Designing multivalent immunogens for alphavirus vaccine optimization.

There is a pressing need for vaccines against mosquito-borne alphaviruses such as Venezualen and eastern equine encephalitis viruses (VEEV, EEEV). We demonstrate an approach to vaccine development based on physicochemical properties (PCP) of amino acids to design a PCP-consensus sequence of the epitope-rich B domain of the VEEV major antigenic E2 protein. The consensus "spike" domain was incorporated into a live-attenuated VEEV vaccine candidate (ZPC/IRESv1). Mice inoculated with either ZPC/IRESv1 or the same virus containing the consensus E2 protein fragment (VEEVconE2) were protected against lethal challenge with VEEV strains ZPC-738 and 3908, and Mucambo virus (MUCV, related to VEEV), and had comparable neutralizing antibody titers against each virus. Both vaccines induced partial protection against Madariaga virus (MADV), a close relative of EEEV, lowering mortality from 60% to 20%. Thus PCP-consensus sequences can be integrated into a replicating virus that could, with further optimization, provide a broad-spectrum vaccine against encephalitic alphaviruses.

The Novel Small Molecule Inhibitor, OSU-T315, Suppresses Vestibular Schwannoma and Meningioma Growth by Inhibiting PDK2 Function in the AKT Pathway Activation.

Activation of PKB/AKT signaling, which requires PDK1 and PDK2 function, drives Vestibular Schwannoma (VS) and meningioma growth. PDK2 function is defined as a molecule that phosphorylates AKT-Ser473. Integrin-Linked Kinase (ILK) functions as PDK2 in PKB/AKT activation in many cancers; therefore, we hypothesized that OSU-T315, a small molecule ILK inhibitor, will inhibit the ILK-PDK2 function in PKB/AKT signaling activation in VS and meningioma cell growth. OSU-T315 decreased cell viability at IC50 < 2µM in VS (HEI193) and meningioma (Ben-Men-1) cell lines, in primary cells at < 3.5µM, while in normal primary Schwann cells at 7.1µM. OSU-T315 inhibits AKT signaling by decreasing phosphorylation at AKT-Ser473, AKT-Thr308, ILK-Ser246 and ILK-Thr173. In addition, OSU-T315 affected the phosphorylation or expression levels of AKT downstream proliferation effectors as well as autophagy markers. Flow cytometry shows that OSU-T315 increased the percentage of cells arrested at G2/M for both, HEI193 (39.99%) and Ben-Men-1 (26.96%) cells, compared to controls (21.54%, 8.47%). Two hours of OSU-T315 treatment increased cell death in both cell lines (34.3%, 9.1%) versus untreated (12.1%, 8.1%). Though longer exposure increased cell death in Ben-Men-1, TUNEL assays showed that OSU-T315 does not induce apoptosis. OSU-T315 was primarily cytotoxic for HEI193 and Ben-Men-1 inducing a dysregulated autophagy. Our studies suggest that OSU-T315 has translational potential as a chemotherapeutic agent against VS and meningioma.

Efficacy of oryzalin and associated histological changes in Cryptosporidium-infected neonatal rats.

This paper reports the anti-cryptosporidial effects of, and concomitant amelioration of the histological changes in the gut of neonatal rats with intestinal cryptosporidiosis treated with the dinitroaniline, oryzalin. The ED50 was determined to be 7 mg/kg using twice daily doses administered for 3 consecutive days. A maximum inhibition of 85.5% was achieved at 25 mg/kg and this inhibition remained constant despite increasing the oryzalin dose to 200 mg/kg. Cryptosporidiosis significantly decreased the intestinal villus/crypt (VC) ratio by approximately 50% (duodenum = 2.3, jejunum = 2.5 and ileum = 1.7) when compared to uninfected untreated controls (duodenum = 4.3, jejunum = 5.9 and ileum = 4.5). Treatment with oryzalin doubled the VC ratio in the duodenum, jejunum and ileum following doses of 5 mg, 50 mg and 200 mg/kg respectively. Oryzalin concentrations in the small intestine contents and plasma were determined, using HPLC, at 0.5, 1 and 2 h after dosing. The much greater dose required to return VC ratios to normal in the ileum (200 mg/kg) compared to the duodenum (6.25 mg/kg) appeared to reflect the decreased concentration of the drug in the distal small intestine. Concentrations of oryzalin equivalent to the in vitro IC50 were maintained for 2 h in the first half of the small intestine following a single dose of 100 mg/kg.

