Volume 386, Issue 2

In order to demonstrate that an existing zinc-finger protein can be simply modified to enhance DNA binding and sequence discrimination in both episomal and chromatin contexts using existing zinc-finger DNA recognition code data, and without recourse to phage display and selection strategies, we have examined the consequences of a single zinc-finger extension to a synthetic three-zinc-finger VP16 fusion protein, on transcriptional activation from model target promoters harbouring the zinc-finger binding sequences. We report a nearly 10-fold enhanced transcriptional activation by the four-zinc-finger VP16 fusion protein relative to the progenitor three-finger VP16 protein in transient assays and a greater than five-fold enhancement in stable reporter-gene expression assays. A marked decrease in transcriptional activation was evident for the four-zinc-finger derivative from mutated regulatory regions compared to the progenitor protein, as a result of recognition site-size extension. This discriminatory effect was shown to be protein concentration-dependent. These observations suggest that four-zinc-finger proteins are stable functional motifs that can be a significant improvement over the progenitor three-zinc-finger protein, both in terms of specificity and the ability to target transcriptional function to promoters, and that single zinc-finger extension can therefore have a significant impact on DNA zinc-finger protein interactions. This is a simple route for modifying or enhancing the binding properties of existing synthetic zinc-finger-based transcription factors and may be particularly suited for the modification of endogenous zinc-finger transcription factors for promoter biasing applications.

The plasma prekallikrein gene is expressed in many different human tissues at distinctly different levels and therefore tissue-specific control of the gene transcription is likely. In this study we demonstrate that transcription of the plasma prekallikrein gene can be initiated at multiple sites, for which at least four different promoters are utilized. A comparison of the genomic and mRNA sequences of mouse plasma prekallikrein revealed that the sequence segment that was formerly regarded as the first exon of the mouse plasma prekallikrein gene consists of three exons, with the first exon localized 14.2 kbp upstream of the translation start. For the rat and human plasma prekallikrein genes, in silico analysis suggested an analogous exon-intron organization. Determination of the transcription start sites showed that in both mouse and human, the proximal and distal regions could be utilized for transcription initiation; however, the proximal region is preferred. A deletion mutation analysis of the proximal promoter region using a 1.7-kbp segment revealed a strong activating region immediately upstream of the known mRNA, followed by both a modest repressor and an enhancer region.

A central feature of the serpin inhibition mechanism is insertion of the reactive center loop into the central β-sheet (β-sheet A). This insertion also occurs when the reactive center loop is cleaved without protease inhibition. Using this effect, we have measured the enthalpy (ΔH) of loop cleavage and insertion for plasminogen activator inhibitor 1 (PAI-1) as -38 kcal/mol. Because loop insertion can be blocked by incorporating a peptide into the central β-sheet, it was possible to assign -7 kcal/mol to loop cleavage and -31 kcal/mol to loop insertion. These values are lower than values reported for the serpins α 1 -proteinase inhibitor and antithrombin of -53 to -63 kcal/mol, respectively, for loop insertion with negligible enthalpy for loop cleavage. A free energy difference of -9 kcal/mol has been reported between the active and spontaneously loop inserted ‘latent forms’ of PAI-1, which is significantly smaller in magnitude than the -31 kcal/mol of enthalpy we measured for loop insertion. Because the enthalpy should relate closely to those regions of PAI-1 that have moved to lower potential energy, a difference distance matrix is presented that identifies regions of PAI-1 that move during loop insertion.

The human malaria parasite Plasmodium falciparum possesses a single gene with high similarity to the metalloproteins arginase and agmatinase. The recombinant protein reveals strict specificity for arginine, and it has been proposed that its function in ornithine production is as a precursor for polyamine biosynthesis. The specific activity of the plasmodial arginase was found to be 31 μmol min -1  mg -1 protein and the k cat was calculated as 96 s -1 . The K m value for arginine and K i value for ornithine were determined as 13 mM and 19 mM, respectively. The active arginase is a homotrimer of ca. 160 kDa. Dialysis of the arginase against EDTA results in monomers of approximately 48 kDa; however, the quaternary structure can be restored by addition of Mn 2+ . Mutagenic analyses of all the amino acid residues proposed to be involved in metal binding led to complex dissociation, except for the His-193-Ala mutant, which was also inactive but retained the trimeric structure. Substitution of His-233, which has been suggested to be in charge of proton shuttling within the active site, disrupted the trimeric structure and thereby the activity of the Pf arginase. Northern blot analysis identified a stage-specific expression pattern of the plasmodial arginase in the ring/young trophozoite stage, which guarantees the provision of ornithine for essential polyamine biosynthesis.

