Regulation of biosynthesis and intracellular localization of rice and tobacco homologues of nucleosome assembly protein 1

The nucleosome assembly protein 1 (NAP1) is considered to be a conserved histone chaperone, facilitating the assembly of nucleosomes in all eukaryotes. However, studies in yeast and animal cells also indicated that NAP1 proteins have diverse functions likely independent of nucleosome-assembly activity. Here, we describe the isolation and characterization of cDNAs encoding NAP1-like proteins from the monocotyledon rice ( Oryza sativa L.) and the dicotyledon tobacco ( Nicotiana tabacum L.). Northern-blot analysis demonstrated that the two rice NAP1-like genes are predominantly expressed in stem tissues such as root and shoot apical meristems as well as in young flowers. During the cell cycle, all four tobacco NAP1-like genes are highly expressed, with one of them showing a slightly increased expression at the G1/S transition. These results are consistent with a role for plant NAP1-like proteins in cell division. In vitro binding assays revealed that different NAP1-like proteins bind, with distinct relative binding strengths, to different classes of histone. Intracellular localization analyses showed that some NAP1-like proteins could be targeted into the nucleus whereas others are exclusively cytoplasm-localized. It is thus likely that different plant NAP1-like proteins have distinct functions in vivo. Plant NAP1-like proteins were observed to concentrate around the metaphase plate and in the phragmoplast, suggesting a role in mitotic events and cytokinesis.     

Characteristics and application of S1–P1 nucleases in biotechnology and medicine

3′–nucleases/nucleotidases of the S1–P1 family (EC 3.1.30.1) are single–strand–specific or non-specific zinc–dependent phosphoesterases present in plants, fungi, protozoan parasites, and in some bacteria. They participate in a wide variety of biological processes and their current biotechnological applications rely on their single–strand preference, nucleotide non-specificity, a broad range of catalytic conditions and high stability. We summarize the present and potential utilization of these enzymes in biotechnology and medicine in the context of their biochemical and structure–function properties. Explanation of unanswered questions for bacterial and trypanosomatid representatives could facilitate development of emerging applications in medicine.     

Receptor-mediated nonproteolytic activation of prorenin and induction of TGF-β1 and PAI-1 expression in renal mesangial cells

While elevated plasma prorenin levels are commonly found in diabetic patients and correlate with diabetic nephropathy, the pathological role of prorenin, if any, remains unclear. Prorenin binding to the (pro)renin receptor [(p)RR] unmasks prorenin catalytic activity. We asked whether elevated prorenin could be activated at the site of renal mesangial cells (MCs) through receptor binding without being proteolytically converted to renin. Recombinant inactive rat prorenin and a mutant prorenin that is noncleavable, i.e., cannot be activated proteolytically, are produced in 293 cells. After MCs were incubated with 10(-7) M native or mutant prorenin for 6 h, cultured supernatant acquired the ability to generate angiotensin I (ANG I) from angiotensinogen, indicating both prorenins were activated. Small interfering RNA (siRNA) against the (p)RR blocked their activation. Furthermore, either native or mutant rat prorenin at 10(-7) M alone similarly and significantly induced transforming growth factor-β(1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin mRNA expression, and these effects were blocked by (p)RR siRNA, but not by the ANG II receptor antagonist, saralasin. When angiotensinogen was also added to cultured MCs with inactive native or mutant prorenin, PAI-1 and fibronectin were further increased significantly compared with prorenin or mutant prorenin alone. This effect was blocked partially by treatment with (p)RR siRNA or saralasin. We conclude that prorenin binds the (p)RR on renal MCs and is activated nonproteolytically. This activation leads to increased expression of PAI-1 and transforming growth factor-β(1) via ANG II-independent and ANG II-dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may play a role in the development of diabetic nephropathy.     

