Analysis of Bone Mineral Profile After Prolonged Every-Other-Day Feeding in C57BL/6J Male and Female Mice

Biological Trace Element Research - Intermitted fasting or every-other-day feeding (EOD) has many positive effects in rodents and humans. Our goal was to describe how EOD influences bone mineral...     

Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants

Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP) neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.     

Diether derivatives of homo- or substituted piperidines as non-imidazole histamine H3 receptor ligands

Synthesis and biological activities of a series of homo- or substituted piperidine unsymmetrical diethers are described. The novel compounds were evaluated for histamine H₃ receptor binding affinities at recombinant human H₃ receptor stably expressed in HEK-293 cells. All diethers showed in vitro affinities in nanomolar concentration range. The most potent compounds are 1-[3-(3-(4-chlorophenoxy)propoxy)propyl]-3-methylpiperidine 11 (K_i = 3.2 nM) and 1-[3-(3-(4-chlorophenoxy)propoxy)propyl]azepane 13 (K_i = 3.5 nM).     

The influence of opioids on matrix metalloproteinase-2 and -9 secretion and mRNA levels in MCF-7 breast cancer cell line

Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in degradation of extracellular matrix, a process that initiates uncontrolled spread of proliferating cancer cells and therefore plays a crucial role in cancer invasion and metastasis. Compounds able to modulate MMP activity may become important tools in cancer research. In the present study we examined the effect of two μ-selective opioids, morphine and endomorphin-2 (EM-2) on the production of MMP-2 and MMP-9 in MCF-7 cells. We report that both opioids time- and concentration-dependently inhibited the expression and secretion of these MMPs. The observed effect was not reversed by naloxone (Nal). Further experiments showed that morphine and EM-2 decreased endothelial nitric oxide synthase (eNOS) mRNA level and nitric oxide (NO) secretion in MCF-7 cells. These findings indicate that attenuation of MMP secretion by opioids was not mediated by opioid receptors but was under the control of nitric oxide system.     

Synthesis of acyloxymethyl ester prodrugs of the transferable protein farnesyl transferase substrate farnesyl methylenediphosphonate

Three isoprenoid diphosphate analogues of farnesyl diphosphate (FPP) where the diphosphate has been replaced by methylene diphosphonate and the negative charges masked by frangible pivaloyloxymethyl (POM) esters were prepared. Farnesyl methylenediphosphonate is a sub-micromolar substrate for protein farnesyl transferase. The tripivaloyloxymethyl esters of isoprenoid methylenediphosphonate have significantly increased lipophilicity and may act as important farnesyl diphosphate prodrugs.     

Influence of Incubation Conditions on Hydrolysis Efficiency and Iodine Enrichment in Baker’s Yeast

The influence of incubation conditions, enzyme type, hydrolysis time, and potassium iodide concentration on hydrolysis and iodine enrichment were studied in supernatant and pellets of Saccharomyces cervisiae hydrolysates. The type of enzyme used and incubation time significantly influence hydrolysis efficiency and protein concentration in supernatant and pellet. The highest protein hydrolysis efficiency was obtained by 24-h incubation with papain. Significantly lower values were observed for pepsin and autolysis. The potassium iodide concentration influences the iodine content of supernatant and pellet, but not hydrolysis. Iodide enrichment of supernatant and pellet depends on the concentration of iodide using during incubation. High concentration of iodide and long incubation times were the conditions for optimal iodide enrichment and high-protein hydrolysates. The optimal hydrolysis efficiency and iodine enrichment were obtained during 24-h incubation with papain in a 4.5-mM potassium iodide medium. The efficiency reached 98.22% with iodine concentrations of 2,664.91 and 9,200.67 μg/g iodine in pellet and supernatant, respectively.     

Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers

Elastin-like polypeptides (ELP) are artificial, genetically encodable biopolymers, belonging to elastomeric proteins, which are widespread in a wide range of living organisms. They are composed of a repeating pentapeptide sequence Val–Pro–Gly–Xaa–Gly, where the guest residue (Xaa) can be any naturally occurring amino acid except proline. These polymers undergo reversible phase transition that can be triggered by various environmental stimuli, such as temperature, pH or ionic strength. This behavior depends greatly on the molecular weight, concentration of ELP in the solution and composition of the amino acids constituting ELPs. At a temperature below the inverse transition temperature (T_t), ELPs are soluble, but insoluble when the temperature exceeds T_t. Furthermore, this feature is retained even when ELP is fused to the protein of interest. These unique properties make ELP very useful for a wide variety of biomedical applications (e.g. protein purification, drug delivery etc.) and it can be expected that smart biopolymers will play a significant role in the development of most new materials and technologies. Here we present the structure and properties of thermally responsive elastin-like polypeptides with a particular emphasis on biomedical and biotechnological application.     

Analysis of O6-methylguanine-DNA methyltransferase methylation status in sporadic colon polyps

Background/aimThe aim of our study was to check how MGMT methylation status together with known factors influenced the risk of colon cancer development.Materials and methodsWe examined patients with colon polyps. Information concerning gender, age, lifestyle, diet, anthropometry and medical information, including cancer and family history of cancer, was analyzed. Polymorphism variety of MGMT gene was investigated in another study. Genetic analysis for MGMT methylation assessment was performed for polyp tissue samples from 143 patients.ResultsPositive methylation MGMT status was found in 55 patients. There was no correlation between gender and MGMT methylation status (p = 0.43). We did not find correlation between patients younger and older than 60 (p = 0.87). There was no correlation between smoking and MGMT methylation status (p = 0.36). We did not find correlation between BMI and MGMT methylation status (p = 0.86). We did not find correlation between MGMT methylation status and colon cancer in familial history (p = 0.45).ConclusionOur study showed no correlations between methylation status of MGMT polymorphisms and clinical features like age, gender, polyp localization, smoking status, or obesity. It has been shown previously that MGMT methylation status may show nonspecific methylation in colon polyps. Gene methylation status in adenoma tissues has also been associated by other authors with the adenoma's size, histology, and degree of atypia. In our study, we evaluated the gene methylation status in colon polyps and found no association with adenoma characteristics. The present study showed no correlation for MGMT methylation in polyps in different regions of colon.     

Alleviation of deleterious effects of protein mutation through inactivation of molecular chaperones

Molecular chaperones recognize and bind destabilized proteins. This can be especially important for proteins whose stability is reduced by mutations. We focused our study on a major chaperone system, RAC-Ssb, which assists folding of newly synthesized polypeptides in the yeast cytosol. A sensitive phenotypic assay, the red color of Ade2 mutants, was used to screen for variants with metabolic activity dependent on RAC-Ssb. None of the Ade2 mutants were found to exhibit lower metabolic activity after inactivation of RAC-Ssb. In order to explicitly test the relationship between protein instability and activity of chaperones, a series of temperature sensitive Ade2 mutants were tested in the presence or absence of RAC-Ssb. The growth of Ade2(ts) mutants at elevated temperatures was enhanced if chaperones were missing. Similar pattern was found for thermally sensitive mutants of several other genes. Because RAC-Ssb normally supports the folding of proteins, it appears paradoxical that catabolic activity of mutants is reduced when these chaperones are present. We suggest that under non-stressful conditions, molecular chaperones are tuned to support folding of native proteins, but not that of mutated ones.     

Design of selective substrates of proteinase 3 using combinatorial chemistry methods

In this study, chemical synthesis of the selective chromogenic/fluorogenic substrates for proteinase 3 is described. The substrates’ sequence was obtained using combinatorial chemistry methods. Deconvolution of the tripeptide library against proteinase 3 with general formula ABZ-X₃-X₂-X₁-ANB-NH₂ yielded the active sequence. Selected peptide was further modified on its C terminus to investigate the impact of chromophore moiety modification on enzyme-substrate interaction. To determine specificity, activity of selected substrates was characterized against proteinase 3 and neutrophil elastase. Finally, the peptide ABZ-Tyr-Tyr-Abu-ANB-NH₂ displayed the highest value of specificity constant (k_cat/K_M = 189 × 10³ M⁻¹ s⁻¹) for proteinase 3. To the best of our knowledge, this is the first short peptide that undergoes selective proteolysis by proteinase 3 and displays no significant hydrolysis in the presence of human neutrophil elastase and cathepsin G.