Domain structure and subunit interactions in the type I DNA methyltransferase M.EcoR124I.

The type IC DNA methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two modification subunits, HsdM, and a single specificity subunit, HsdS. Studies have been largely restricted to the HsdM subunit or to the intact methyltransferase since the HsdS subunit is insoluble when over-expressed independently of HsdM. Two soluble fragments of the HsdS subunit have been cloned, expressed and purified; a 25 kDa N-terminal fragment (S3) comprising the N-terminal target recognition domain together with the central conserved domain, and a 8.6 kDa fragment (S11) comprising the central conserved domain alone. Analytical ultracentrifugation shows that the S3 subunit exists principally as a dimer of 50 kDa. Gel retardation and competition assays show that both S3 and S11 are able to bind to HsdM, each with a subunit stoichiometry of 1:1. The tetrameric complex (S3/HsdM)(2) is required for effective DNA binding. Cooperative binding is observed and at low enzyme concentration, the multisubunit complex dissociates, leading to a loss of DNA binding activity. The (S3/HsdM)(2) complex is able to bind to both the EcoR124I DNA recognition sequence GAAN(6)RTCG and a symmetrical DNA sequence GAAN(7)TTC, but has a 30-fold higher affinity binding for the latter DNA sequence. Exonuclease III footprinting of the (S3/HsdM)(2) -DNA complex indicates that 29 nucleotides are protected on each strand, corresponding to a region 8 bp on both the 3' and 5' sides of the recognition sequence bound by the (S3/HsdM)(2) complex.

Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas.

The melanoma differentiation associated gene-7 (mda-7) has a potential inhibitory role in melanoma progression, although the mechanisms underlying this effect are still unknown. mda-7 mRNA has been found to be present at higher levels in cultured normal melanocytes compared with metastatic melanoma cell lines. Furthermore, levels of mda-7 message have shown an inverse correlation with melanoma progression in human tumor samples, suggesting that mda-7 may be a novel tumor suppressor gene. We have designed this study to investigate MDA-7 protein expression in different stages of melanoma progression and to examine its antiproliferative effects in vitro. Our data demonstrate that MDA-7 protein can be found in normal melanocytes and early stage melanomas. It is also observed in smooth muscle cells in the skin. However, in keeping with a possible role as a tumor suppressor, MDA-7 expression is decreased in more advanced melanomas, with nearly undetectable levels in metastatic disease. We also investigated antitumor effects of overexpressed MDA-7 on human melanoma cells in vitro. Our results demonstrate that Ad-mda-7 induces apoptosis and G2/M cell cycle arrest in melanoma cells, but not in normal human melanocytes.

Solution structure and backbone dynamics of the DNA-binding domain of mouse Sox-5.

The fold of the murine Sox-5 (mSox-5) HMG box in free solution has been determined by multidimensional NMR using (15)N-labeled protein and has been found to adopt the characteristic twisted L-shape made up of two wings: the major wing comprising helix 1 (F10--F25) and helix 2 (N32--A43), the minor wing comprising helix 3 (P51--Y67) in weak antiparallel association with the N-terminal extended segment. (15)N relaxation measurements show considerable mobility (reduced order parameter, S(2)) in the minor wing that increases toward the amino and carboxy termini of the chain. The mobility of residues C-terminal to Q62 is significantly greater than the equivalent residues of non-sequence-specific boxes, and these residues show a weaker association with the extended N-terminal segment than in non-sequence boxes. Comparison with previously determined structures of HMG boxes both in free solution and complexed with DNA shows close similarity in the packing of the hydrophobic cores and the relative disposition of the three helices. Only in hSRY/DNA does the arrangement of aromatic sidechains differ significantly from that of mSox-5, and only in rHMG1 box 1 bound to cisplatinated DNA does helix 1 have no kink. Helix 3 in mSox-5 is terminated by P68, a conserved residue in DNA sequence-specific HMG boxes, which results in the chain turning through approximately 90 degrees.