Induction of α1-antitrypsin mRNA and cloning of its cDNA

Local inflammation was inflicted in a baboon by turpentine administration in order to induce the plasma level of α1-antitrypsin, an acute phase protein synthesized in the liver. Comparison of the α1-antitrypsin mRNA activity in the induced and non-induced baboon liver indicated that the “acute phase” response to chemical-inflicted inflammation is mediated through an increase in the steady-state level of cellular mRNA. Alpha-1-antitrypsin was then enriched from the induced baboon liver to a purity of greater than 90% by specific immunoprecipitation of polysomes. Double-stranded DNA was synthesized from the enriched mRNA and inserted into the $\text{Pst}$ I site of pBR322. Recombinant clones containing α1-antitrypsin cDNA sequences were identified by hybridselected translation and confirmed by DNA sequence analysis.     

Transforming growth factor-β1 regulation of laminin γ1 and fibronectin expression and survival of mouse mesangial cells

The transforming growth factor-beta (TGF-β) 1 is a mediator of extracellular matrix (ECM) gene expression in mesangial cells and the development of diabetic glomerulopathy. Here, we investigate the effects of TGF-β1 on laminin γ1 and fibronectin polypeptide expression and cell survival in mouse mesangial cells (MES-13). TGF-β1 (10 ng/ml) stimulates laminin-γ1 and fibronectin expression ~two-fold in a time-dependent manner (0–48 h). TGF-β1 treatment also retards laminin-γ1 mobility on SDS-gels, and tunicamycin, an inhibitor of the N-linked glycosylation, blocks the mobility shift. TGF-β1 increases the binding of laminin γ1 to WGA-agarose and the binding is abolished by tunicamycin suggesting that laminin γ1 is modified by N-linked glycosylation. TGF-β1 also elevates fibronectin glycosylation but its mobility is not altered. The degradation of laminin γ1 and fibronectin proteins is reduced by their glycosylation. In addition, TGF-β1 enhances mesangial cell viability and metabolic activities initially (0–24 h); however, eventually leads to cell death (24–48 h). TGF-β1 elevates pro-apoptotic caspase-3 activity and decrease cell cycle progression factor cyclin D1 expression, which parallels cell death. These results indicate that TGF-β1 plays an important role in ECM expression, protein glycosylation and demise of mesangial cells in the diabetic glomerular mesangium. (Mol Cell Biochem 278: 165–175, 2005)     

PLK1 and β-TrCP-Dependent Ubiquitination and Degradation of Rap1GAP Controls Cell Proliferation

Rap1GAP is a GTPase-activating protein (GAP) that specifically stimulates the GTP hydrolysis of Rap1 GTPase. Although Rap1GAP is recognized as a tumor suppressor gene and downregulated in various cancers, little is known regarding the regulation of Rap1GAP ubiquitination and degradation under physiological conditions. Here, we demonstrated that Rap1GAP is ubiquitinated and degraded through proteasome pathway in mitosis. Proteolysis of Rap1GAP requires the PLK1 kinase and β-TrCP ubiquitin ligase complex. We revealed that PLK1 interacts with Rap1GAP in vivo through recognition of an SSP motif within Rap1GAP. PLK1 phosphorylates Ser525 in conserved ₅₂₄DSGHVS₅₂₉ degron of Rap1GAP and promotes its interaction with β-TrCP. We also showed that Rap1GAP was a cell cycle regulator and that tight regulation of the Rap1GAP degradation in mitosis is required for cell proliferation.     

The development of the antigenic structures of human α1-antichymotrypsin and C 1 q

Summary The cross-reactions of human ₁-antichymotrypsin and C 1 q with their homologues in the plasmas of the chimpanzee, several Old World monkeys and nine non-primate eutheria were investigated by standard procedures. The results show that cross-reactions are limited to the first species mentioned. Comparative Ouchterlony tests and absorption controls revealed the presence of two (human) determinants on both human and chimpanzee molecules, while the cercopithecoids analyzed carried only one of them on their homologue. The results are discussed briefly with reference to earlier findings from this laboratory.

