The effects of both synthetic and biologically produced angiotensin II (AII) upon the process of glolerular filtration were examined in the plasma-expanded (2.5% body wt) Munich-Wistar rat, by micropuncture evaluation of pressures, nephron plasma flow (rpf) and filtration rate (sngfr). Plasma expansion was chosen as a control condition because (a) response to AII was uniform and predictable, (b) endogenous generation of AII was presumably suppressed, and (c) the high control values for rpf permitted accurate determination of values for the glomerular permeability coefficient (LpA) before and during AII infusion. With subpressor quantities of synthetic Asn-1, Val-5 AII (less than 5 ng/100 g body wt/min), sngfr fell from 47.7 in the control group to 39.8 nl/min/g kidney (P less than 0.005). The rpf fell to 60% of control values (P less than 0.001). Measurement of glomerular capillary (PG) and Bowman's space (Pt) hydrostatic pressures in surface glomeruli with a servo-nulling device permitted evaluation of the hydrostatic pressure gradient (deltaP = PG - Pi). DeltaP increased from 38.1 +/- 1.2 in control to 45.9 +/- 1.3 mm Hg after Asn-1, Val-5 AII and essentially neutralized the effect of decreased rpf in sngfr. The sngfr then fell as a result of a decreased in LpA from 0.063 +/- 0.008 in control to 0.028 +/- 0.004 nl/s/g kidney/mm Hg after Asn-1, Val-5 AII (P less than 0.02). Lower doses of Asp-1, Ile-5 AII (less than 3 ng/100 g body wt/min) had no effect on sngfr, rpf, deltaP, and afferent and efferent vascular resistance, but significantly elevated systemic blood pressure, suggesting peripheral effects on smooth muscle at this low dose. LpA was 0.044 +/- 0.007 nl/s/g kidney/mm Hg after low-dose Asp-1, Ile-5 AII, and 0.063 +/- 0.008 in the control group (0.02 greater than P greater than 0.1). Higher, equally pressor doses of native AII (5 ng/100 g body wt/min) produced effects almost identical to similar quantites of synthetic Asn-1, Val-5 AII upon rpf, deltaP, sngfr, and renal vascular resistance. LpA again fell to 0.026 +/- 0.004 nl/s/g kidney/mn Hg, a value almost identical to that after the synthetic AII. Paired studies with Asp-1, Ile-5 AII also demonstrated a consistent reduction in LpA.

Although several variants of DNA repair genes have been identified, their functional significance has not been determined. Using samples collected from 135 cancer-free women, this study evaluated whether amino acid substitution variants of DNA repair genes contribute to ionizing radiation (IR) susceptibility as measured by prolonged cell cycle G2 delay. PCR-restriction fragment length polymorphism (RFLP) assays were used to determine four genotypes: X-ray repair cross complementing group 1 (XRCC1, exon 6, C/T, 194 Arg/Trp and exon 10, G/A, 399 Arg/Gln), XRCC group 3 (XRCC3, exon 7, C/T, 241 Thr/Met) and apurinic/apyrimidinic endonuclease 1 (APE1, exon 5, T/G, 148 <em>Asp</em>/Glu). Fluorescence-activated cell sorter (FACS) analysis was used to measure cell cycle delay. APE1 (exon 5) genotype was significantly associated with mitotic delay (P = 0.01), with the Glu/Glu genotype having prolonged delay compared with the other two genotypes. The mitotic delay index (mean +/- SD) in women with the APE1 codon 148 <em>Asp</em>/<em>Asp</em>, <em>Asp</em>/Glu and Glu/Glu genotypes was 30.95 +/- 10.15 (n = 49), 30.65 +/- 10.4 (n = 60) and 39.56 +/- 13.12 (n = 21), respectively. There was a significant interaction between family history (FH) and APE1 (exon 5) genotype (P = 0.007) as well as FH and XRCC1 (exon 10) genotype (P = 0.005) in mitotic delay. Lastly, prolonged cell cycle delay was significantly associated with number of variant alleles when APE1 <em>Asp</em>148Glu and XRCC1 Arg399Gln genotypes were evaluated in a four-level model (chi(2) for linear trend = 10.9; P = 0.001). These results suggest that amino acid substitution variants of XRCC1 and APE1 may contribute to IR hypersensitivity.

