Polyarginine-containing peptides represent potent inhibitors of furin, a mammalian endoprotease that plays an important role in metabolism, activation of pathogenic toxins, and viral proliferation. The therapeutic use of D-polyarginines is especially interesting because they are not cleaved by furin and possess inhibitory potency almost equal to L-polyarginines. In this study we attempted to determine the important elements within polyarginines that contribute to effective inhibition. Structure-function analyses of polyarginine peptides showed that inhibition by polyarginine-containing peptides appeared to depend on the total number of basic charges of the positively charged inhibitors bound to the negatively charged substrate binding pocket; peptide positioning did not appear to be rigorously determined. Screening of L-and D-decapeptide positional scanning combinatorial peptide libraries indicated a preference for basic residues in nearly all positions, similar to previous results with hexapeptide libraries. Length and terminal modification studies showed that the most potent D-polyarginine tested was nona-D-arginine (D9R) amide with a K i of 1.3 nM. D9R amide was shown to protect RAW264.7 cells against anthrax toxemia with an IC 50 of 3.7 M. Because of its high stability, specificity, low toxicity, small molecular weight, and extremely low K i against furin, D9R amide or its derivatives may represent promising compounds for therapeutic use.Furin is a mammalian subtilisin/Kex2p-like endoprotease that is involved in the processing of many precursor proteins (reviewed in Refs. 1-3). The enzyme has a ubiquitous tissue distribution and cycles between the trans-Golgi network, the cell surface, and the endosomes. Furin plays a role in embryogenesis and homeostasis (4) and is also responsible for processing bacterial toxin precursors and virus envelope glycoprotein precursors (5, 6). Because of its involvement in bacterial and viral pathogenesis, furin represents an attractive target for therapeutic drugs.Polyarginines are known to be potent, small inhibitors of furin. L6R (hexa-L-arginine), 1 for example, exhibits the low inhibition constant (K i ) of 114 nM (7), and the D-forms of these polyarginines were also shown to be inhibitory. Moreover, D6R amide has been shown to block the activation of Pseudomonas aeruginosa exotoxin A (8) and to protect against anthrax toxemia both in vivo and in vitro (9).The structure of mouse furin has been recently determined (10) and reveals that the active site of the enzyme contains an extended substrate-binding groove that is lined with many negatively charged residues: these include Asp-258 and Asp-306 (surrounding the S1 subsite); Asp-154 and Asp-191, which form the surface of the S2 pocket; Glu-236 and Glu-264 (S4 subsite); Glu-257 and Glu-264 (Glu-264 takes part in forming the S4 and S5 subsites); and Glu-230 and Asp-233 (S6 subsite). No basic residues are present in the general area between the S5 and S1 subsites; basic residues are found only on the outer edge of the S1Ј sub...

Despite the extensive research efforts over the past 25 years that have focused on HIV, there is still no cure for AIDS. However, tremendous progress in the understanding of the structure and biology of the HIV virus led to the development of safe and potent HIV-based transgene delivery vectors. These genetic vehicles are referred to as lentiviral vectors. They appear to be better suited for particular applications, such as transgene delivery into stem cells, compared to other viral- and non-viral vectors. This is because Lentivirus-based vectors can efficiently infect nondividing and slowly dividing cells. In the present review article, the current state of understanding of HIV-1 is discussed and the main characteristics that had an impact on vector design are outlined. A historical view on the vector concept is presented to facilitate discussion of recent results in vector engineering in a broader context. Subsequently, a state of the art overview concerning vector construction and vector production is given. This review also touches upon the subject of lentiviral vector safety and related topics that can be helpful in addressing this issue are discussed. Finally, examples of Lentivirus-based gene delivery systems and their applications are presented, with emphasis on animal transgenesis and human gene therapy.

No abstract

No abstract

Roles played by homocysteine and choline in the regulation of MS (methionine synthase) have been examined in fungi. The Aspergillus nidulans metH gene encoding MS was cloned and characterized. Its transcription was not regulated by methionine, but was enhanced by homocysteine and repressed by choline and betaine. MS activity levels were regulated in a similar way. The repression by betaine was due to its metabolic conversion to choline, which was found to be very efficient in A. nidulans. Betaine and choline supplementation stimulated growth of leaky metH mutants apparently by decreasing the demand for methyl groups and thus saving methionine and S -adenosylmethionine. We have also found that homocysteine stimulates transcription of MS-encoding genes in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

No abstract

No abstract

Background Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. However, establishing its molecular diagnosis remains challenging. Chromosomal breakage analysis is the gold standard diagnostic test for this disease. Nevertheless, molecular analysis is always required for the identification of pathogenic alterations in the FA genes. Results We report here on a family with FA diagnosis in two siblings. Mitomycin C (MMC) test revealed high level of chromosome breaks and radial figures. In both children, array—Comparative Genomic Hybridization (aCGH) showed maternally inherited 16q24.3 deletion, including FANCA gene, and next generation sequencing (NGS) disclosed paternally inherited novel variants in the FANCA gene—Asn1113Tyr and Ser890Asn. A third sibling was shown to be a carrier of FANCA deletion only. Conclusions Although genetic testing in FA patients often requires a multi-method approach including chromosome breakage test, aCGH, and NGS, every effort should be made to make it available for whole FA families. This is not only to confirm the clinical diagnosis of FA in affected individuals, but also to enable identification of carriers of FA gene(s) alterations, as it has implications for diagnostic and genetic counselling process.