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Zusammenfassung Die Kreuzreaktionen des menschlichen ₁-Antichymotrypsin und des C 1 q mit seinen Homologen im Plasma des Schimpansen, einiger Altweltaffen und demjenigen von 9 Nichtprimaten (Eutheria) wurden mit Standardmethoden untersucht. Die Ergebnisse zeigen, daß Kreuzreaktionen auf die zuerst genannten Species beschränkt sind. Vergleichende Ouchterlony-Tests und Absorptionskontrollen ließen die Anwesenheit zweier (menschlicher) Determinaten auf den Molekülen des Menschen und des Schimpansen erkennbar werden, während die untersuchten Cercopithecoidea nur eine dieser Determinanten besitzen. Die Ergebnisse werden kurz im Zusammenhang mit früheren Befunden aus unserem Laboratorium diskutiert.

     

Aggresome formation and segregation of inclusions influence toxicity of α-synuclein and synphilin-1 in yeast

PD (Parkinson's disease) is a neurodegenerative disorder, caused by a selective loss of dopaminergic neurons in the substantia nigra, which affects an increasing number of the elderly population worldwide. One of the major hallmarks of PD is the occurrence of intracellular protein deposits in the dying neurons, termed Lewy bodies, which contain different proteins, including aggregated α-synuclein and its interacting protein synphilin-1. During the last decade, a number of groups developed yeast models that reproduced important features of PD and allowed the deciphering of pathways underlying the cytotoxicity triggered by α-synuclein. Here, we review the recent contributions obtained with yeast models designed to study the presumed pathobiology of synphilin-1. These models pointed towards a crucial role of the sirtuin Sir2 and the chaperonin complex TRiC (TCP-1 ring complex)/CCT (chaperonin containing TCP-1) in handling misfolded and aggregated proteins.     

Synthesis and properties of alkylglucosides with mild detergent action: improved synthesis and purification of β-1-octyl-, -nonyl-, and -decyl-glucose. Synthesis of β-1-undecylglucose and β-1-dodecylmaltose

Medium chain β-1-alkylglycosides show interesting mild detergent properties. Therefore, their synthesis and purification have been investigated and improved so as to permit preparation of 50–100 g amounts. Preparatory methods are presented for the already known compounds β-1-octyl-, β-1-nonyl and β-1-decyl-glucose and for the new compounds β-1-undecylglucose and β-1-dodecylmaltose. Some relevant properties such as melting point, optical rotation, critical micelle concentration and NMR-spectra have been determined. They illustrate the suitability of this class of detergents for membrane research.     

Design of a controlled release system of OP-1 and TGF-β1 based in microparticles of sodium alginate and release characterization by HPLC-UV

A new system for sustained release of growth factors, such as osteogenic protein 1 (OP-1) and transforming growth factor β1 (TGF-β1), intended to repair and promote dental tissue regeneration in rats was designed and characterized in this work. The release system was made with microparticles of sodium alginate, produced by ionic gelling dripping technique. The release profiles of OP-1 and TGF-β1 from biopolymer matrix were determined by high-performance liquid chromatography (HPLC), and with this purpose, an HPLC-UV method was developed. About 80% of each growth factor was released in the first 24 h, reaching almost 100% in 168 h. The system was tested during the tissue repair in rat molars in comparison with calcium hydroxide and both growth factors not encapsulated. The dentin sialoprotein (DSP) was used as a repair marker. It was detected by immunohistochemistry, after 14- and 28-d post-treatment. X ² test (p ≤ 0.001) and Fisher exact test (p ≤ 0.05) were applied for assessment of the amount of immunostaining. The treatment with encapsulated OP-1 showed an increased DSP immunostaining after 14 d and did not find any significant difference with the immunostaining observed for calcium hydroxide treatment. Treatment with TGF-β1 did not show significant difference with calcium hydroxide. Treatment with both factors OP-1 and TGF-β1 showed higher DSP immunostaining in comparison with calcium hydroxide treatment. In conclusion, the combination of both growth factors encapsulated showed more DSP immunostaining in comparison with each one separated, either encapsulated or not.     