Photoacoustic imaging is an emerging modality that overcomes to a great extent the resolution and depth limitations of optical imaging while maintaining relatively high-contrast. However, since many diseases will not manifest an endogenous photoacoustic contrast, it is essential to develop exogenous photoacoustic contrast agents that can target diseased tissue(s). Here we present a novel photoacoustic contrast agent, Indocyanine Green dye-enhanced single walled carbon nanotube (SWNT-ICG). We conjugated this contrast agent with cyclic Arg-Gly-Asp (RGD) peptides to molecularly target the alpha(v)beta(3) integrins, which are associated with tumor angiogenesis. Intravenous administration of this tumor-targeted contrast agent to tumor-bearing mice showed significantly higher photoacoustic signal in the tumor than in mice injected with the untargeted contrast agent. The new contrast agent gave a markedly 300 times higher photoacoustic contrast in living tissues than previously reported SWNTs, leading to subnanomolar sensitivities. Finally, we show that the new contrast agent can detect approximately 20 times fewer cancer cells than previously reported SWNTs.

Drug-induced hepatotoxicity represents a major clinical problem and an impediment to new medicine development. Serum biomarkers hold the potential to provide information about pathways leading to cellular responses within inaccessible tissues, which can inform the medicinal chemist and the clinician with respect to safe drug design and use. Hepatocyte apoptosis, necrosis, and innate immune activation have been defined as features of the toxicological response associated with the hepatotoxin acetaminophen (APAP). Within this investigation, we have unambiguously identified and characterized by liquid chromatography-tandem mass spectrometry differing circulating molecular forms of high-mobility group box-1 protein (HMGB1) and keratin-18 (K18), which are linked to the mechanisms and pathological changes induced by APAP in the mouse. Hypoacetylated HMGB1 (necrosis indicator), caspase-cleaved K18 (apoptosis indicator), and full-length K18 (necrosis indicator) present in serum showed strong correlations with the histological time course of cell death and was more sensitive than alanine aminotransferase activity. We have further identified a hyperacetylated form of HMGB1 (inflammatory indicator) in serum, which indicated that hepatotoxicity was associated with an inflammatory response. The inhibition of APAP-induced apoptosis and K18 cleavage by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone are associated with increased hepatic damage, by a shift to necrotic cell death only. These findings illustrate the initial verification of K18 and HMGB1 molecular forms as serum-based sensitive tools that provide insights into the cellular dynamics involved in APAP hepatotoxicity within an inaccessible tissue. Based on these findings, potential exists for the qualification and measurement of these proteins to further assist in vitro, in vivo, and clinical bridging in toxicological research.

There is a growing consensus that corneal myofibroblasts are derived from adjacent stromal keratocytes which undergo an orderly phenotypic transition from quiescent keratocyte to activated fibroblast to myofibroblast. Both in vivo and in vitro studies have shown this transition to be dependent, in part, on transforming growth factor beta (TGFbeta). In many fibroblastic cells autocrine production of platelet derived growth factor (PDGF) is known to mediate the growth up-regulation by TGFbeta. In this study, blocking antibodies to PDGF significantly reduced by 80% (P<0.025) the TGFbeta1 stimulated cell cycle entry of serum-free cultured rabbit corneal keratocytes. AntiPDGF treatment also markedly reduced the TGFbeta1-induced intracellular actin filament re-organization, fibronectin fibril assembly, and focal contact formation as well as reducing by 80% the expression of alpha-smooth muscle (alpha-SM) specific isoform of actin characteristic of myofibroblast differentiation. Although PDGF treatment of quiescent keratocytes produced an activated, fibroblastic cell type, PDGF stimulated keratocytes exhibited the same temporal, myofibroblastic differentiation response to TGFbeta1 as did quiescent keratocytes. Furthermore, blocking TGFbeta1 induction of myofibroblast differentiation with the Arg-Gly-Asp containing peptide, GRGDdSP, for 3 days followed by allowing progression of myofibroblast differentiation by removing GRGDdSP did not change the temporal response or tyrosine phosphorylation cascade (2-72 hr) leading to myofibroblast differentiation. Nor did PDGF treatment of keratocytes reverse the RGD blockade of TGFbeta1 induced myofibroblast differentiation. Overall these cumulative findings indicate that myofibroblast differentiation in the rabbit corneal keratocyte requires synergistic growth factor/integrin signaling involving TGFbeta, PDGF, and the fibronectin receptor. Additionally, the similar TGFbeta1 temporal response of PDGF-stimulated compared to nai;ve keratocytes suggests that myofibroblast differentiation does not require transition through a fibroblast phenotype.