Riboflavin synthase catalyses a mechanistically complex dismutation affording riboflavin and 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione from 6,7-dimethyl-8-ribityllumazine. A pentacyclic adduct (compound 2 ) of two substrate molecules was used as substrate for pre-steady-state kinetic analysis. Whereas the wild-type enzyme catalyses the decomposition of compound 2 into a mixture of riboflavin and 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione, as well as into two equivalents of 6,7-dimethyl-8-ribityllumazine, a H102Q mutant enzyme predominantly catalyses the former reaction. Stopped-flow experiments with this mutant enzyme failed to identify a reaction intermediate between compound 2 and riboflavin. However, the apparent rate constants for the formation of riboflavin as observed by stopped-flow and quenched-flow experiments were significantly different, thus suggesting that the reaction proceeds via a significantly populated intermediate, the absorbance of which is similar to that of compound 2 . An F2A mutant enzyme converts compound 2 predominantly into 6,7-dimethyl-8-ribityllumazine. Stopped-flow experiments using compound 2 as substrate indicated a slight and rapid initial increase in absorbance at 310 nm, followed by a slower decrease. This finding, in conjunction with different apparent rates for the formation of 6,7-dimethyl-8-ribityllumazine, suggests the involvement of a significantly populated intermediate in the transition between compound 2 and 6,7-dimethyl-8-ribityllumazine, the optical spectrum of which is similar to that of compound 1 .

The antimicrobial activity of bovine lactoferrin (bLF) is attributed to lactoferricin, which is situated in the N1-domain of bLF. Recently, another antimicrobial domain consisting of residues 268–284, designated lactoferrampin (LFampin), has been identified in the N1-domain of bLF, which exhibited antimicrobial activity against Candida albicans and several bacteria. In the present study, the candidacidal activity of a series of peptides spanning this antimicrobial domain was investigated in relation to the charge and the capacity to form a helical conformation in hydrophobic environments. C-Terminal truncation of LFampin resulted in a drastic decrease in candidacidal activity. Positively charged residues clustered at the C-terminal side of the LFampin domain appeared to be crucial for the candidacidal activity. The ability to adopt helical conformations did not change when LFampin was truncated at the C-terminal side. N-Terminally truncated LFampin peptides, truncated up to the sequence 270–284, were more reluctant to adopt a helical conformation. Therefore, we conclude that the C-terminal part of LFampin 265–284, which is the most active peptide, is crucial for its candidacidal activity, due to the presence of clustered positive charges, and that the N-terminal part is essential for activity as it facilitates helix formation.

Rab38 is a new member of the Rab small G protein family that regulates intracellular vesicle trafficking. Rab38 is expressed in melanocytes and it has been clarified that a point mutation in the postulated GTP-binding domain of Rab38 is the gene responsible for oculocutaneous albinism in chocolate mice. However, basic information regarding recombinant protein production, intracellular location, and tissue-specific expression pattern has not yet been reported. We produced recombinant Rab38 using a baculovirus/insect cell-protein expression system. A combination of Triton X-114 phase separation and nickel-affinity chromatography yielded exclusively prenylated Rab38 that bound [α- 32 P]-GTP. The mRNA and the native protein were expressed in a tissue-specific manner, e.g., in the lung, skin, stomach, liver, and kidney. Freshly isolated rat alveolar type II cells were highly positive for the mRNA signal, but the signal was rapidly lost over time. Immunofluorescence staining demonstrated that expressed GST-tagged Rab38 was mainly co-localized with endoplasmic reticulum-resident protein and also partly with intermittent vesicles between the endoplasmic reticulum and the Golgi complex. These results indicate that Rab38 is expressed non-ubiquitously in specific tissues and regulates early vesicle transport relating to the endoplasmic reticulum, and hence suggest that Rab38 abnormality may cause multiple organ diseases as well as oculocutaneous albinism.