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5.

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4x10(7) M(-1), increasing somewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K(-1) mol(-1), a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about -2 kJ K(-1) mol(-1), in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.

The energetics of HMG box interactions with DNA. Thermodynamic description of the box from mouse Sox-5.

The structural energetics of the HMG box from the DNA-binding protein mouse Sox-5 were examined calorimetrically. It was found that this box, notwithstanding its small size (molecular mass about 10 kDa), does not behave as a single cooperative unit and, on heating, the box reversibly unfolds in two separate stages. The first transition (tt approximately 34 degrees C) involves about 40% of the total enthalpy and the second (tt approximately 46 degrees C) the remainder. Both transitions proceed with significant heat capacity increment, showing that they are associated with the unfolding of two sub-domains having non-polar cores. According to heat capacity, ellipticity, fluorescence and NMR criteria, this HMG box is in a fully compact native state only below 5 degrees C. HMG boxes consist of two approximately orthogonal wings: the minor wing comprises helix 3 and its associated antiparallel N-terminal strand, whilst the major wing is composed of helices I and II. Analysis of the fluorescence and NMR spectra for this box obtained at different temperatures shows that the lower melting transition can be assigned to the minor wing and the upper transition to the major wing. Under physiological conditions (37 degrees C), the minor wing is considerably unfolded, whilst the major wing is essentially fully folded. DNA binding in vivo therefore involves refolding of the minor wing.

Template-dependent initiation of Sindbis virus RNA replication in vitro.

Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

Mapping human telomere regions with YAC and P1 clones: chromosome-specific markers for 27 telomeres including 149 STSs and 24 polymorphisms for 14 proterminal regions.

A YAC library enriched for telomere clones was constructed and screened for the human telomere-specific repeat sequence (TTAGGG). Altogether 196 TYAC library clones were studied: 189 new TYAC clones were isolated, 149 STSs were developed for 132 different TY-ACs, and 39 P1 clones were identified using 19 STSs from 16 of the TYACs. A combination of mapping methods including fluorescence in situ hybridization, somatic cell hybrid panels, clamped homogeneous electric fields, meiotic linkage, and BLASTN sequence analysis was utilized to characterize the resource. Forty-five of the TYACs map to 31 specific telomere regions. Twenty-four linkage markers were developed and mapped within 14 proterminal regions (12 telomeres and 2 terminal bands). The polymorphic markers include 12 microsatellites for 10 telomeres (1q, 2p, 6q, 7q, 10p, 10q, 13q, 14q, 18p, 22q) and the terminal bands of 11q and 12p. Twelve RFLP markers were identified and meiotically mapped to the telomeres of 2q, 7q, 8p, and 14q. Chromosome-specific STSs for 27 telomeres were identified from the 196 TYACs. More than 30,000 nucleotides derived from the TYAC vector-insert junction regions or from regions flanking TYAC microsatellites were compared to reported sequences using BLASTN. In addition to identifying homology with previously reported telomere sequences and human repeat elements, gene sequences and a number of ESTs were found to be highly homologous to the TYAC sequences. These genes include human coagulation factor V (F5), Weel protein tyrosine kinase (WEE1), neurotropic protein tyrosine kinase type 2 (NTRE2), glutathione S-transferase (GST1), and beta tubulin (TUBB). The TYAC/P1 resource, derivative STSs, and polymorphisms constitute an enabling resource to further studies of telomere structure and function and a means for physical and genetic map integration and closure.

The DNA bend angle and binding affinity of an HMG box increased by the presence of short terminal arms.

