Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy

To study the effect of troponin (Tn) T mutations that cause familial hypertrophic cardiomyopathy (FHC) on cardiac muscle contraction, wild-type, and the following recombinant human cardiac TnT mutants were cloned and expressed: I79N, R92Q, F110I, E163K, R278C, and intron 16(G(1) --> A) (In16). These TnT FHC mutants were reconstituted into skinned cardiac muscle preparations and characterized for their effect on maximal steady state force activation, inhibition, and the Ca(2+) sensitivity of force development. Troponin complexes containing these mutants were tested for their ability to regulate actin-tropomyosin(Tm)-activated myosin-ATPase activity. TnT(R278C) and TnT(F110I) reconstituted preparations demonstrated dramatically increased Ca(2+) sensitivity of force development, while those with TnT(R92Q) and TnT(I79N) showed a moderate increase. The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity. ATPase activation was also impaired by TnT(F110I), while its inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca(2+) sensitivity of force; however, it produced an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca(2+) regulation of force development caused by these mutations would likely cause altered contractility and contribute to the development of FHC.     

NMR structure of stem-loop SL2 of the HIV-1 psi RNA packaging signal reveals a novel A-U-A base-triple platform

The genome of the human immunodeficiency virus type-1 (HIV-1) contains a stretch of approximately 120 nucleotides known as the psi-site that is essential for RNA packaging during virus assembly. These nucleotides have been proposed to form four stem-loops (SL1-SL4) that have both independent and overlapping functions. Stem-loop SL2 is important for efficient recognition and packaging of the full-length, unspliced viral genome, and also contains the major splice-donor site (SD) for mRNA splicing. We have determined the structure of the 19-residue SL2 oligoribonucleotide by heteronuclear NMR methods. The structure is generally consistent with the most recent of two earlier secondary structure predictions, with residues G1-G2-C3-G4 and C6-U7 forming standard Watson Crick base-pairs with self-complementary residues C16-G17-C18-C19 and A12-G13, respectively. However, residue A15, which is located near the center of the stem, does not form a predicted bulge, and residues A5 and U14 do not form an expected Watson-Crick base-pair. Instead, these residues form a novel A5-U14-A15 base-triple that appears to be stabilized by hydrogen bonds from A15-H61 and -H62 to A5-N1 and U14-O2, respectively; from A5-H61 to U14-O2, and from C16-H42 to U14-O2'. A kink in the backbone allows the aromatic rings of the sequential U14-A15 residues to be approximately co-planar, adopting a stable "platform motif" that is structurally similar to the A-A (adenosine) platforms observed in the P4-P6 ribozyme domain of the Tetrahymena group I intron. Platform motifs generally function in RNA by mediating long-range interactions, and it is therefore possible that the A-U-A base-triple platform mediates long-range interactions that either stabilize the psi-RNA or facilitate splicing and/or packaging. Residue G8 of the G8-G9-U10-G11 tetraloop is stacked above the U7-A12 base-pair, and the remaining tetraloop residues are disordered and available for potential interactions with either other RNA or protein components.     

Activation pattern and toxicity of the Cry11Bb1 toxin of Bacillus thuringiensis subsp. medellin

Bacillus thuringiensis protoxins undergo proteolytic processing in the midgut of susceptible insects to become active. The ability to process the Cry11Bb1 protoxin by trypsin and Culex quinquefasciatus larval gut extracts was tested. The protease activity indicated by the appearance of proteolytic products increased with an increment in pH, with the highest activity being observed at pH 10.6. A time course study showed the proteolysis of the 94-kDa Cry11Bb protein ending with the production of fragments of relative molecular mass of 30 and 35 kDa within 5 min. In vitro, gut proteases extract cleaved the solubilized toxin between Ser59 and Ile60 and between Ala395 and Asn396, generating a 30-kDa N-terminal and a 35-kDa C-terminal fragment, respectively. Similarly, mosquito larvae processed in vivo the parasporal inclusions, generating the same fragments as those observed in vitro. The Cry11Bb1 protoxin activated with trypsin or gut proteases showed larvicidal activity against C. quinquefasciatus first instar larvae. The data suggest that gut proteases participate in the activation of CryllBbl protoxin, generating at least two different fragments on which the activity could reside.     

