Alcohol oxidation by Candida guilliermondii yeasts grown on hexadecanol

A number of enzyme systems involved in the first steps of hexadecane oxidation can be induced by hexadecanol, an intermediate product of hexadecane degradation. It has also been found that, in Candida guilliermondii cells and in their mitochondrial fraction, an oxidase system is induced when the cells are grown on hexadecanol. This system is similar to that in cells grown on hexadecane; it oxidises higher alcohols at a high rate and is not inhibited by the inhibitors of the man phosphorylating respiration chain. The membrane-bound alcohol dehydrogenase and aldehyde dehydrogenase activities resistant to pyrazole, an inhibitor of cytosol ethanol dehydrogenase, are induced together with the oxidase activity when the cells are grown on hexadecanol as well as on hexadecane. The oxidation of higher alcohols by whole cells is entirely inhibited by azide although their oxidation by mitochondria is resistant to the action of azide; apparently, azide inhibits the transport of alcohols into the cell.     

Esterase activity of hydrocarbon-oxidizing bacteria

The activity of esterase was studied in bacteria oxidizing hydrocarbons and belonging to the genera Rhodococcus, Arthrobacter and Pseudomonas. Indophenyl acetate was used as a substrate of the reaction catalysed by the enzyme. Exocellular esterases were not found. Endocellular esterases differed in their activity and thermostability both among the genera and among species of one and the same genus.     

Functional role of carbohydrate residues in human immunoglobulin G and therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies (TMA) provide an important means for treating diseases that were previously considered untreatable. Currently more than 40 full-size TMAs created primarily based on immunoglobulin G1 are widely used for treating various illnesses. Glycosylation of TMA is among other numerous factors that affect their biological activity, effector functions, immunogenicity, and half-life in the patient’s serum. The importance of carbohydrate residues for activity of human serum immunoglobulin and TMA produced in animal cells is considered in this review, with emphasis given to N-glycosylation of the Fc fragment of the antibody.     

The discovery of Hsp70 domain with cell-penetrating activity

Chaperone Hsp70 can cross the plasma membrane of living cells using mechanisms that so far have not received much research attention. Searching the part of the molecule that is responsible for transport ability of Hsp70, we found a cationic sequence composed of 20 amino acid residues on its surface, KST peptide, which was used in further experiments. We showed that KST peptide enters living cells of various origins with the same efficiency as the full-length chaperone. KST peptide is capable of carrying cargo with a molecular weight 30 times greater than its own into cells. When we compared the membrane-crossing activity of KST peptide in complex with Avidin (KST–Av complex) with that of similarly linked canonical TAT peptide, we found that TAT peptide penetrated SK-N-SH human neuroblastoma cells at a similar rate and efficiency as the KST peptide. Furthermore, KST peptide can carry protein complexes consisting of a specific antibody coupled to the peptide through the Avidin bridge. An antibody to Hsp70 delivered to SK-N-SH cells with high expression level of Hsp70 reduced the protective power of the chaperone and sensitized the cells to the pro-apoptotic effect of staurosporine. We studied the mechanisms of penetration of KST–Av and full-length Hsp70 inside human neuroblastoma SK-N-SH and human erythroleukemia K-562 cells and found that both used an active intracellular transport mechanism that included vesicular structures and negatively charged lipid membrane domains. Competition analysis of intracellular transport showed that the chaperone reduced intracellular penetration of KST peptide and conversely KST peptide prevented Hsp70 transport in a dose-dependent manner.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-014-0554-z) contains supplementary material, which is available to authorized users.     

New viral vector for efficient production of target proteins in plants

A new potato virus X (PVX)-based viral vector for superproduction of target proteins in plants has been constructed. The triple gene block and coat protein gene of PVX were substituted by green fluorescent protein. This reduced viral vector was delivered into plant cells by agroinjection (injection of Agrobacterium tumefaciens cells, carrying viral vector cDNA within T-DNA, into plant leaves), and this approach allowed to dramatically reduce the size of the vector genome. The novel vector can be used for production of different proteins including pharmaceuticals in plants.     

mRNA Translation Regulation by the Gly-Ala Repeat of Epstein-Barr Virus Nuclear Antigen 1

