Although metastatic breast cancer is responsive to radioimmunotherapy (RIT), a systemic targeted radiation modality, complete and permanent remissions are not typical with single-modality treatment. Antiangiogenic agents, which target normal, proliferating endothelial cells, have the potential to provide relatively nontoxic continuous inhibition of tumor growth by blocking new blood vessel growth and may synergize with RIT to increase efficacy. This study was designed to determine whether, and how, the cyclic Arg-Gly-Asp peptide Cilengitide (EMD 121974), which targets the alpha(v)beta(3) integrin receptor expressed on neovasculature, could increase systemic RIT efficacy of therapy in a human breast cancer tumor model having mutant p53 and expressing bcl-2. HBT 3477 breast cancer tumor response in nude mice was compared between groups of untreated mice (n = 24), Cilengitide-treated mice (n = 18), RIT (200-260 mu Ci (90)Y-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-peptide ChL6; n = 46), and combined modality RIT (CMRIT) using RIT and six doses of Cilengitide (250 microg/dose; n = 41). Tumor size, survival, body weight, and blood counts were monitored for efficacy and toxicity of therapy. To clarify the mechanism of synergistic effect, tumors were evaluated at selected time points through 6 days for apoptosis, proliferation, and microvessel density. Cilengitide alone did not alter tumor growth when compared with untreated mice, but CMRIT with Cilengitide increased efficacy of treatment, with the cure rate for mice that received 260 mu Ci RIT increasing from 15 to 53% (P = 0.011). Lower-dose RIT (200 mu Ci) combined with Cilengitide resulted in less increase in cures (36 compared with 25% for RIT alone; P = 0.514). Combined analysis for high- and low-dose groups demonstrated increased efficacy of CMRIT (P = 0.020). Analysis of tumors from CMRIT mice indicated significantly increased apoptosis of tumor and endothelial cells 5 days after RIT compared with tumors from mice given RIT alone. Proliferation was decreased in CMRIT tumors compared with RIT tumors at 6 days (ANOVA, P < 0.05). Microvessel density in tumors from RIT and CMRIT mice was not different. No increased toxicity attributable to Cilengitide was observed based upon pooled blood sample and no statistical increase in mortality. In conclusion, CMRIT, combining Cilengitide and RIT, significantly increased the efficacy of therapy and increased apoptosis compared with single-modality therapy with either agent, in an aggressive, well-studied breast cancer model. The enhanced therapeutic synergy is of particular note, having been achieved without additional toxicity.

Many biologically important peptide sequences contain proline. It confers unique conformational constraints on the peptide chain in that the side-chain is cyclized back onto the backbone amide position. Inside an alpha-helix the possibility of making hydrogen bonds to the preceding turn is lost and a kink will be introduced. The conformational restrictions imposed by proline motifs in a peptide chain appear to imply important structural or biological functions as can be deduced from their often remarkably high degree of conservation as found in many proteins and peptides, especially cytokines, growth factors, G-protein-coupled receptors, V3 loops of the HIV envelope glycoprotein gp 120, and neuro- and vasoactive peptides. Only a limited number of peptidases are known to be able to hydrolyze proline adjacent bonds. Their activity is influenced by the isomeric state (cis-trans) as well as the position of proline in the peptide chain. The three proline specific metallo-peptidases (aminopeptidase P, carboxypeptidase P and prolidase) are activated by Mn2+, whereas the three serine type peptidases cleaving a post proline bond (prolyl oligopeptidase, dipeptidyl peptidase IV, and prolylcarboxypeptidase) share the sequential order of the catalytic Ser-Asp-His triade, which differentiates them from the chymotrypsin (His-Asp-Ser) and subtilisin (Asp-His-Ser) families. An endo or C terminal Pro-Pro bond and an endo pre-Pro peptide bond possess a high degree of resistance to any mammalian proteolytic enzyme.

BACKGROUND

The Rho small GTPase regulates myosin II activity through the phosphorylation of the myosin light chain (MLC) by activating Rho-kinase, which is a target of Rho. Several lines of evidence point to an important role of Rho in the action of lysophosphatidic acid (LPA) and thrombin in provoking neurite retraction in N1E-115 neuroblastoma cells.