Left helix of polyproline II type and genesis of β-structures in spidroins 1 and 2 and their recombinant analogs

The distribution of secondary structure elements along the polypeptide chains of spider silk proteins spidroins 1 and 2 and their recombinant analogs has been studied by statistical methods. It was found that these proteins as monomers contain only traces of β-structure, while the Ala-rich and the Gly-rich regions are predicted as α-helices and as left-handed helices of polyproline II type. Analysis of literature and our CD data shows that the major polypeptide chain conformation of spidroins 1 and 2 and their recombinant analogs in aqueous solutions is the polyproline II helix, with some α-helices and a very small share of β-structures. The transition to the state with extended conformations, which are characteristic of mature silk fibers, requires dehydration of the polypeptide backbone. Thus, the genesis of β-structure in spider web proteins is determined by the conditions of transitions between the main regular backbone conformations.     

Discovery and optimization of adamantyl carbamate inhibitors of 11β-HSD1

Synthesis of 2-adamantyl carbamate derivatives of piperidines and pyrrolidines led to the discovery of 9a with an IC₅₀ of 15.2 nM against human 11β-HSD1 in adipocytes. Optimization for increased adipocyte potency, metabolic stability and selectivity afforded 11k and 11l, both of which were >25% orally bioavailable in rat.     

Synthesis and biological activity of aryl S-β-glycosides of 1-thio-N-acetylmuramyl-L-alanyl-D-isoglutamine

Phenyl, p-tolyl, and p-tert-butylphenyl β-1-thio-N-acetylglucosaminides were synthesized by the treatment of thiophenols with peracetate of α-D-glucosaminyl chloride in the presence of triethylamine or under the conditions of phase-transfer catalysis with quaternary ammonium salts. The compounds synthesized were used for obtaining of glycosides of 4,6-O-isopropylidene-N-acetylmuramic acid, which were coupled with L-Ala-D-Glu(NH₂)-OBzl and then deprotected to obtain the target aryl β-thioglycosides of N-acetylmuramyl-L-analyl-D-isoglutamine (MDP). The aryl β-thioglycosides of MDP were found to stimulate an antibacterial resistance toward Staphylococcus aureus in mice. The reliable induction of the spontaneous activity of natural killers in the population of blood mononuclear cells was observed only for phenyl β-thio-MDP at a dose of 200 μg/ml. Original Russian Text ? A.E. Zemlyakov, V.N. Tsikalova, L.R. Azizova, V.Ya. Chirva, E.L. Mulik, M.V. Shkalev, O.V. Kalyuzhin, M.V. Kiselevsky, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 2, pp. 245–251.     

Mapping of the regulatory type Iα and catalytic β subunits of cAMP-dependent protein kinase and interleukin 1α and 1β in the pig

The genes coding for the regulatory type I subunit (PRKAR1A) and the catalytic subunit (PRKACB) of cAMP-dependent protein kinase and the genes for interleukin 1 (IL1A) and interleukin 1 (IL1B) were localized in the pig by means of radioactive in situ hybridization. PRKAR1A was mapped to 12p1.4 and PRKARB to 6q3.1 q3.3. The genes for IL1A and IL1B were both assigned to Chromosome (Chr) 3, in the region q1.2 q1.3 and q1.1 q1.4, respectively. The cDNA nucleotide sequences of these porcine genes were compared with those of human, mouse, and cattle. The location of the genes was discussed in relation to the position of their homologous loci in these mammalian species.     