In the present study we examined the trafficking pathways of connexin49 (Cx49) fused to green fluorescent protein (GFP) in polar and non-polar cell lines. The Cx49 gene was isolated from ovine lens by RT-PCR. Cx49 cDNA was fused to GFP and the hybrid cDNA was transfected into several cell lines. After transfection of Cx49-GFP cDNA into HeLa cells, it was shown using the double whole-cell patch-clamp technique that the expressed fusion protein was still able to form conducting gap junction channels. Synthesis, assembly, and turnover of the Cx49-GFP hybrid protein were investigated using a pulse-chase protocol. A major 78-kDa protein band corresponding to Cx49-GFP could be detected with a turnover of 16–20 h and a half-life time of 10 h. The trafficking pathways of Cx49-GFP were monitored by confocal laser microscopy. Fusion proteins were localized in subcellular compartments, including the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment, the Golgi apparatus, and the trans-Golgi network, as well as vesicles traveling towards the plasma membrane. Time-dependent sequential localization of Cx49-GFP in the ER and then the Golgi apparatus supports the notion of a slow turnover of Cx49-GFP compared to other connexins analyzed so far. Gap junction plaques resembling the usual punctuate distribution pattern could be demonstrated for COS-1 and MDCK cells. Basolateral distribution of Cx49-GFP was observed in polar MDCK cells, indicating specific sorting behavior of Cx49 in polarized cells. Together, this report describes the first characterization of biosynthesis and trafficking of lens Cx49.

While the role of C2-ceramide in the induction of programmed cell death (PCD) in animal systems has been well documented, little is known of its role in plant cells. Here we show that C2-ceramide induces PCD in Arabidopsis suspension cultures, which is preceded by the generation of a calcium transient and an increase in reactive oxygen species (ROS). Inhibition of the calcium transient prevented cell death, whereas inhibition of ROS had no effect on cell survival. These observations suggest that calcium signalling plays a role in ceramide-induced PCD but is independent of the generation of ROS.

Microvascular endothelial cells from human neonatal foreskin were grown in vitro until a three-dimensional network of capillary-like structures was formed. All stages of the angiogenic cascade could be observed in this in vitro model, including the formation of an internal lumen. The microscopy focused on morphology, formation of an internal lumen, role of the extracellular matrix, polarity of the cells, and the time-course of the angiogenic cascade. Bright-field microscopy revealed cells arranged circularly side by side and the internal lumen of capillary-like structures was verified by electron microscopy. Immunolabeling revealed a peritubular localization of collagen IV. Reporter gene expression after the formation of capillary-like structures was marginally higher than control expression, but clearly lower than the expression of cells at the stage of proliferation. Highest transfection efficiencies were obtained using vectors with the CMV promoter and the long fragment of the Ets-1 promoter. This is a first study of transfection efficiencies mapped for stages of in vitro angiogenesis. We describe here the morphological features of a long-term in vitro model of angiogenesis of human microvascular endothelial cells that could be used for transfection studies, without the provision of an extracellular matrix substrate. The cells self-create their own extracellular matrix to proliferate and form a three-dimensional network of capillary-like structures with an internal lumen.

A new tissue kallikrein-like protease, blarinasin, has been purified from the salivary glands of the short-tailed shrew Blarina brevicauda . Blarinasin is a 32-kDa N-glycosylated protease with isoelectric values ranging between 5.3 and 5.7, and an optimum pH of 8.5 for enzyme activity. The cloned blarinasin cDNA coded for a pre-pro-sequence and a mature peptide of 252 amino acids with a catalytic triad typical for serine proteases and 43.7–54.0% identity to other mammalian tissue kallikreins. Blarinasin preferentially hydrolysed Pro-Phe-Arg-4-methylcoumaryl-7-amide (MCA) and N- tert -butyloxycarbonyl-Val-Leu-Lys-MCA, and preferentially converted human high-molecular-weight kininogen (HK) to bradykinin. The activity of blarinasin was prominently inhibited by aprotinin ( K i =3.4 nM). A similar kallikrein-like protease, the lethal venom blarina toxin, has previously been purified from the salivary glands of the shrew Blarina and shows 67.9% identity to blarinasin. However, blarinasin was not toxic in mice. Blarinasin is a very abundant kallikrein-like protease and represents 70–75% of kallikrein-like enzymes in the salivary gland of B. brevicauda .

Hepatitis C virus (HCV) infection is one of the world's major health problems, and the identification of efficient HCV inhibitors is a major goal. Here we report the isolation of efficient anti-HCV internal ribosome entry site (IRES) RNA molecules identified by a new in vitro selection method. The newly developed procedure consists of two sequential steps that use distinct criteria for selection: selection for binding and selection for cleaving. The selection protocol was applied to a population of more than 10 15 variants of an anti-hepatitis C virus ribozyme covalently linked to an aptamer motif. The ribozyme was directed against positions 357 to 369 of the HCV IRES, and the cleavage substrate was a 691-nucleotide-long RNA fragment that comprises the entire HCV IRES domain. After six selection cycles, seven groups of RNA variants were identified. A representative of each group was tested for its capacity to inhibit IRES activity using in vitro translation assays. All selected RNAs promoted significant inhibition, some by as much as 95%.