The HMG box of human LEF-1 (hLEF-1, formerly TCF1alpha) has been expressed in four forms: a parent box of 81 amino acids and constructs having either a 10 amino acid C-terminal extension, a 9 amino acid N-terminal extension, or both. These four species have been compared for DNA binding and bending ability using a 28 bp recognition sequence from the TCR alpha-chain enhancer. In the bending assay, whereas the parent box and that with the N-terminal extension bent the DNA by 57/58 degrees, the box extended at the C-terminus bent the DNA by 77/78 degrees, irrespective of the presence or absence of the N-terminal extension. A 6- fold increase in DNA affinity also resulted from addition of both terminal extensions. These observations redefine the functional boundaries of the HMG box. The structure of a mouse LEF-1/DNA complex recently published [Love et al. (1995) Nature 376, 791-795] implies that the higher DNA affinity and in particular the increased bend angle observed are consequences, at least in part, of the C-terminal extension spanning the major groove on the inside of the DNA bend.

A combined analysis of D22S278 marker alleles in affected sib-pairs: support for a susceptibility locus for schizophrenia at chromosome 22q12. Schizophrenia Collaborative Linkage Group (Chromosome 22).

Several groups have reported weak evidence for linkage between schizophrenia and genetic markers located on chromosome 22q using the lod score method of analysis. However these findings involved different genetic markers and methods of analysis, and so were not directly comparable. To resolve this issue we have performed a combined analysis of genotypic data from the marker D22S278 in multiply affected schizophrenic families derived from 11 independent research groups worldwide. This marker was chosen because it showed maximum evidence for linkage in three independent datasets (Vallada et al., Am J Med Genet 60:139-146, 1995; Polymeropoulos et al., Neuropsychiatr Genet 54:93-99, 1994; Lasseter et al., Am J Med Genet, 60:172-173, 1995. Using the affected sib-pair method as implemented by the program ESPA, the combined dataset showed 252 alleles shared compared with 188 alleles not share (chi-square 9.31, 1df, P = 0.001) where parental genotype data was completely known. When sib-pairs for whom parental data was assigned according to probability were included the number of alleles shared was 514.1 compared with 437.8 not shared (chi-square 6.12, 1df, P = 0.006). Similar results were obtained when a likelihood ratio method for sib-pair analysis was used. These results indicate that may be a susceptibility locus for schizophrenia at 22q12.

The DNA sequence specificity of HMG boxes lies in the minor wing of the structure.

To establish the basis of sequence-specific DNA recognition by HMG boxes we separately transferred the minor and major wings from the sequence-specific HMG box of TCF1 alpha into their equivalent position in the non-sequence-specific box 2 of HMG1. Thus chimera THT1 contains the minor wing (of 11 N-terminal and 25 C-terminal residues) from the HMG box of TCF1 alpha and the major wing (the 45 residue central section) from HMG1 box 2, whilst the situation is reversed in chimera HTH1. The structural integrity of the two chimeric proteins was established by CD, NMR and their binding to four-way junction DNA. Gel retardation and circular permutation assays showed that only chimera THT1, containing the TCF1 alpha minor wing, formed a sequence-specific complex and bent the DNA. The bend angle was estimated to be 59 degrees for chimera THT1 and 52 degrees for the HMG box of TCF1 alpha. Our results, in combination with mutagenesis and other data, suggests a model for the DNA binding of HMG boxes in which the N-terminal residues and part of helix 1 contact the minor groove on the outside of a bent DNA duplex.

DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5.

Sox-5 is one of a family of genes which show homology to the HMG box region of the testis determining gene SRY. We have used indirect immunofluorescence to show that Sox-5 protein is localized to the nucleus of post-meiotic round spermatids in the mouse testis. In vitro footprinting and gel retardation assays demonstrate that Sox-5 binds specifically to the sequence AACAAT with moderately high affinity (Kd of approximately 10(-9) M). Moreover, interaction of Sox-5 with its target DNA induces a significant bend in the DNA, characteristic of HMG box proteins. Circular dichroism spectroscopy of the Sox-5 HMG box and its specific complex with DNA shows an alteration in the DNA spectrum, perhaps as a consequence of DNA bending, but none in the protein spectrum on complex formation. The dependence of the change in the CD spectrum with protein to DNA ratio demonstrates the formation of a 1:1 complex. Analysis of the structure of the Sox-5 HMG box by 2D NMR suggests that both the location of helical secondary structure as well as the tertiary structure is similar to that of HMG1 box 2.