RESULTS

Here we examined whether Rho-kinase and myosin II are involved in neurite retraction in N1E-115 cells. We showed that the expression of constitutively active forms of Rho-kinase induced neurite retraction in NIE-115 cells and MLC phosphorylation in NIH 3T3 cells, whereas the expression of dominant negative forms of Rho-kinase inhibited the LPA-induced neurite retraction in N1E-115 cells and the serum-induced MLC phosphorylation in NIH 3T3 cells. The expression of mutant MLCT18D,S19D (substitution of Thr and Ser by Asp), which is known to lead to the activation of myosin ATPase and a conformational change of myosin II when reconstituted with myosin heavy chains in vitro, also promoted neurite retraction.

CONCLUSIONS

These results indicate that Rho-kinase is involved in the LPA-induced neurite retraction downstream of Rho, and that myosin II activation promotes neurite retraction downstream of Rho and Rho-kinase.

Endothelial cells and activated platelets express integrin-type receptors responsible for adhesion to fibrinogen. We have located distinct integrin-directed endothelial cell and platelet attachment sites on immobilized fibrinogen using a combination of synthetic peptides, fibrinogen fragments, and specific anti-peptide monoclonal antibodies. Endothelial cells exclusively recognize an Arg-Gly-Asp-containing site near the C-terminus of the alpha chain (alpha residues 572-574) but fail to recognize the Arg-Gly-Asp sequence in the N-terminal region of the same chain (alpha residues 95-97). In contrast, platelets do not require either Arg-Gly-Asp sequence for binding to intact fibrinogen and are capable of recognizing, in addition to the alpha 572-574 sequence, a site at the C-terminus of the gamma chain (gamma residues 400-411). These data suggest a molecular mechanism whereby platelets and endothelial cells interact with distinct sites on the fibrinogen molecule during hemostasis and wound healing.

Direct sequencing of amplified genomic DNA has been used to investigate the molecular basis of haemophilia B and thus identify specific amino acids that are essential for maintenance of structure or function of factor IX. Substitution of Cys 336, Asn 120 results in loss of circulating factor IX antigen and deletion of Arg 37 in gross reduction of circulating protein and loss of activity, while substitution of Arg -4, Arg 333, Asp 64 and Pro 55 cause loss of function without marked reduction in protein serum levels. Frameshift or point mutations resulting in marked loss of coding information are found in patients who develop antibodies to administered factor IX. An enhanced rate of mutation is evident at two CpG dinucleotides in the factor IX gene, which accounts for approximately 25% of all point mutations causing haemophilia B known to date. Direct sequencing of mutations also permits, for the first time, rapid and unequivocal prenatal and carrier diagnoses, in all cases, by eliminating the need for informative segregation of markers.

Synthetic peptides corresponding to the pseudosubstrate domains of protein kinase C (PKC) have been used as specific inhibitors of PKC in in vitro assays and permeabilized cell systems. However, their use in vivo was hampered by the impermeability of the plasma membrane for such peptides. Here, we show that N-myristoylation of the PKC pseudosubstrate nonapeptide Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln permits its use as an inhibitor of PKC in intact cells. The myristoylated peptide, myr-psi PKC, inhibits phosphorylation of the myristoylated alanine-rich C kinase substrate protein, as induced by 12-O-tetradecanoyl-phorbol-13-acetate, and the activation of phospholipase D by bradykinin, which strictly depends on PKC. Half-maximal inhibition is obtained at concentrations of 8 and 20 microM, respectively. An N-myristoylated peptide derived from an inhibitor protein of the cAMP-dependent protein kinases, Myr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile, was ineffective. These results show that myr-psi PKC is a selective and cell-permeable inhibitor of PKC.