Expression of oncogenic epidermal growth factor receptor family kinases induces paclitaxel resistance and alters beta-tubulin isotype expression

Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII and HER2 are more resistant to paclitaxel-mediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of beta-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed beta-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII- and HER2-expressing cells demonstrated equivalent total beta-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5-fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2. 95-fold) mRNA. Expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. Expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both beta-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes.     

The NMR structure of the nucleocapsid protein from the mouse mammary tumor virus reveals unusual folding of the C-terminal zinc knuckle

The nucleocapsid protein (NC) from the mouse mammary tumor virus (MMTV) has been overexpressed in Escherichia coli and purified to homogeneity for structural studies by nuclear magnetic resonance (NMR) spectroscopy. The protein contains two copies of a conserved zinc-coordinating "CCHC array" or "zinc knuckle" motif common to the nucleocapsid proteins of nearly all known retroviruses. The residues comprising and adjacent to the zinc knuckles were assigned by standard two-dimensional (1)H and three-dimensional (1)H-(15)N NMR methods; the rotational dynamic properties of the protein were determined from (15)N relaxation experiments, and distance restraints derived from the nuclear Overhauser effect (NOE) data were used to calculate the three-dimensional structure. The (1)H-(1)H NOE and (15)N relaxation data indicate that the two zinc knuckles do not interact with each other, but instead behave as independently folded domains connected by a flexible 13-residue linker segment. The proximal zinc knuckle folds in a manner that is essentially identical to that observed previously for the two zinc knuckles of the human immunodeficiency virus type 1 nucleocapsid protein and for the moloney murine leukemia virus nucleocapsid zinc knuckle domain. However, the distal zinc knuckle of MMTV NC exhibits a rare three-dimensional fold that includes an additional C-terminal beta-hairpin. A similar C-terminal reverse turn-like structure was observed recently in the distal zinc knuckle of the Mason-Pfizer monkey virus nucleocapsid protein [Gao, Y., et al. (1998) Protein Sci. 7, 2265-2280]. However, despite a high degree of sequence homology, the conformation and orientation of the beta-hairpin in MMTV NC is significantly different from that of the reverse turn in MPMV NC. The results support the conclusion that structural features of NC zinc knuckle domains can vary significantly among the different genera of retroviridae, and are discussed in terms of the recent and surprising discovery that MMTV NC can facilitate packaging of the HIV-1 genome in chimeric MMTV mutants.     

Identifying novel regulators of shoot meristem development

New organs are initiated throughout the life span of higher plants. This process occurs at the shoot meristem, which is initiated during embryogenesis and is later responsible for generating the above-ground portion of the plant. The shoot meristem can be thought of as having two zones, a central zone containing meristematic cells in an undifferentiated state, and a surrounding peripheral zone where cells enter a specific developmental pathway toward a differentiated state. Recent advances have revealed several genes that specifically regulate meristem development inArabidopsis. However, extensive mutagenesis by several labs have identified only a handful, of loci that appear to specifically regulate shoot meristem development. We have undertaken an enhancer/suppressor mutagenesis of an existing meristem mutant (clv1) and have identified novel regulators of meristem development. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Widespread Recombination,Reassortment, and Transmission of Unbalanced Compound Viral Genotypes in Natural Arenavirus Infections

Arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. Arenaviruses package a large (L) and small (S) genome segment in their virions. For segmented RNA viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. Although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. Here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. From 48 infected animals, we determined the complete or near complete sequence of 210 genome segments that grouped into 23 L and 11 S genotypes. The majority of snakes were multiply infected, with up to 4 distinct S and 11 distinct L segment genotypes in individual animals. This S/L imbalance was typical: in all cases intrahost L segment genotypes outnumbered S genotypes, and a particular S segment genotype dominated in individual animals and at a population level. We corroborated sequencing results by qRT-PCR and virus isolation, and isolates replicated as ensembles in culture. Numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring 2 intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. Overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. This diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. Thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential.     

Binding Interactions of Keratin-Based Hair Fiber Extract to Gold,Keratin, and BMP-2

Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines) have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2) has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (K_D) of 1.8 × 10⁻⁴ M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals) were at work. The association of BMP-2 to kerateine was found to be greater (K_D = 1.1 × 10⁻⁷ M), within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS) shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (K_D = 3.2 × 10⁻⁵ M). BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks), suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5), below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the development of better, more clinically relevant BMP-2-conjugated systems for bone repair and regeneration.