Five-stranded β-sheet sandwiched with two α-helices: A structural link between restriction endonucleases EcoRI and EcoRV

Examination of crystal structures of restriction endonucleases EcoRI and EcoRV complexes with their cognate DNA revealed a common structural element, which forms the core of both proteins. This element consists of a five-stranded β-sheet and two α-helices packed against it and could be described as α–β sandwich in which helices and β-strands lie in two stacked layers. While the spatial structure of this α–β sandwich is conserved in both enzymes, there are no detectable similarities between amino acid sequences except of a few residues involved in active site formation. Probably, other restriction endonucleases which have similar organization of the active site might possess similar structural element regardless of DNA sequence recognized and recognition elements in the enzyme used. © 1994 Wiley-Liss, Inc.     

Cleavage of the Carboxyl-Terminus of LEACS2, a Tomato 1-Aminocycl opropane-1-Carboxylic Acid Synthase Isomer, by a 64-kDa Tomato Metalloprotease Produces a Truncated but Active Enzyme

l-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS (LeACS2) into 52-, 50- and 49-kDa truncated isoforms in ripening tomato (Lycopersicon esculentum Mill. cv.Cooperation 903) fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys438, Glu447, Lys448, Asn456, Ser460, Ser462, Lys463, and Leu474, but does not cleave the Nterminus of LeACS2. Four C-terminus-deleted (26-50 amino acids) LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain (H14) and Ser460 for this metalloprotease.Furhermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.     

Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I

Mammalian cleavage factor I (CF I_m) is composed of two polypeptides of 25 kDa and either a 59 or 68 kDa subunit (CF I_m25, CF I_m59, CF I_m68). It is part of the cleavage and polyadenylation complex responsible for processing the 3′ ends of messenger RNA precursors. To investigate post-translational modifications in factors of the 3′ processing complex, we systematically searched for enzymes that modify arginines by the addition of methyl groups. Protein arginine methyltransferases (PRMTs) are such enzymes that transfer methyl groups from S-adenosyl methionine to arginine residues within polypeptide chains resulting in mono- or dimethylated arginines. We found that CF I_m68 and the nuclear poly(A) binding protein 1 (PABPN1) were methylated by HeLa cell extracts in vitro. By fractionation of these extracts followed by mass spectral analysis, we could demonstrate that the catalytic subunit PRMT5, together with its cofactor WD45, could symmetrically dimethylate CF I_m68, whereas pICln, the third polypeptide of the complex, was stimulatory. As sites of methylation in CF I_m68 we could exclusively identify arginines in a GGRGRGRF or “GAR” motif that is conserved in vertebrates. Further in vitro assays revealed a second methyltransferase, PRMT1, which modifies CF I_m68 by asymmetric dimethylation of the GAR motif and also weakly methylates the C-termini of both CF I_m59 and CF I_m68. The results suggest that native—as compared with recombinant—protein substrates may contain additional determinants for methylation by specific PRMTs. A possible involvement of CF I_m methylation in the context of RNA export is discussed.     

Role of recombination occurring during DNA replication

Unequal crossing over between direct DNA repeats of sister chromosomes occurs during DNA replication in Escherichia coli. Such exchanges yield tandem duplications and thereby increase the expression of the genes involved. Nonhomologous cohesion of sister chromosomes and unequal crossing over were assumed to take place when the replication fork stops. When the replication forks moves continuously, homologous exchanges between sister chromosomes ensure their postreplication repair.     

Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases

We have developed a specific and efficient method for complete removal of polyhistidine purification tags (HisTags) from the N-termini of target proteins. The method is based on the use of the aminopeptidase dipeptidyl peptidase I (DPPI), either alone or in combination with glutamine cyclotransferase (GCT) and pyroglutamyl aminopeptidase (PGAP). In both cases, the HisTag is cleaved off by DPPI, which catalyzes a stepwise excision of a wide range of dipeptides from the N-terminus of a peptide chain. Some sequences, however, are resistant to DPPI cleavage and a number of mature proteins have nonsubstrate N-termini which protects them against digestion. For such proteins, HisTags composed of an even number of residues can be cleaved off by treatment with DPPI alone. When the target protein is unprotected against DPPI, a blocking group is generated enzymatically from a glutamine residue inserted between the HisTag and the target protein. A protein with a HisTag-Gln extension is incubated with both DPPI and GCT. As above, the polyhistidine sequence is cleaved off by DPPI, but when the glutamine residue appears in the N-terminus, it is immediately converted into a pyroglutamyl residue by an excess of GCT and further DPPI digestion is prevented. The desired sequence is finally obtained by excision of the pyroglutamyl residue with PGAP. All the enzymes employed can bind to immobilized metal affinity chromatography (IMAC) matrices, and in this paper we demonstrate a simple and highly effective process combining IMAC purification of His-tagged proteins, our aminopeptidase-based method for specific excision of HisTags and use of subtractive IMAC for removing processing enzymes. Typical recoveries were 75-90% for the enzymatic processing and subtractive IMAC. The integrated process holds promises for use in large-scale production of pharmaceutical proteins because of a simple overall design, use of robust and inexpensive matrices, and use of enzymes of either recombinant or plant origin.     

