Chromatographic determination of angiotensin-converting enzyme and angiotensinase activity

An analytical method utilizing an automatic amino acid analyzer is described for the separation, identification, and measurement of 5 to 50 nmol of angiotensin I, angiotensin II, [Des-Phe8]angiotensin II, Phe-His-Leu, His-Leu, isoleucine, leucine, tyrosine, and phenylalanine. Aminex A-5 cation-exchange resin (0.9 x 15 cm) is sequentially eluted with three sodium citrate buffers: pH 3.25, 0.2 N; pH 4.85, 0.54 N, and pH 6.5, 0.39 N at 60 and 80 degrees C. Reaction with ninhydrin is used for detection. This chromatographic system was used to determine angiotensin-converting enzyme activity and the angiotensinase activity of rabbit brain endopeptidase B. In each assay, the unhydrolyzed substrate and both products were measured simultaneously in one step without pretreatment of the hydrolysate. Products were recovered in 1:1 molar ratios and the overall recovery of an hydrolyzed substrate of products was quantitative.     

A reactive peptidic linker for self-assembling hybrid quantum dot-DNA bioconjugates

Self-assembly of proteins, peptides, DNA, and other biomolecules to semiconductor quantum dots (QD) is an attractive bioconjugation route that can circumvent many of the problems associated with covalent chemistry and subsequent purification. Polyhistidine sequences have been shown to facilitate self-assembly of proteins and peptides to ZnS-overcoated CdSe QDs via complexation to unoccupied coordination metal sites on the nanocrystal surface. We describe the synthesis and characterization of a thiol-reactive hexahistidine peptidic linker that can be chemically attached to thiolated-DNA oligomers and mediate their self-assembly to CdSe-ZnS core-shell QDs. The self-assembly of hexahistidine-appended DNA to QDs is probed with gel electrophoresis and fluorescence resonance energy transfer techniques, and the results confirm high-affinity conjugate formation with control over the average molar ratio of DNA assembled per QD. To demonstrate the potential of this reactive peptide linker strategy, a prototype QD-DNA-dye molecular beacon is self-assembled and tested against both specific and nonspecific target DNAs. This conjugation route is potentially versatile, as altering the reactivity of the peptide linker may allow targeting of different functional groups such as amines and facilitate self-assembly of other nanoparticle-biomolecule structures.     

Novel instrument for high-resolution ultrasound flow imaging

In multigate systems employed in Doppler flow-imaging equipment, the velocities of targets located at different ranges along the ultrasound beam axis are simultaneously detected. Typically, to reduce the computation efforts, this processing is aimed at extracting a single parameter (e.g., the mean velocity) from each signal. In this article, a novel ultrasound multigate instrument is presented capable of providing a detailed analysis of Doppler signals originated from 64 different range cells. By means of a dedicated high-speed fast-Fourier transform processor, these signals are transformed into the frequency domain without loss of information. The implementation of the new multigate system is described here, and the pieces of information which it can provide are discussed. Significant results obtained in vitro and in vivo demonstrate that the new instrument is capable of accurately reproducing the actual velocity profiles of the interrogated flow. © 1997 John Wiley & Sons, Inc. Int J Imaging Syst Technol, 8, 438–443, 1997     

Controlled DNA-templated metal deposition: towards ultra-thin nanowires

In this paper, we report the metallization of a dsDNA template using a novel photography-derived two-step strategy in which dsDNA is first complexed with Ag(I) ions and then irradiated with UV light at 254 nm. The nucleobases act as light harvesters and sensitizers, triggering the photoreduction of the complexed silver ions. This process yields a silver nanoparticles blueprint along the DNA strand. The silver latent image is then developed by depositing metallic nickel through an electroless plating process. This photography-derived procedure generates very homogeneous and evenly distributed strings of silver-core/nickel-shell nanoparticles. Although still discontinuous, we believe that such chains can serve as the base for obtaining continuous metal nanowires. Furthermore, this process can most likely be extended to other plating metals, resulting in a broadly general procedure for metallizing DNA with a variety of different materials. Because of the intrinsic simplicity in using light as the key step, this methodology might be amenable to large-scale development, eventually leading to a very efficient molecular-photolithography process.     

