A kinesin heavy chain (KIF5A) mutation in hereditary spastic paraplegia (SPG10)

We have identified a missense mutation in the motor domain of the neuronal kinesin heavy chain gene KIF5A, in a family with hereditary spastic paraplegia. The mutation occurs in the family in which the SPG10 locus was originally identified, at an invariant asparagine residue that, when mutated in orthologous kinesin heavy chain motor proteins, prevents stimulation of the motor ATPase by microtubule-binding. Mutation of kinesin orthologues in various species leads to phenotypes resembling hereditary spastic paraplegia. The conventional kinesin motor powers intracellular movement of membranous organelles and other macromolecular cargo from the neuronal cell body to the distal tip of the axon. This finding suggests that the underlying pathology of SPG10 and possibly of other forms of hereditary spastic paraplegia may involve perturbation of neuronal anterograde (or retrograde) axoplasmic flow, leading to axonal degeneration, especially in the longest axons of the central nervous system.     

Integration and excision of the Mycobacterium tuberculosis prophage-like element,phiRv1

The genomes of Mycobacterium tuberculosis H37Rv and CDC1551 each contain two prophage-like elements, phiRv1 and phiRv2. The phiRv1 element is not only absent from Mycobacterium bovis BCG but is in different locations within the two sequenced M. tuberculosis genomes; in both cases phiRv1 is inserted into a REP13E12 repeated sequence, which presumably contains the bacterial attachment site, attB, for phiRv1. Although phiRv1 is probably too small to encode infectious phage particles, it may nevertheless have an active integration/excision system and be capable of moving from one chromosomal position to another. We show here that the M. tuberculosis H37Rv phiRv1 element does indeed encode an active site-specific recombination system in which an integrase of the serine recombinase family (Rv1586c) catalyses integration and excision and a small, basic phiRv1-encoded protein (Rv1584c) controls the directionality of re-combination. Integration-proficient plasmid vectors derived from phiRv1 efficiently transform BCG, can utilize four of the seven REP13E12 sites present in BCG as attachment sites, and can occupy more than one site simultaneously.     

Combinatorial library of indinavir analogues: replacement for the aminoindanol at P2'

A 1X22X41 combinatorial library or 902 compounds of indinavir analogues was synthesized on the solid support to identify a replacement for the aminoindanol moiety at P2'. 2,6-Dimethyl-4-hydroxy phenol was discovered to be a good replacement for aminoindanol.     

Characterization of a genetically engineered elastin-like polypeptide for cartilaginous tissue repair

Elastin-like polypeptides (ELPs) are artificial polypeptides with unique properties that make them attractive as a biomaterial for tissue-engineered cartilage repair. ELPs are composed of a pentapeptide repeat, Val-Pro-Gly-Xaa-Gly (Xaa is any amino acid except Pro), that undergo an inverse temperature phase transition. They are soluble in aqueous solution below their transition temperature (T(t)) but aggregate when the solution temperature is raised above their T(t). This study investigates the rheological behavior of an un-cross-linked ELP, below and above its T(t), and also examines the ability of ELP to promote chondrogenesis in vitro. A thermally responsive ELP with a T(t) of 35 degrees C was synthesized using recombinant DNA techniques. The complex shear modulus of the ELP increased by 3 orders of magnitude as it underwent its inverse temperature phase transition, forming a coacervate, or gel-like, ELP phase. Values for the complex shear moduli of the un-cross-linked ELP coacervate are comparable to those reported previously for collagen, hyaluronan, and cross-linked synthetic hydrogels. Cell culture studies show that chondrocytes cultured in ELP coacervate maintain a rounded morphology and their chondrocytic phenotype, characterized by the synthesis of a significant amount of extracellular matrix composed of sulfated glycosaminoglycans and collagen. These results suggest that ELPs demonstrate great potential for use as in situ forming scaffolds for cartilaginous tissue repair.     

Cloning of two tryptophan hydroxylase genes expressed in the diencephalon of the developing zebrafish brain

The monoamine serotonin (5-HT) exerts key neuromodulatory activities in all animal phyla, but the development and function of the serotonergic system is still incompletely understood. The zebrafish Danio rerio is an excellent model to approach this question since it is amenable to a combination of genetic, molecular and embryological studies. In order to characterize the organization of serotonergic neurons in the zebrafish we cloned two cDNAs encoding distinct forms of tryptophan hydroxylase (Tph), the rate-limiting enzyme in serotonin synthesis. We report here the pattern of expression of these two genes in relation with immunoreactive TH and 5-HT nuclei in the developing zebrafish embryo and early larva. tphD1 expression starts at 22 h post-fertilization (hpf) in the epiphysis and in basal spinal cells. Expression persists in the epiphysis until at least 4 days (dpf). Between 48 hpf and 3 dpf, tphD1 expression is initiated in retinal amacrine cells and in restricted preoptic and posterior tubercular nuclei within the basal diencephalon. At 3 and 4 dpf, tphD1 expression is newly initiated in the caudal hypothalamus and in branchial arches-associated neurons. tphD2 mRNA is detected transiently (between 30 somites and 32 hpf) in a restricted preoptic nucleus. All sites of tphD1 or D2 expression within the anterior central nervous system are also immunoreactive for 5-HT, but are not positive for TH. However, neither tphD gene is expressed in raphe nuclei, suggesting that additional tph gene(s) exist in zebrafish to account for 5-HT synthesis in that location. The co-expression of tphD1, tphD2 and 5-HT in the zebrafish diencephalon appears in striking contrast to the situation in mammals, where diencephalic serotonin results from re-uptake rather than from local production.     

Rapid method for measuring ScFv thermal stability by yeast surface display

We have characterized a simplified method to determine the relative thermal stability of single-chain antibodies by following the irreversible denaturation of scFv fusions on the surface of yeast by flow cytometry. The method was highly reproducible and correlated well with other methods used to monitor thermal denaturation of the soluble proteins. We found a range of thermal stabilities for wild-type single-chain antibodies with half-maximum denaturation temperatures between 43 and 61 degrees C. The ability to quantitate thermal stability of antibodies or other proteins that are immobilized on the surface of yeast allows rapid comparisons of primary structural information with stability. Thermal denaturation could be a useful parameter to consider in the choice of scFv fragments for various applications.     

Synthesis and biological activity of piperazine-based dual MMP-13 and TNF-alpha converting enzyme inhibitors

A series of novel MMP-13 and TNF-alpha converting enzyme inhibitors based on piperazine 2-hydroxamic acid scaffolds are described. The TACE, MMP-1 and MMP-13 activity of these inhibitors as well as the effect of substitution of the piperazine nitrogen and the P-1' benzyloxy tailpiece is discussed. Moderate in vivo activity is observed with several members of this group.     

4-(2-pyridyl)piperazine-1-carboxamides: potent vanilloid receptor 1 antagonists

A series of 4-(2-pyridyl)piperazine-1-carboxamide analogues based on the lead compound 1 was synthesized and evaluated for VR1 antagonist activity in capsaicin-induced (CAP) and pH (5.5)-induced (pH) FLIPR assays in a rat VR1-expressing HEK293 cell line. Potent VR1 antagonists were identified through SAR studies. From these studies, 18 was found to be very potent in the in vitro assay [IC(50)=4.8 nM (pH) and 35 nM (CAP)] and orally available in rat (F%=15.1).