Rag-dependent and Rag-independent mechanisms of Notch1 rearrangement in thymic lymphomas of Atm−/− and scid mice

The pathways of thymic lymphomagenesis are classified as Rag-dependent or -independent according to their dependence on recombination-activating gene (Rag1/2) proteins. The role of the two-lymphoma pathways in oncogene rearrangements and the connection between lymphoma pathways and rearrangement mechanisms, however, remain obscure. We compared the incidence and latency of thymic lymphomas, and associated rearrangements of the representative oncogene Notch1 among Rag2^?/?, ataxia telangiectasia mutated (Atm)^?/?, and severe combined immune deficiency (scid) mice combined with Rag2 deficiency. Contrary to expectations, Rag2^?/? mice were prone to thymic lymphoma development, suggesting the existence of a Rag2-independent lymphoma pathway in Rag2^?/? mice. The lymphoma incidence in Rag2^?/?Atm^?/? mice was lower than that in Atm^?/? mice, but higher than that in Rag2^?/? mice, indicating that Atm^?/? mice develop lymphomas through both pathways. Scid mice developed lymphomas with an incidence and latency similar to Rag2^?/?scid mice, suggesting that Rag2-mediated V(D)J recombination-driven events are not necessarily required for lymphomagenesis in scid mice. Notch1 rearrangement mechanisms were classified as Rag2-dependent or Rag2-independent based on the presence of recombination signal-like sequences at rearranged sites. In Rag2^?/? lymphomas, Notch1 must be rearranged independently of Rag2 function, implying that Rag2^?/? mice are susceptible to lymphomagenesis due to the presence of other rearrangement mechanisms. The results in Atm^?/? mice suggest that Notch1 was rearranged through both lymphoma pathways. In scid mice, the frequency of Rag2-mediated rearrangements was relatively low compared with that in wild-type mice, suggesting that the Rag2-independent lymphoma pathway prevails in the development of thymic lymphomas in scid mice. Thus, two rearrangement mechanisms underlie the lymphoma pathways and constitute the mechanistic bases for lymphomagenesis, thereby providing the molecular criteria for distinguishing between Rag2-dependent and Rag2-independent lymphoma pathways.     

Physiology and pathology of nuclear phospholipase C β1

The existence and function of inositide signaling in the nucleus is well documented and we know that the existence of the inositide cycle inside the nucleus has a biological role. An autonomous lipid-dependent signaling system, independently regulated from its plasma membrane counterpart, acts in the nucleus and modulates cell cycle progression and differentiation.We and others focused on PLCβ1, which is the most extensively investigated PLC isoform in the nuclear compartment. PLCβ1 is a key player in the regulation of nuclear inositol lipid signaling, and, as discussed above, its function could also be involved in nuclear structure because it hydrolyses PtdIns(4,5)P2, a well accepted regulator of chromatin remodelling. The evidence, in a number of patients with myelodysplastic syndromes, that the mono-allelic deletion of PLCβ1 is associated with an increased risk of developing acute myeloid leukemia paves the way for an entirely new field of investigation. Indeed the genetic defect evidenced, in addition to being a useful prognostic tool, also suggests that altered expression of this enzyme could have a role in the pathogenesis of this disease, by causing an imbalance between proliferation and apoptosis. The epigenetics of PLCβ1 expression in MDS has been reviewed as well.     

Comparison of human and chimpanzee ξ1 blobin genes

Summary The DNA base sequences of the entire chimpanzee 1 globin gene and an additional 1 kb of DNA flanking both the human and chimpanzee genes have been determined. Whereas the human 1 gene contains a termination codon in the sixth position, the chimpanzee gene appears to be functional. This finding confirms Proudfoot et al.'s suggestion that the human 1 gene was recently inactivated. Like the corresponding human 1 and 2 genes, the first and second introns of the chimpanzee 1 gene are occupied largely by tandem repeats of short oligonucleotides. These tandem repeats have undergone several rearrangements since the divergence of the human and chimpanzee 1 genes.