The location of the structural gene for aggregation substance on the sex pheromone plasmid pAD1 of Enterococcus faecalis was determined using an oligonucleotide deduced from the N-terminal amino acid sequence of the purified protein. The nucleotide sequence was determined for the corresponding region and two open reading frames (ORFs) could be identified. ORF1 codes for a small (Mr 13,160) acidic protein of unknown function. The gene for aggregation substance (named asa1) was found to code for a protein of 1296 amino acids (Mr 142,248). The protein has a signal peptide of 43 amino acids (the resulting Mr for mature aggregation substance is 137,429) and contains in its C-terminal region a proline-rich sequence, previously characterized as being involved in cell wall association, which is followed by a membrane anchor. The membrane anchor showed significant similarity to that of other Gram-positive organisms, but no other similarities to surface proteins from Gram-positive bacteria were found. In particular, no repeats on the DNA or protein level could be detected for pAD1-specific aggregation substance. The protein contains the amino acid motifs Arg-Gly-Asp-Ser and Arg-Gly-Asp-Val (once each), which, it is proposed, play a crucial role in adherence to eukaryotic cells.

Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAs(Arg) including four isoaccepting molecules both in vivo and in vitro. The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2',3'-cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAs(Arg), the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNA(Arg)-cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAs(Arg), which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.

A major effect in the energetics of protein folding is the loss of conformational entropy of the side-chains. The definition of entropy as the Boltzmann sampling over all states (S = -R sigma p(i) ln p(i)) requires evaluation of the probability (p(i)) of the system being in rotameric state i. The principle of this paper is to obtain an estimate of p(i) from the observed distribution of exposed side-chain rotamers in 50 non-homologous protein crystal structures. However because of limited data we show that for all side-chains except Asn, Asp and Glu the side-chain distribution is independent of burial and accordingly all data were pooled in the calculation of p(i). For Asn, Asp and Glu side-chains with relative accessibility>> 60% were used. The scale includes effects due to the symmetry of side-chains such as Phe and the free rotation of side-chain amide, carboxyl and hydroxyl groups. An empirical scale for the loss of side-chain conformational entropy during protein folding is thereby obtained. Values of the change in free energy due to entropy (-T delta S) on burying a side-chain range from 0 for Ala, Gly and Pro to +2.1 kcal/mol for Gln (T = 300 K). We explore the consistency of a simple model for protein folding that includes side-chain entropy, main-chain entropy, hydrophobicity and hydrogen bonding. The stability of site-directed mutations is discussed in terms of conformational entropy.

This study focuses on the molecular level interpretation of the selective gas-phase cleavage at aspartic acid residues (Asp) in protonated peptides. A phi3P+CH2C(=O)group (phi = 2,4,6-trimethoxyphenyl) is attached to the N-terminal nitrogen of the selected peptides LDIFSDF and LDIFSDFR, via solid-phase synthesis, to "mimic" the tightly held charge of a protonated arginine (Arg) residue. Collision-induced dissociation in a quadrupole ion trap instrument and surface-induced dissociation in a dual quadrupole instrument were performed for electrospray-generated ions of the fixed-charge peptide derivatives. Selective cleavages at Asp-Xxx are observed for those ions with charge provided only by the fixed charge or for those with a fixed charge and one Arg plus one added proton. This supports a previously proposed mechanism which suggests that the cleavages at Asp-Xxx, initiated by the acidic hydrogen of the Asp residue, become significant when ionizing protons are strongly bound by Arg in the protonated peptides. It is clear that the fixed charge is indeed serving as a "mimic" of protonated Arg and that a protonated Arg side chain is not required to interact with the Asp to induce cleavage at Asp-Xxx. When the number of protons exceeds the number of Arg in a peptide containing Arg and Asp, nonselective cleavages occur. The fragmentation efficiency of the peptides is consistent with the idea that these nonselective cleavages are promoted by a mobile proton. The peptide with a fixed charge and one added proton, [phi3P+CH2C(=O)-LDIFSDF + H]2+, fragments much more efficiently than the corresponding peptide with a fixed charge, an Arg and one added proton, [phi3P+CH2C(=O)-LDIFSDFR + H]2+; both of these fragment more efficiently than the peptide with a fixed charge and no added proton, phi3P+CH2C(=O)-LDIFSDF. MS/MS/MS (i.e., MS3) experimental results for bn ions formed at Asp-Xxx from phi3P+CH2C(=O)-LDIFSDF and its H/D exchange derivative, phi3P+CH2C(=O)-LDIFSDF-d11, are consistent with the bn ions formed at Asp-Xxx having a succinic anhydride cyclic structure. MS/MS experiments were also carried out for phi3P+CH2C(=O)-AAAA, a peptide derivative containing active hydrogens only at amide nitrogens plus the C-terminus, and its active H/D exchange product, phi3P+CH2C(=O)-AAAA-d5. The results show that a hydrogen originally located at an amide nitrogen is transferred away in the formation of a cyclic charge remote b ion.

