Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene

A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to a proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAH cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM- phenotype. Together with the other mutations recently reported in the PAH gene, the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level.     

Protease Ti from Escherichia coli requires ATP hydrolysis for protein breakdown but not for hydrolysis of small peptides

Protease Ti, a new ATP-dependent protease in Escherichia coli, degrades proteins and ATP in a linked process, but these two hydrolytic functions are catalyzed by distinct components of the enzyme. To clarify the enzyme's specificity and the role of ATP, a variety of fluorogenic peptides were tested as possible substrates for protease Ti or its two components. Protease Ti rapidly hydrolyzed N-succinyl(Suc)-Leu-Tyr-amidomethylcoumarin (AMC) (Km = 1.3 mM) which is not degraded by protease La, the other ATP-dependent protease in E. coli. Protease Ti also hydrolyzed, but slowly, Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. However, it showed little or no activity against basic or other hydrophobic peptides, including ones degraded rapidly by protease La. Component P, which contains the serine-active site, by itself rapidly degrades the same peptides as the intact enzyme. Addition of component A, which contains the ATP-hydrolyzing site and is necessary for protein degradation, had little or no effect on peptide hydrolysis. N-Ethylmaleimide, which inactivates the ATPase, did not inhibit peptide hydrolysis. In addition, this peptide did not stimulate the ATPase activity of component A (unlike protein substrates). Thus, although the serine-active site on component P is unable to degrade proteins, it is fully functional against small peptides in the absence of ATP. At high concentrations, Suc-Leu-Tyr-AMC caused a complete inhibition of casein breakdown, and diisopropylfluorophosphate blocked similarly the hydrolysis of both protein and peptide substrates. Thus, both substrates seem to be hydrolyzed at the same active site on component P, and ATP hydrolysis by component A either unmasks or enlarges this proteolytic site such that large proteins can gain access to it.     

Chemical synthesis in protein engineering: total synthesis, purification and covalent structural characterization of a mitogenic protein, human transforming growth factor-alpha

Successful approaches to protein engineering required that the desired analogs be easily and rapidly obtained in sufficient quantities and purities for unambiguous structural and functional characterizations. Chemical synthesis is the method of choice for engineering small peptides. We now demonstrate that with improved methodologies and instrumentation, total chemical synthesis can be used to produce a small protein in a form suitable for engineering studies. Active human transforming growth factor-alpha (TGF-alpha), a 50 amino acid long protein with three disulfide bonds, has been synthesized and purified in multiple tens of mg amounts in less than 7 days. The purified human TGF-alpha migrated as a single band on SDS-polyacrylamide gels, ran as a single sharp major band at pI = 6.2 on isoelectric focusing gels, displayed an MW = 5546.2 (Th.5546.3) by mass spectrometry, contained three disulfide bonds and had EGF receptor binding, mitogenic and soft agar colony formation activities. The locations of disulfide bonds were found to be analogous to those found in epidermal growth factor (EGF) and in human TGF-alpha expressed in bacteria.     

Modulation of RNA transport by polyvinylpyrrolidone

A number of studies have documented substantial loss of nuclear protein during aqueous nuclear isolation procedures. This loss can, to some extent, be counteracted by addition of impermeable macromolecules like polyvinylpyrrolidone, which prevent nuclear swelling. While nucleic acids appear to be much less susceptible to leakage during isolation, the effects of these additives on RNA release duringin vitro incubation have not been examined. Here we show that addition of polyvinylpyrrolidone results in significant decreases in RNA transport; inhibition becomes maximal at 50–75 M addition.     

Influence of water limitation on endogenous oxidative stress and cell death within unsaturated Pseudomonas putida biofilms

Here we examined how water limitation (matric stress) and high osmolarity (solute stress) influence the extent of endogenous oxidative stress and cell death patterns within Pseudomonas putida biofilms. The temporal dynamics and spatial organization of reactive oxygen species (ROS) accumulation and dead cells in biofilms developed under water‐replete and solute stress conditions were similar to each other. Arrays of dead cells, typically one cell width in diameter, were distributed throughout the biofilm and occasionally they spanned the entire depth of the biofilm. These arrays of dead cells were not observed under water‐limiting conditions, although the extent of ROS accumulation and cell death was substantially greater. Despite the greater death rate under water‐limiting conditions, culturable population sizes were transiently maintained at levels comparable to those under water‐replete and solute stress conditions. There was greater spatial stratification of dead cells under water‐limiting than water‐replete conditions with viable cells primarily located at the air interface, which could facilitate cell dispersal following a wetting event. Under water‐limiting conditions, ROS accumulation is greater in an ΔalgD mutant compared with the wild type, suggesting that the exopolysaccharide alginate attenuates the extent of dehydration‐mediated oxidative stress. We conclude that endogenous ROS accumulation is correlated with cell death within P. putida biofilms, although mechanisms contributing to their accumulation may differ under water‐replete and water‐limiting conditions.     