Protein palmitoylation is the most common posttranslational lipid modification; its reversibility mediates protein shuttling between intracellular compartments. A large family of DHHC (Asp-His-His-Cys) proteins has emerged as protein palmitoyl acyltransferases (PATs). However, mechanisms that regulate these PATs in a physiological context remain unknown. In this study, we efficiently monitored the dynamic palmitate cycling on synaptic scaffold PSD-95. We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors. A dendritically localized DHHC2 but not the Golgi-resident DHHC3 mediates this activity-sensitive palmitoylation. Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect. These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

Hepatitis B e antigen (HBeAg) constitutes the nucleocapsid of hepatitis B virus (HBV) and occurs in association with plasma proteins, particularly with IgG, in the serum of persons infected with the virus. A polypeptide with an approximate m.w. of 15,500 (P15.5) is obtained either from HBeAg in the serum or from the nucleocapsid of HBV. P15.5 preparations from serum and virus resembled closely each other in the amino acid composition. The C-terminus amino acid sequence of P15.5 from serum was determined to be -Thr-Thr-Val-Val, whereas that from the virus ended with -Thr-Thr. The same sequence of four amino acid residues was found on the gene coding for the nucleopeptide of hepatitis B virus with a molecular size of 19,000 daltons (P19). P15.5 identified on the nucleotide sequence of P19 was composed of 149 amino acid residues with a calculated molecular size of 16,770 daltons. The gene coding for P19 had two -Asp-Pro-connections. By splitting these connections in P19 and P15.5 preparations with formic acid, smaller polypeptides were obtained with sizes predicted from the nucleotide sequence and with the N-terminus amino acid of proline as expected. One of two monoclonal antibodies raised against the core of Dane particles (HBcAg) bound with P15.5 preparations purified from serum and HBV. The IgG fraction from a human serum containing antibodies to HBcAg but not to HBeAg bound with P15.5 also. On the basis of the results obtained, the IgG molecules associated with P15.5 in the serum of persons infected with HBV may well represent the antibodies against HBcAg with limited specificities.

CXCR4 is a G-coupled receptor for the stromal cell-derived factor (SDF-1) chemokine, and a CD4-associated human immunodeficiency virus type 1 (HIV-1) coreceptor. These functions were studied in a panel of CXCR4 mutants bearing deletions in the NH(2)-terminal extracellular domain (NT) or substitutions in the NT, the extracellular loops (ECL), or the transmembrane domains (TMs). The coreceptor activity of CXCR4 was markedly impaired by mutations of two Tyr residues in NT (Y7A/Y12A) or at a single Asp residue in ECL2 (D193A), ECL3 (D262A), or TMII (D97N). These acidic residues could engage electrostatical interactions with basic residues of the HIV-1 envelope protein gp120, known to contribute to the selectivity for CXCR4. The ability of CXCR4 mutants to bind SDF-1 and mediate cell signal was consistent with the two-site model of chemokine-receptor interaction. Site I involved in SDF-1 binding but not signaling was located in NT with particular importance of Glu(14) and/or Glu(15) and Tyr(21). Residues required for both SDF-1 binding and signaling, and thus probably part of site II, were identified in ECL2 (Asp(187)), TMII (Asp(97)), and TMVII (Glu(288)). The first residues () of NT also seem required for SDF-1 binding and signaling. A deletion in the third intracellular loop abolished signaling, probably by disrupting the coupling with G proteins. The identification of CXCR4 residues involved in the interaction with both SDF-1 and HIV-1 may account for the signaling activity of gp120 and has implications for the development of antiviral compounds.

We have analyzed by gene amplification and sequencing mutations in the quinolone resistance-determining regions of the gyrA, gyrB, and parC genes of fluoroquinolone-resistant Streptococcus pneumoniae mutants obtained during therapy or in vitro. Mutations leading to substitutions in ParC were detected in the two mutants obtained in vivo, BM4203-R (substitution of a histidine for an aspartate at position 84 [Asp-84->>His]; Staphylococcus aureus coordinates) and BM4204-R (Ser-80->>Phe), and in two mutants obtained in vitro (Ser-80->>Tyr). An additional mutant obtained in vitro, BM4205-R3, displayed a higher level of fluoroquinolone resistance and had a mutation in gyrA leading to a Ser-84->>Phe change. We could not detect any mutation in the three remaining mutants obtained in vitro. Total DNA from BM4203-R, BM4204-R, and BM4205-R3 was used to transform S. pneumoniae CP1000 by selection on fluoroquinolones. For the parC mutants, transformants with phenotypes indistinguishable from those of the donors were obtained at frequencies (5 x 10(-3) to 8 x 10(-3)) compatible with monogenic transformation. By contrast, transformants were obtained at a low frequency (4 x 10(-5)), compatible with the transformation of two independent genes, for the gyrA mutant. Resistant transformants of CP1000 were also obtained with an amplified fragment of parC from BM4203-R and BM4204-R but not with a gyrA fragment from BM4205-R3. All transformants had mutations identical to those in the donors. These data strongly suggest that ParC is the primary target for fluoroquinolones in S. pneumoniae and that BM4205-R3 is resistant to higher levels of the drugs following the acquisition of two mutations, including one in gyrA.