A 6.4-kb DNA fragment containing the DNA gyrase gyrA and gyrB genes was cloned and sequenced from the quinolone-susceptible Staphylococcus aureus type strain ATCC 12600. An expression plasmid was constructed by inserting the cloned genes into the Escherichia coli-S. aureus shuttle vector pAT19, and deletion plasmids carrying only functional gyrA and gyrB genes were derived from this plasmid. An efficient transformation system for S. aureus RN4220 was established by using these plasmids. Quinolone-resistant mutants of S. aureus RN4220 were isolated by three-step selection with quinolones. The first- and second-step mutants were considered to be transport mutants, and the third-step mutants were divided into five groups with respect to their resistance patterns and transformation results with gyrA and gyrB genes. Sequencing analysis of the resulting mutant gyrase genes showed that they had the following point mutations: group 1, Ser-84 (TCA) to Leu (TTA) in GyrA; group 2, Ser-84 (TCA) to Ala (GCA), Ser-85 (TCT) to Pro (CCT), or Glu-88 (GAA) to Lys (AAA) in GyrA; group 3, Asp-437 (GAC) to Asn (AAC) in GyrB; group 4, Arg-458 (CGA) to Gln (CAA) in GyrB; and group 5, Ser-85 (TCT) to Pro (CCT) in GyrA and Asp-437 (GAC) to Asn (AAC) in GyrB. When the gyrA and/or gyrB mutants were transformed with the wild-type gyrA and/or gyrB plasmids, they became quinolone susceptible, but transformants with the plasmids having the same mutations on the gyrA and/or gyrB genes did not confer susceptibility. These results indicate that mutations in both gyrA and gyrB can be responsible for quinolone resistance in S. aureus.

The synthetic cell attachment-promoting peptides from fibronectin (Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.)., 309:30-33) were found to detach cultured cells from the substratum when added to the culture in a soluble form. Peptides ranging in length from tetrapeptide to heptapeptide and containing the active L-arginyl-glycyl-L-aspartic acid (Arg-Gly-Asp) sequence had the detaching activity, whereas a series of different peptides with chemically similar structures had no detectable effect on any of the test cells. The Arg-Gly-Asp-containing peptides caused detachment of various cell lines of different species and histogenetic origin. Studies with defined substrates showed that the active peptides could inhibit the attachment of cells to vitronectin in addition to fibronectin, indicating that vitronectin is recognized by cells through a similar mechanism as fibronectin. The peptides did not inhibit the attachment of cells to collagen. However, cells cultured on collagen-coated plastic for 24-36 h, as well as cells with demonstrable type I or type VI collagen in their matrix, were susceptible to the detaching effect of the peptides. These results indicate that the recognition mechanism(s) by which cells bind to fibronectinand vitronectin plays a major role in the substratum attachment of cells and that collagens may not be directly involved in cell-substratum adhesion. Since vitronectin is abundant in serum, it is probably an important component in mediating the attachment of cultured cells. The independence of the effects of the peptide on the presence of serum and the susceptibility of many different cell types to detachment by the peptide show that the peptides perturb an attachment mechanism that is intrinsic to the cells and fundamentally significant to their adhesion.