Cell-penetration by Co(III)cyclen-based peptide-cleaving catalysts selective for pathogenic proteins of amyloidoses

Derivatives of the Co(III) complex of 1,4,7,10-tetraazacyclododecane (cyclen) with various organic pendants have been reported as target-selective peptide-cleaving catalysts, which can be exploited as catalytic drugs. In order to provide a firm basis for the catalytic drugs based on Co(III)cyclen, the ability of the Co(III)cyclen-containing peptide-cleaving catalysts to penetrate animal cells such as mouse fibroblast NIH-3T 3 or human embryonic kidney (HEK) 293 cells is demonstrated in the present study. Since the catalysts destroy pathogenic proteins for amyloidoses, results of the present study are expected to initiate extensive efforts to obtain therapeutically safe catalytic drugs for amyloidoses such as Alzheimer’s disease, type 2 diabetes mellitus, Parkinson’s disease, Huntington’s disease, mad cow disease, and so on.     

Carbon nanotube clusters as universal bacterial adsorbents and magnetic separation agents

The magnetic susceptibility and high bacterial affinity of carbon nanotube (CNT) clusters highlight their great potential as a magnetic bio‐separation agent. This article reports the CNT clusters' capability as “universal” bacterial adsorbents and magnetic separation agents by designing and testing a multiwalled carbon nanotube (MWNT) cluster‐based process for bacterial capturing and separation. The reaction system consisted of large clusters of MWNTs for bacterial capture and an external magnet for bio‐separation. The designed system was tested and optimized using Escherichia coli as a model bacterium, and further generalized by testing the process with other representative strains of both gram‐positive and gram‐negative bacteria. For all strains tested, bacterial adsorption to MWNT clusters occurred spontaneously, and the estimated MWNT clusters' adsorption capacities were nearly the same regardless of the types of strains. The bacteria‐bound MWNT clusters also responded almost instantaneously to the magnetic field by a rare‐earth magnet (0.68 Tesla), and completely separated from the bulk aqueous phase and retained in the system. The results clearly demonstrate their excellent potential as highly effective “universal” bacterial adsorbents for the spontaneous adsorption of any types of bacteria to the clusters and as paramagnetic complexes for the rapid and highly effective magnetic separations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Structural Basis for Asymmetric Association of the βPIX Coiled Coil and Shank PDZ

βPIX (p21-activated kinase interacting exchange factor) and Shank/ProSAP protein form a complex acting as a protein scaffold that integrates signaling pathways and regulates postsynaptic structure. Complex formation is mediated by the C-terminal PDZ binding motif of βPIX and the Shank PDZ domain. The coiled-coil (CC) domain upstream of the PDZ binding motif allows multimerization of βPIX, which is important for its physiological functions. We have solved the crystal structure of the βPIX CC-Shank PDZ complex and determined the stoichiometry of complex formation. The βPIX CC forms a 76-Å-long parallel CC trimer. Despite the fact that the βPIX CC exposes three PDZ binding motifs in the C-termini, the βPIX trimer associates with a single Shank PDZ. One of the C-terminal ends of the CC forms an extensive β-sheet interaction with the Shank PDZ, while the other two ends are not involved in ligand binding and form random coils. The two C-terminal ends of βPIX have significantly lower affinity than the first PDZ binding motif due to the steric hindrance in the C-terminal tails, which results in binding of a single PDZ domain to the βPIX trimer. The structure shows canonical class I PDZ binding with a β-sheet interaction extending to position − 6 of βPIX. The βB-βC loop of Shank PDZ undergoes a conformational change upon ligand binding to form the β-sheet interaction and to accommodate the bulky side chain of Trp − 5. This structural study provides a clear picture of the molecular recognition of the PDZ ligand and the asymmetric association of βPIX CC and Shank PDZ.     

Molecular diversity at the major cluster of disease resistance genes in cultivated and wild Lactuca spp.

Diversity was analyzed in wild and cultivated Lactuca germplasm using molecular markers derived from resistance genes of the NBS-LRR type. Three molecular markers, one microsatellite marker and two SCAR markers that amplified LRR-encoding regions, were developed from sequences of resistance gene homologs at the main resistance gene cluster in lettuce. Variation for these markers were assessed in germplasm including accessions of cultivated lettuce, Lactuca sativa L. and three wild Lactuca spp., L. serriola L., L. saligna and L. virosa L. Diversity was also studied within and between natural populations of L. serriola from Israel and California; the former is close to the center of diversity for Lactuca spp. while the latter is an area of more recent colonization. Large numbers of haplotypes were detected indicating the presence of numerous resistance genes in wild species. The diversity in haplotypes provided evidence for gene duplication and unequal crossing-over during the evolution of this cluster of resistance genes. However, there was no evidence for duplications and deletions within the LRR-encoding regions studied. The three markers were highly correlated with resistance phenotypes in L. sativa. They were able to discriminate between accessions that had previously been shown to be resistant to all known isolates of Bremia lactucae. Therefore, these markers will be highly informative for the establishment of core collections and marker-aided selection. A hierarchical analysis of the population structure of L. serriola showed that countries, as well as locations, were significantly differentiated. These differences may reflect local founder effects and/or divergent selection. Received: 7 March 1999 / Accepted: 25 March 1999