Evidence for a Conserved Function of Eukaryotic Pantothenate Kinases in the Regulation of Mitochondrial Homeostasis and Oxidative Stress

Human PANK1, PANK2, and PANK3 genes encode several pantothenate kinase isoforms that catalyze the phosphorylation of vitamin B5 (pantothenic acid) to phosphopantothenate, a critical step in the biosynthesis of the major cellular cofactor, Coenzyme A (CoA). Mutations in the PANK2 gene, which encodes the mitochondrial pantothenate kinase (PanK) isoform, have been linked to pantothenate-kinase associated neurodegeneration (PKAN), a debilitating and often fatal progressive neurodegeneration of children and young adults. While the biochemical properties of these enzymes have been well-characterized in vitro, their expression in a model organism such as yeast in order to probe their function under cellular conditions have never been achieved. Here we used three yeast mutants carrying missense mutations in the yeast PanK gene, CAB1, which are associated with defective growth at high temperature and iron, mitochondrial dysfunction, increased iron content, and oxidative stress, to assess the cellular function of human PANK genes and functional conservation of the CoA-controlled processes between humans and yeast. Overexpression of human PANK1 and PANK3 in these mutants restored normal cellular activity whereas complementation with PANK2 was partial and could only be achieved with an isoform, PanK2^mtmΔ, lacking the mitochondrial transit peptide. These data, which demonstrate functional conservation of PanK activity between humans and yeast, set the stage for the use of yeast as a model system to investigate the impact of PKAN-associated mutations on the metabolic pathways altered in this disease.     

Seventeen copies of the human 37 kDa laminin receptor precursor/p40 ribosome-associated protein gene are processed pseudogenes arisen from retropositional events

A cDNA coding for a 37 kDa polypeptide has been identified in several species as both the potential precursor of the 67 kDa laminin receptor (37LRP) and a putative ribosome-associated protein (p40). Interestingly, increased expression of this polypeptide (37LRP/p40) is consistently observed in invasive and metastatic cancer cells and is associated with poor prognosis. Southern-blot analysis of human genomic DNA predicted multiple copies of the 37LRP/p40 gene. In this study, we report that the number of copies of this sequence in the human genome is 26 +/- 2. We have sequenced and analyzed 19 genomic clones corresponding to the 37LRP/p40 gene and found that they were all processed pseudogenes. They all lack intronic sequences and show multiple genetic alterations leading in some cases to the appearance of stop codons. Moreover, they all bear characteristic features of retroposons as the presence of a poly(A)-tail at their 3' end and short direct repeated flanking DNA sequences. None of the pseudogenes analyzed present cis-elements in their 5' flanking region such as TATA or GC boxes. Our date reveal that over 50% of the 37LRP/p40 gene copies are pseudogenes most probably generated by retropositional events. The finding of multiple pseudogenes for the 37LRP/p40 suggests that the accumulation of several copies of this gene might have given a survival advantage to the cell in the course of evolution.     

Simultaneous onset of primary cutaneous B-cell lymphoma and human herpesvirus 8-associated Kaposi's sarcoma

We report the simultaneous occurrence of Kaposi's sarcoma (KS) and primary cutaneous B-cell lymphoma (CBCL) of the leg in a 79-year-old woman, seronegative for HIV-1, HTLV-1 and HTLV-2. The CBCL underwent complete clinical remission after local radiotherapy, whilst the KS became disseminated within a year following diagnosis. However, 2 years after the diagnosis of KS, the patient died with neurological symptoms. These were presumed to be due to involvement of the central nervous system by lymphoma, although in the absence of an autopsy, this could not be proven. Skin biopsies from the original KS and CBCL lesions, as well as short-term culture of spindle cells from the KS lesion and peripheral blood mononuclear cells (PBMC), were studied by semiquantitative polymerase chain reaction (PCR) using primers specific for DNA sequences of a novel gamma-herpesvirus-8 (HHV-8). PCR studies were strongly positive for the virus on KS cells and PBMC; conversely, a low viral load was found on CBCL cells. A high titre of serum IgG antibodies reacting with the nuclei of the HHV-8 positive cell line BCP-1 was found. These data suggest that reactivation of latent infection with HHV-8 had occurred in this patient, and that HHV-8 is directly involved in KS, but not in CBCL of the leg, an aggressive variant of CBCL.