Half of the survivors of bacterial meningitis experience motor deficits, seizures, hearing loss or cognitive impairment, despite adequate bacterial killing by antibiotics. We demonstrate that the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (z-VAD-fmk) prevented hippocampal neuronal cell death and white blood cell influx into the cerebrospinal fluid compartment in experimental pneumococcal meningitis. Hippocampal neuronal death was due to apoptosis derived from the inflammatory response in the cerebrospinal fluid. Apoptosis was induced in vitro in human neurons by inflamed cerebrospinal fluid and was blocked by z-VAD-fmk. As apoptosis drives neuronal loss in pneumococcal meningitis, caspase inhibitors might provide a new therapeutic option directed specifically at reducing brain damage.

We have isolated cDNAs from maize (ZGB1) and Arabidopsis (AGB1) encoding proteins homologous to beta subunits of guanine nucleotide-binding protein (G protein). The predicted ZGB1 and AGB1 gene products are 76% identical to each other and 41% or more identical to animal G protein beta subunits. Both predicted proteins contain seven repeats of the so-called "WD-40" motif, where WD is Trp-Asp. RNA blot analysis indicates that ZGB1 mRNA is present in the root, leaf, and tassel and that AGB1 mRNA is expressed in the root, leaf, and flower. DNA blot hybridizations indicate that maize and Arabidopsis genomes contain no other genes that are highly similar to ZGB1 and AGB1, respectively, suggesting that the newly isolated G protein beta-subunit homologues are likely to have unique functions. Furthermore, these G protein beta-subunit homologues are conserved among other plant species and may play important role(s) in plant signaling.

The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IXalpha (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-R(c)-like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion.

Glutamine tract expansion triggers nine neurodegenerative diseases by conferring toxic properties to the mutant protein. In SCA1, phosphorylation of ATXN1 at Ser776 is thought to be key for pathogenesis. Here, we show that replacing Ser776 with a phosphomimicking Asp converted ATXN1 with a wild-type glutamine tract into a pathogenic protein. ATXN1[30Q]-D776-induced disease in Purkinje cells shared most features with disease caused by ATXN1[82Q] having an expanded polyglutamine tract. However, in contrast to disease induced by ATXN1[82Q] that progresses to cell death, ATXN1[30Q]-D776 failed to induce cell death. These results support a model where pathogenesis involves changes in regions of the protein in addition to the polyglutamine tract. Moreover, disease initiation and progression to neuronal dysfunction are distinct from induction of cell death. Ser776 is critical for the pathway to neuronal dysfunction, while an expanded polyglutamine tract is essential for neuronal death.

The contiguous sequence of 1,003,450 bp spanning map positions 64% to 92% of the genome of Synechocystis sp. strain PCC6803 has been deduced. Computer analysis of the sequence predicts that this region contains at least 818 potential ORFs, in which 255 (31%) were either genes that had already been identified or their homologues, 84 (10%) were homologues to registered hypothetical genes, and 149 (18%) showed weak similarities to reported genes. The remaining 330 ORFs showed no apparent similarity to any reported genes or carried no significant protein motifs. The potential ORFs as a whole occupied 86% of the sequenced region, implying compact arrangement of genes in the genome. As to the structural RNA genes, one rRNA operon consisting of 5,028 bp and at least 11 species of tRNA genes were identified. It is noteworthy that 10 out of the 11 tRNA species showed significant sequence similarities to tRNAs reported in plant chloroplasts. As other notable unique sequences, three classes of IS-like elements each with characteristics typical of IS elements were identified, and a typical unit of WD(Trp-Asp)-repeats which have only been detected in the regulatory proteins of eukaryotes was identified within the large 5,079-bp ORF located at map position 69%.

Absorbance changes in the infrared and visible spectral range were measured in parallel during the photocycle of light-adapted bacteriorhodopsin, which is accompanied by a vectorial proton transfer. A global fit analysis yielded the same rate constants for the chromophore reactions, for protonation changes of protein side groups, and for the backbone motion. From this result we conclude that all reactions in various parts of the protein are synchronized to each other and that no independent cycles exist for different parts. The carbonyl vibration of Asp-85, indicating its protonation, appears with the same rate constant as the Schiff base deprotonation. The carbonyl vibration of Asp-96 disappears, indicating most likely its deprotonation, with the same rate constant as for the Schiff base reprotonation. This result supports the proposed mechanism in which the protonated Schiff base, a deprotonated aspartic acid (Asp-85) on the proton-release pathway, and a protonated aspartic acid (Asp-96) on the proton-uptake pathway act as internal catalytic proton-binding sites.