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).

Pertussis toxin catalyzes the transfer of ADP-ribose from NAD to the guanine nucleotide-binding regulatory proteins Gi, Go, and transducin. Based on a partial amino acid sequence for a tryptic peptide of ADP-ribosylated transducin, asparagine had been characterized as the site of pertussis toxin-catalyzed ADP-ribosylation. Subsequently, cDNA data for the alpha subunit of transducin indicated that the putative asparagine residue was, in fact, not present in the protein. To determine the amino acid that served as the ADP-ribose acceptor, radiolabel from [adenine-U-14C]NAD was incorporated, in the presence of pertussis toxin, into the alpha subunit of transducin (0.3 mol/mol). An ADP-ribosylated, tryptic peptide was purified and fully sequenced by automated Edman degradation. The amino acid sequence, Glu-Asn 343-Leu-Lys-Asp 346-X-Gly 348-Leu-Phe, corresponds to the cDNA sequence coding the carboxyl-terminal nonapeptide, Glu 342-Phe 350, which includes by cDNA sequence cysteine at position 347. Neither Asn 343 nor Asp 346 appeared to be modified; residue 347 adhered to the sequencing resin. Cysteine, the missing residue, was eluted from the sequencing resin with acetic acid along with 76% of the peptide-associated radioactivity, half of which, presumably ADP-ribosylcysteine, eluted from an anion exchange column between NAD and ADP-ribose; the other half had a retention time corresponding to 5'-AMP. We conclude that Cys 347 and not Asn 343 or Asp 346 is the site of pertusis toxin-catalyzed ADP-ribosylation in transducin.

Protein tyrosine phosphatases (PTPases) and kinases coregulate the critical levels of phosphorylation necessary for intracellular signalling, cell growth and differentiation. Yersinia, the causative bacteria of the bubonic plague and other enteric diseases, secrete an active PTPase, Yop51, that enters and suppresses host immune cells. Though the catalytic domain is only approximately 20% identical to human PTP1B, the Yersinia PTPase contains all of the invariant residues present in eukaryotic PTPases, including the nucleophilic Cys 403 which forms a phosphocysteine intermediate during catalysis. We present here structures of the unliganded (2.5 A resolution) and tungstate-bound (2.6 A) crystal forms which reveal that Cys 403 is positioned at the centre of a distinctive phosphate-binding loop. This loop is at the hub of several hydrogen-bond arrays that not only stabilize a bound oxyanion, but may activate Cys 403 as a reactive thiolate. Binding of tungstate triggers a conformational change that traps the oxyanion and swings Asp 356, an important catalytic residue, by approximately 6 A into the active site. The same anion-binding loop in PTPases is also found in the enzyme rhodanese.

The extended-spectrum beta-lactamase CTX-M-15 confers resistance to ceftazidime, unlike the majority of CTX-M-type enzymes. Kinetic parameters were determined from purified CTX-M-15 and CTX-M-3, which differ by the single amino acid substitution Asp-240 to Gly, according to the Ambler numbering of class A beta-lactamases. Relative molecular masses of CTX-M-15 and CTX-M-3 were approximately 29 kDa and pI values were 8.9 and 8.4, respectively. CTX-M-15 had higher affinities for beta-lactams (lower K(m) values) than those of CTX-M-3 but catalytic efficiency (k(cat)/K(m) values) was variable depending on the beta-lactam substrate. Only CTX-M-15 showed a measurable catalytic efficiency for ceftazidime. Clavulanic acid and tazobactam were good inhibitors of both enzymes. MICs of beta-lactams for Escherichia coli reference strains expressing cloned beta-lactamase genes in the same genetic background were similar except for ceftazidime. This work underlines the fact that some CTX-M enzymes may hydrolyse ceftazidime and thus confer resistance to this expanded-spectrum cephalosporin in Enterobacteriaceae.