Invasive cleavage reactions on DNA-modified diamond surfaces

Recently developed DNA-modified diamond surfaces exhibit excellent chemical stability to high-temperature incubations in biological buffers. The stability of these surfaces is substantially greater than that of gold or silicon surfaces, using similar surface attachment chemistry. The DNA molecules attached to the diamond surfaces are accessible to enzymes and can be modified in surface enzymatic reactions. An important application of these surfaces is for surface invasive cleavage reactions, in which target DNA strands added to the solution may result in specific cleavage of surface-bound probe oligonucleotides, permitting analysis of single nucleotide polymorphisms (SNPs). Our previous work demonstrated the feasibility of performing such cleavage reactions on planar gold surfaces using PCR-amplified human genomic DNA as target. The sensitivity of detection in this earlier work was substantially limited by a lack of stability of the gold surface employed. In the present work, detection sensitivity is improved by a factor of approximately 100 (100 amole of DNA target compared with 10 fmole in the earlier work) by replacing the DNA-modified gold surface with a more stable DNA-modified diamond surface.     

Economic Integration in Tanzania (1970–2011): A Biophysical Assessment

This article assesses the impact of economic integration on Tanzania's sociometabolic profile for the years 1970–2011, which witnessed an opening and further integration of Tanzania's economy through increased trade and foreign investment, through a time‐series economy‐wide material flows analysis (EW‐MFA). The EW‐MFA results show that contrary to the trade patterns of many developing countries, increased economic integration has resulted in Tanzania becoming a net importer of resources across all material categories when measured by the physical trade balance indicator. Additionally, the article discusses the conceptual and empirical challenges of measuring ecologically unequal exchange with EW‐MFAs for developing countries whose export profiles are dominated by lightweight, high‐value precious stones and metals. It also assesses the degree to which the Tanzanian economy has undergone dematerialization over the past 40 years of economic integration.     

DNA sequence selectivity of human topoisomerase I‐mediated DNA cleavage induced by camptothecin

In probing the mechanism of inhibition of hypoxia inducible factor (HIF-1) by campothecins, we investigated the ability of human topoisomerase I to bind and cleave HIF-1 response element (HRE), which contains the known camptothecin-mediated topoisomerase I cleavage site 5′-TG. We observed that the selection of 5′-TG by human topoisomerase I and topotecan depends to a large extent on the specific flanking sequences, and that the presence of a G at the −2 position (where cleavage occurs between −1 and +1) prevents the HRE site from being a preferred site for such cleavage. Furthermore, the presence of −2 T/A can induce the cleavage at a less preferred TC or TA site. However, in the absence of a more preferred site, the HRE site is shown to be cleaved by human topoisomerase I in the presence of topotecan. Thus, it is implied that the −2 base has a significant influence on the selection of the camptothecin-mediated Topo I cleavage site, which can overcome the preference for +1G. While the cleavage site recognition has been known to be based on the concerted effect of several bases spanning the cleavage site, such a determining effect of an individual base has not been previously recognized. A possible base-specific interaction between DNA and topoisomerase I may be responsible for this sequence selectivity.     

蛋白质内含子介导的蛋白纯化系统在变性条件下的应用

由蛋白质内含子介导的亲和蛋白质纯化系统(IMPACT)已得到广泛应用,通过其纯化得到的目的蛋白不含蛋白纯化标签以及多余的氨基酸残基,且操作简单成本低廉。这些优点使得其相对于其他蛋白纯化系统有着无与伦比的优势。但是现有报道都局限于非变性条件下使用,这往往会限制其在一些包涵体蛋白变性条件下使用。以一已知表达形成包涵体形式的丝素蛋白为例,研究IMPACT系统在时变性条件下使用变性剂浓度、温度和诱导断裂还原剂浓度。实验表明,在4 M尿素,100 mM DTT室温作用下蛋白质内含子会获得最大断裂效率(80%)。柱上在线断裂实验表明,其最终蛋白得率超过65%。     

ALTERATIONS WITH AGING OF THE ENDOCRINE AND NEUROENDOCRINE CIRCADIAN SYSTEM IN HUMANS

This was an open-label study in 19 children aged 9–13 years, weighing 27–44 kg, with bronchial asthma. Twenty-four-hour steady-state concentrations of theophylline and its metabolites 1,3-dimethyl uric acid, 3-methyl xanthine and 1-methyl uric acid were assessed after daily dosing of 600 mg (ca18 mg/kg/day) of the sustainedrelease theophylline micro-pellet sprinkle system BY158K, for 4 days. The dosing regimen used was an unequal twice-daily dose of 200 mg in the morning after breakfast and 400 mg in the evening after dinner. Twenty-four-hour peak expiratory flow (PEF) profiles were compared before treatment and at steady-state, along with lung function parameters after bronchial provocation. Mean values±SD (n=16) of the steady-state characteristics were C_min6.8±2.1 mg/1, C_max14.5±4.8 mg/1 and C_av10.S±2.9 mg/1, the plateau time was 11.7±4.8 hr and peak-trough fluctuation and swing were 72±21 and 118±52%, respectively. There was an excellent reproducibility of theophylline pre-dose levels at corresponding time points of the 24-hr sampling period [r=0.864 (p< 0.001)]. Mean values±SD of the 24 hr average serum metabolite levels were 0.9±0.2 mg/1 for 1, 3-dimethyl uric acid, 0.6±0.1 mg/1 for 3-methyl xanthine and 0.4±0.1 mg/1 for 1-methyl uric acid. Lung function (n=17) following bronchial provocation, improved in 10 children after theophylline treatment of 4 days, remained stable in 2 patients and deteriorated in 5 patients. Serum theophylline profiles and PEF profiles ran largely in parallel over the 24-hr period. Six children exhibited typical theophylline induced side-effects, headache (n=3), nausea (n=4), dizziness (n=l), vomiting (n=4), sleep disturbances (n=1), pallor (n=1) and tremor(n=1), necessitating in 3 children one dose omission/reduction (n=2) or subsequent dose reduction (n=1). It has been shown that a twice daily dosing regimen with unequal doses of anhydrous theophylline (BY158K) is well suited to this population of fast metabolisers. The patients were well protected throughout the day, including the critical early morning hours.