The broadly neutralizing anti-HIV antibody 4E10 recognizes an epitope very close to the virus membrane on the glycoprotein gp41. It was previously shown that epitope recognition improves in a membrane context and that 4E10 binds directly, albeit weakly, to lipids. Furthermore, a crystal structure of Fab 4E10 complexed to an epitope peptide revealed that the centrally placed, protruding H3 loop of the antibody heavy chain does not form peptide contacts. To investigate the hypothesis that the H3 loop apex might interact with the viral membrane, two Trp residues in this region were substituted separately or in combination with either Ala or Asp by site-directed mutagenesis. The resultant IgG variants exhibited similar affinities for an epitope peptide as WT 4E10 but lower apparent affinities for both viral membrane mimetic liposomes and Env(-) virus. Variants also exhibited lower apparent affinities for Env(+) virions and failed to significantly neutralize a number of 4E10-sensitive viruses. For the extremely sensitive HXB2 virus, variants did neutralize, but at 37- to >250-fold lower titers than WT 4E10, with Asp substitutions exerting a greater effect on neutralization potency than Ala substitutions. Because reductions in lipid binding reflect trends in neutralization potency, we conclude that Trp residues in the antibody H3 loop enable membrane proximal epitope recognition through favorable lipid interactions. The requirement for lipophilic residues such as Trp adjacent to the antigen binding site may explain difficulties in eliciting 4E10-like neutralizing antibody responses by immunization and helps define a unique motif for antibody recognition of membrane proximal antigens.

Four complementation groups of temperature-sensitive (ts) mutants of Sindbis virus that fail to make RNA at the nonpermissive temperature are known, and we have previously shown that group F mutants have defects in nsP4. Here we map representatives of groups A, B, and G. Restriction fragments from a full-length clone of Sindbis virus, Toto1101, were replaced with the corresponding fragments from the various mutants. These hybrid plasmids were transcribed in vitro by SP6 RNA polymerase to produce infectious RNA transcripts, and the virus recovered was tested for temperature sensitivity. After each lesion was mapped to a specific region, cDNA clones of both mutants and revertants were sequenced in order to determine the precise nucleotide change responsible for each mutation. Synthesis of viral RNA and complementation by rescued mutants were also examined in order to study the phenotype of each mutation in a uniform genetic background. The single mutant of group B, ts11, had a defect in nsP1 (Ala-348 to Thr). All of the group A and group G mutants examined had lesions in nsP2 (Ala-517 to Thr in ts17, Cys-304 to Tyr in ts21, and Gly-736 to Ser in ts24 for three group A mutants, and Phe-509 to Leu in ts18 and Asp-522 to Asn in ts7 for two group G mutants). In addition, ts7 had a change in nsP3 (Phe-312 to Ser) which also rendered the virus temperature sensitive and RNA-. Thus, changes in any of the four nonstructural proteins can lead to failure to synthesize RNA at a nonpermissive temperature, indicating that all four are involved in RNA synthesis. From the results presented here and from previous results, several of the activities of the nonstructural proteins can be deduced. It appears that nsP1 may be involved in the initiation of minus-strand RNA synthesis. nsP2 appears to be involved in the initiation of 26S RNA synthesis, and in addition it appears to be a protease that cleaves the nonstructural polyprotein precursors. It may also be involved in shutoff of minus-strand RNA synthesis. nsP4 appears to function as the viral polymerase or elongation factor. The functions of nsP3 are as yet unresolved.