Solution structure of a DNA-binding domain from HMG1.

We have determined the tertiary structure of box 2 from hamster HMG1 using bacterial expression and 3D NMR. The all alpha-helical fold is in the form of a V-shaped arrowhead with helices along two edges and one rather flat face. This architecture is not related to any of the known DNA binding motifs. Inspection of the fold shows that the majority of conserved residue positions in the HMG box family are those involved in maintaining the tertiary structure and thus all homologous HMG boxes probably have essentially the same fold. Knowledge of the tertiary structure permits an interpretation of the mutations in HMG boxes known to abrogate DNA binding and suggests a mode of interaction with bent and 4-way junction DNA.

Interhospital transfer in the management of acute trauma.

During an 8-month period, 538 injured patients were transferred from primary hospitals to a referral hospital for further management of their injuries. Delay at the primary hospital was identified in 20% of all transfers and in 40% of patients transferred for management of head injury or multisystem injury. Delay at the primary hospital resulted in a median time from injury to arrival at the second hospital of 4 h. Defects in clinical management during transport included inexperienced escorts, inadequate airway control, ventilation, fluid resuscitation and stabilization of chest injuries. Nearly half of transfers were inappropriate because of the relatively minor nature of the injuries. Most of these had solitary musculoskeletal injuries to the extremities. These patients reflect the marked deficiency of specialist orthopaedic services in western Sydney during the study. Development of a metropolitan regional system of trauma care in western Sydney requires urgent action towards reducing the frequency of transfer, minimizing delays in transfer and maximizing basic resuscitation of seriously injured patients. Some designation of hospital roles is required and needs to be accompanied by a prehospital triage process. The population also has a right to expect adequate specialty services at suburban hospitals to enable treatment of minor and moderate single system injuries. Future trauma system developments should adequately reflect population growth and technological advances in clinical care.

Coordinate replication of dispersed repetitive sequences in Physarum polycephalum.

The synchronous macroplasmodial growth phase of the slime mould Physarum polycephalum was used to study the in vivo replication of large chromosomal DNA segments. Newly replicated DNA was isolated at various points in S-phase by its preferential association with the nuclear matrix. This DNA was then used to probe cosmid clones of the Physarum genome. The results indicate that certain dispersed repetitive sequences in the genome are coordinately replicated. The observed pattern of replication may be due either to the presence of a replication origin within each repetitive sequence or to the systematic arrangement of these sequences around a replication origin. The latter appears more likely since the repetitive sequences are probably not randomly scattered within the genome.

Uptake and metabolism of beta-carotene and retinal by C3H/10T1/2 cells.

Both beta-carotene (beta-C), a vitamin A precursor, and vitamin A itself have been shown to reversibly inhibit neoplastic transformation in 10T1/2 cells during the progression phase of carcinogenesis. In order to determine whether the activity of beta-C in these cells may be attributed to conversion to vitamin A or is intrinsic to the carotenoid molecule, the uptake and metabolism of beta-C, and of retinal, the immediate product of dioxygenase-cleavage of beta-C, was studied in 10T1/2 cells. Cellular uptake of 2.6 nmol/10(6) cells occurred 24 h after treatment with 10(-5) M beta-C. Thereafter, cell levels remained relatively stable between 1 and 2 nmol/10(6) cells over the 1-week treatment period. Upon removal of beta-C from the medium, cellular levels decreased by approximately 80% in 2 weeks, then stabilized. Retinal was rapidly and quantitatively converted to retinol by 10T1/2 cells, suggesting that the inhibitory action of retinal on neoplastic transformation in these cells is due to its conversion to retinol, and that any enzymatic conversion of beta-C to retinal by these cells would be expected to result in retinol as the end product. Using [14C]beta-C, we found no evidence for formation of [14C]retinol, [14C]retinal or [14C]retinoic acid using sensitive HPLC. We therefore conclude that beta-C has intrinsic chemopreventive activity in 10T1/2 cells, perhaps due to its anti-oxidant properties.