The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5′ untranslated region (UTR) of the c-myc mRNA to the 5′ UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA-1) and latency-associated nuclear antigen 1 (LANA-1), from Kaposi''s sarcoma-associated herpesvirus (KSHV), are major latency proteins of these two gammaherpesviruses that are essential for maintaining viral episomes in infected cells (21, 22). Independent studies suggest that both proteins have evolved mechanisms to remain largely invisible to the immune system, which could otherwise eliminate latently infected cells (8, 9, 19, 25). These mechanisms act in cis and are mediated via an internal repeat region. In the case of EBNA-1 this region consists of an N-terminal glycine-alanine repeat (GAr), and for LANA-1 the region consists of a glutamine-glutamate-aspartate central repeat (QED-CR). Although the two domains do not share amino acid homology, both retard their own synthesis to reduce the production of defective ribosomal products that can be processed for the major histocompatibility complex (MHC) class I-restricted antigen presentation pathway (23, 24), highlighting the importance of translation control in regulating MHC class I-restricted antigen presentation. To compensate for their low rates of synthesis, both proteins also have slow turnover rates (4, 8).Regulation of translation for most prokaryotic and eukaryotic mRNAs occurs at the level of initiation, but there are examples where regulation of protein synthesis depends on the elongation stage (17). The two main types of translation initiation are the classic cap-dependent and the less frequent cap-independent translation mechanisms (5, 7, 11, 14, 16). In the former, the preinitiation complex is formed around the cap structure in the 5′ untranslated region (UTR) of the message, whereas in the latter the 40S subunit is directed toward the mRNA via an internal ribosome entry site (IRES). The mechanism of GAr- and LANA-1-mediated control of translation seems different from other types of viral regulation in several aspects. The EBNA-1 GAr is 60 to 300 amino acids long, depending on virus isolate, and is positioned in the N-terminal part of the protein. The GAr message is GC rich but does not activate protein kinase R and eukaryotic initiation factor 2α phosphorylation (25). The fact that the GAr has to be encoded to suppress translation, coupled with the restricted use of GGG and GGA codons to express Gly and of GCA to express Ala in the GAr (GAT, GAG, and CAG for aspartic acid, glutamic acid, and glutamine, respectively, in the LANA sequence), could suggest that codon exhaustion might explain the effect of these repeats. However, manipulations of sequence order, orientation, and composition of the QED-CR and GAr domains and the observation that antibodies directed toward the GAr can stimulate translation in vitro instead favor a direct role for the amino acid sequence (8, 25).Here, we have studied GAr-mediated regulation of translation in vitro and in vivo. The results presented suggest that, once synthesized, the nascent GAr peptide sequence prevents the assembly of the following upstream ribosomes. This knowledge should further understanding of how amino acid repeat sequences can affect mRNA translation in cis and should shed light on a novel type of viral control of mRNA translation and its implications in regulating MHC class I-restricted antigen presentation.     

Mammalian end binding proteins control persistent microtubule growth

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.     

Tumor-supportive and Osteoclastogenic Changes Induced by Breast Cancer-derived Factors Are Reversed by Inhibition of γ-Secretase

During breast cancer metastasis to bone, tumor cells home to bone marrow, likely targeting the stem cell niche, and stimulate osteoclasts, which mediate osteolysis required for tumor expansion. Although osteoblasts contribute to the regulation of the hematopoietic stem cell niche and control osteoclastogenesis through production of proresorptive cytokine RANKL (receptor activator of NF-κB ligand), their role in cancer metastases to bone is not fully understood. C57BL/6J mouse bone marrow cells were treated for 3–12 days with ascorbic acid (50 μg/ml) in the presence or absence of 10% medium conditioned by breast carcinoma cells MDA-MB-231, 4T1, or MCF7. Treatment with cancer-derived factors resulted in a sustained 40–60% decrease in osteoblast differentiation markers, compared with treatment with ascorbic acid alone, and induced an osteoclastogenic change in the RANKL/osteoprotegerin ratio. Importantly, exposure of bone cells to breast cancer-derived factors stimulated the subsequent attachment of cancer cells to immature osteoblasts. Inhibition of γ-secretase using pharmacological inhibitors DAPT and Compound E completely reversed cancer-induced osteoclastogenesis as well as cancer-induced enhancement of cancer cell attachment, identifying γ-secretase activity as a key mediator of these effects. Thus, we have uncovered osteoblasts as critical intermediary of premetastatic signaling by breast cancer cells and pinpointed γ-secretase as a robust target for developing therapeutics potentially capable of reducing both homing and progression of cancer metastases to bone.