Knowledge of the tertiary structure of the proteinase from human immunodeficiency virus HIV-1 is important to the design of inhibitors that might possess antiviral activity and thus be useful in the treatment of AIDS. The conserved Asp-Thr/Ser-Gly sequence in retroviral proteinases suggests that they exist as dimers similar to the ancestor proposed for the pepsins. Although this has been confirmed by X-ray analyses of Rous sarcoma virus and HIV-1 proteinases, these structures have overall folds that are similar to each other only where they are also similar to the pepsins. We now report a further X-ray analysis of a recombinant HIV-1 proteinase at 2.7 A resolution. The polypeptide chain adopts a fold in which the N- and C-terminal strands are organized together in a four-stranded beta-sheet. A helix precedes the single C-terminal strand, as in the Rous sarcoma virus proteinase and also in a synthetic HIV-1 proteinase, in which the cysteines have been replaced by alpha-aminobuytric acid. The structure reported here provides an explanation for the amino acid invariance amongst retroviral proteinases, but differs from that reported earlier in some residues that are candidates for substrate interactions at P3, and in the mode of intramolecular cleavage during processing of the polyprotein.

Nonsteroidal anti-inflammatory drugs, which inhibit cyclooxygenase (COX) activity, are powerful antineoplastic agents that exert their antiproliferative and proapoptotic effects on cancer cells by COX-dependent and/or COX-independent pathways. Celecoxib, a COX-2-specific inhibitor, has been shown to reduce the number of adenomatous colorectal polyps in patients with familial adenomatous polyposis. Here, we show that celecoxib induces apoptosis in the colon cancer cell line HT-29 by inhibiting the 3-phosphoinositide-dependent kinase 1 (PDK1) activity. This effect was correlated with inhibition of the phosphorylation of the PDK1 downstream substrate Akt/protein kinase B (PKB) on two regulatory sites, Thr(308) and Ser(473). However, expression of a constitutive active form of Akt/PKB (myristoylated PKB) has a low protective effect toward celecoxib-induced cell death. In contrast, overexpression of constitutive active mutant of PDK1 (PDK1(A280V)) was as potent as the pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, to impair celecoxib-induced apoptosis. By contrast, cells expressing a kinase-defective mutant of PDK1 (PDK1(K114G)) remained sensitive to celecoxib. Furthermore, in vitro measurement reveals that celecoxib was a potential inhibitor of PDK1 activity with an IC(50) = 3.5 microm. These data indicate that inhibition of PDK1 signaling is involved in the proapoptotic effect of celecoxib in HT-29 cells.

In the past few years, enormous progress has been made in understanding cytokinin perception and signalling. Three cytokinin receptor proteins, which are hybrid histidine kinases, have been identified in Arabidopsis. These receptors may transduce signals in a quantitative rheostat-like fashion, thus permitting long-lasting and continuously variable signalling that is directly dependent on the hormone concentration. Evidence has been provided that downstream signalling is transmitted through a His-to-Asp phospho-relay involving phosphotransmitter and response regulator proteins, typical of two-component systems. On the basis of mutant analysis, protein-protein interaction studies and target gene identification, a cellular network is emerging that links cytokinin activity to both developmental and physiological processes.

MSCs are nonhematopoietic stem cells capable of differentiating into various mesoderm-type cells. MSCs have been considered to be a potential vehicle for cell-based gene therapy because MSCs are relatively easily expanded in vitro and have the propensity to migrate to and proliferate in the tumor tissue after systemic administration. Here, we demonstrated the tropism of mouse MSCs to tumor cells in vitro and multiple tumor tissues in the lung after i.v. injection of green fluorescent protein-positive MSCs in vivo. We transduced CX3CL1 (fractalkine), an immunostimulatory chemokine, to the mouse MSCs ex vivo using an adenoviral vector with the Arg-Gly-Asp-4C peptide in the fiber knob. Intravenous injection of CX3CL1-expressing MSCs to the mice bearing lung metastases of C26 and B16F10 cells strongly inhibited the development of lung metastases and thus prolonged the survival of these tumor-bearing mice. This antitumor effect depended on both innate and adaptive immunity. These results suggest that MSCs can be used as a vehicle for introducing biological agents into multiple lung tumor tissues. Disclosure of potential conflicts of interest is found at the end of this article.