolic syndrome, systemic inflammation and innate immune system. It has been suggested recently that the decrease in renal function may cause an increase in serum chemerin concentration. In this paper we investigated the effect of kidney transplantation on elevated serum chemerin concentration in dialyzed patients with end stage renal disease (ESRD). Twenty five ESRD patients were tested before and 3months after the kidney transplantation. The control group was comprised of twenty one healthy subjects. Serum chemerin concentrations were measured using commercial ELISA kit, and were related to clinical status, and biomarkers of renal function. We have shown that the kidney transplantation resulted in the decrease of the serum chemerin concentration. Concomitantly, serum creatinine, blood urea nitrogen, phosphate and C-reactive protein concentrations were significantly reduced, while estimated glomerular filtration rate (eGFR), calcium and hemoglobin substantially increased. Univariate regression analysis showed that serum chemerin concentration was positively correlated with serum creatinine and phosphate concentrations and negatively correlated with eGFR. The results presented here indicate that the serum chemerin concentration in patients with ESRD normalizes after the kidney transplantation, and provide additional evidence that serum chemerin concentration is related to renal function.     

A Novel Insight into the Oxidoreductase Activity of Helicobacter pylori HP0231 Protein

Background

The formation of a disulfide bond between two cysteine residues stabilizes protein structure. Although we now have a good understanding of the Escherichia coli disulfide formation system, the machineries at work in other bacteria, including pathogens, are poorly characterized. Thus, the objective of this work was to improve our understanding of the disulfide formation machinery of Helicobacter pylori, a leading cause of ulcers and a risk factor for stomach cancer worldwide.

Methods and Results

The protein HP0231 from H. pylori, a structural counterpart of E. coli DsbG, is the focus of this research. Its function was clarified by using a combination of biochemical, microbiological and genetic approaches. In particular, we determined the biochemical properties of HP0231 as well as its redox state in H. pylori cells.

Conclusion

Altogether our results show that HP0231 is an oxidoreductase that catalyzes disulfide bond formation in the periplasm. We propose to call it HpDsbA.     

The 3' splice site of influenza A segment 7 mRNA can exist in two conformations: a pseudoknot and a hairpin

The 3' splice site of influenza A segment 7 is used to produce mRNA for the M2 ion-channel protein, which is critical to the formation of viable influenza virions. Native gel analysis, enzymatic/chemical structure probing, and oligonucleotide binding studies of a 63 nt fragment, containing the 3' splice site, key residues of an SF2/ASF splicing factor binding site, and a polypyrimidine tract, provide evidence for an equilibrium between pseudoknot and hairpin structures. This equilibrium is sensitive to multivalent cations, and can be forced towards the pseudoknot by addition of 5 mM cobalt hexammine. In the two conformations, the splice site and other functional elements exist in very different structural environments. In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop. The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site.     

PTEN as a prognostic and predictive marker in postoperative radiotherapy for squamous cell cancer of the head and neck

Background

Tumor suppressor PTEN is known to control a variety of processes related to cell survival, proliferation, and growth. PTEN expression is considered as a prognostic factor in some human neoplasms like breast, prostate, and thyroid cancer.

Methodology/Principal Findings

In this study we analyzed the influence of PTEN expression on the outcome of a randomized clinical trial of conventional versus 7-days-a-week postoperative radiotherapy for squamous cell cancer of the head and neck. The patients with cancer of the oral cavity, oropharynx, and larynx were randomized to receive 63 Gy in fractions of 1.8 Gy given 5 days a week (CF) or 7 days a week (p-CAIR). Out of 279 patients enrolled in the study, 147 paraffin blocks were available for an immunohistochemical assessment of PTEN. To evaluate the prognostic value of PTEN expression and the effect of fractionation relative to PTEN, the data on the outcome of a randomized clinical trial were analyzed. Tumors with a high intensity of PTEN staining had significant gain in the loco-regional control (LRC) from p-CAIR (5-year LRC 92.7% vs. 70.8%, for p-CAIR vs. CF, p = 0.016, RR = 0.26). By contrast, tumors with low intensity of PTEN did not gain from p-CAIR (5-year LRC 56.2% vs. 47.2%, p = 0.49, RR = 0.94). The intensity of PTEN highly affected the LRC in a whole group of 147 patients (5-year LRC 80.9% vs. 52.3% for high vs. low PTEN, p = 0.0007, RR = 0.32). In multivariate Cox analysis, including neck node involvement, EGFR, nm23, Ki-67, p53, cyclin D1, tumor site and margins, PTEN remained an independent predictor of LRC (RR = 2.8 p = 0.004).

Conclusions/Significance

These results suggest that PTEN may serve as a potent prognostic and predictive marker in postoperative radiotherapy for high-risk squamous cell cancer of the head and neck.     

Differential phosphorylation of thylakoid proteins in mesophyll and bundle sheath chloroplasts from maize plants grown under low or high light

In C4 plants, such as maize, the photosynthetic apparatus is partitioned over two cell types called mesophyll (M) and bundle sheath (BS), which have different structure and specialization of the photosynthetic thylakoid membranes. We characterized protein phosphorylation in thylakoids of the two cell types from maize grown under either low or high light. Western blotting with phosphothreonine antibodies and ProQ phosphostaining detected light-dependent changes in the protein phosphorylation patterns. LC-MS/MS with alternating CID and electron transfer dissociation sequencing of peptide ions mapped 15 protein phosphorylation sites. Phosphorylated D2, CP29, CP26, Lhcb2 proteins, and ATPsynthase were found only in M membranes. A previously unknown phosphorylation site was mapped in phosphoenolpyruvate carboxykinase from the BS cells. Phosphorylation stoichiometry was calculated from the ratios of normalized ion currents for phosphorylated to nonphosphorylated peptide pairs from the D1, D2, CP43, and PbsH proteins of photosystem II (PSII). Every PSII in M thylakoids contained on average 1.5 ± 0.1 or 2.3 ± 0.2 phosphoryl groups in plants grown under either low or high light, while in BS membranes the corresponding numbers were 0.25 ± 0.1 or 0.7 ± 0.2, respectively. It is suggested that the phosphorylation level, as well as turnover of PSII depend on the structure of thylakoids.