We have investigated low molecular weight antibacterial proteins from the Cecropia moth. Hyalophora cecropia. In addition to the previously described cecropins A and B, five new antibacterial proteins were discovered, the cecropins C, D, E and F, and the factor G. A scheme for the purification of these factors is presented. Cecropin D is a major cecropin, its amino acid sequence, WNPFKELEKVGQRVRDAVISAGPAVATVAQATALAK, shows homology to cecropin A and B. Like these cecropins, cecropin D has a block C-terminal. The previously tentative C-terminal sequence of cecropin A is also confirmed. It is concluded that the three major cecropins, A, B and D, are products of three different genes that are derived from a common ancestor. The cecropins C, E and F were present in very low amounts, and thus their primary structures could not be fully elucidated. Cecropin C has an amino acid sequence that up to residue 37 is identical to the sequence of A, though it lacks the C-terminal blocking group. It may be a precursor or degradation product of cecropin A. The minor cecropin E shows a similar relation to cecropin D. Cecropin F has a single amino acid replacement (17 Asp leads to Asn) compared to cecropin D, and is probably a product of an allele that is present at a low frequency in the population. The primary structure of the factor G could not be determined, however its amino acid composition is different from that of the cecropins. All the major cecropins were found to be efficient against several gram-positive and gram-negative bacterial strains. No significant difference was found between them in their activity against Escherichia coli, though against some less susceptible bacteria the most basic cecropins were more effective, the activity falling in the series B greater than A much greater than D.

A new candidate hormone--designated peptide YY (PYY)--has been isolated from extracts of porcine upper intestinal tissue by using a novel chemical assay method. This peptide has been detected in the extracts by the presence of its unusual COOH-terminal tyrosine amide structure. The peptide consists of a linear chain of 36 amino acids and its complete amino acid sequence is: Tyr-Pro-Ala-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Ser-Arg-Tyr -Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2. This peptide has a distinct sequence homology to the pancreatic polypeptide. It is therefore proposed that PYY and the pancreatic polypeptide together form a new peptide family. PYY has been found to strongly inhibit both secretin- and cholecystokinin-stimulated pancreatic secretion in the anesthetized cat.

The alphav-integrins, cell adhesion molecules that are highly expressed on activated endothelial cells and tumor cells but not on dormant endothelial cells or normal cells, present an attractive target for tumor imaging and therapy. We previously coupled a cyclic Arg-Gly-Asp (RGD) peptide, c(RGDyK), with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and labeled the RGD-DOTA conjugate with 64Cu (half-life, 12.8 h; 19% beta+) for solid tumor targeting, with high tumor-to-background contrast. The rapid tumor washout rate and persistent liver and kidney retention of this tracer prompted us to optimize the tracer for improved pharmacokinetic behavior. In this study, we introduced a polyethylene glycol (PEG; molecular weight, 3,400) moiety between DOTA and RGD and evaluated the 64Cu-DOTA-PEG-RGD tracer for microPET imaging in brain tumor models.

METHODS

DOTA was activated in situ and conjugated with RGD-PEG-NH2 under slightly basic conditions. alphavbeta3-Integrin-binding affinity was evaluated with a solid-phase receptor-binding assay in the presence of 125I-echistatin. Female nude mice bearing subcutaneous U87MG glioblastoma xenografts were administered 64Cu-DOTA-PEG-RGD, and the biodistributions of the radiotracer were evaluated from 30 min to 4 h after injection. microPET (20 min of static imaging at 1 h after injection) and then quantitative autoradiography were used for tumor visualization and quantification. The same tracer was also applied to an orthotopic U87MG model for tumor detection.

RESULTS

The radiotracer was synthesized with a high specific activity (14,800-29,600 GBq/mmol [400-800 Ci/mmol]). The c(RGDyK)-PEG-DOTA ligand showed intermediate binding affinity for alphavbeta3-integrin (50% inhibitory concentration, 67.5 +/- 7.8 nmol/L [mean +/- SD]). The pegylated RGD peptide demonstrated rapid blood clearance (0.57 +/- 0.15 percentage injected dose [%ID]/g [mean +/- SD] at 30 min after injection and 0.03 +/- 0.02 %ID/g at 4 h after injection). Activity accumulation in the tumor was rapid and high at early time points (2.74 +/- 0.45 %ID/g at 30 min after injection), and some activity washout was seen over time (1.62 +/- 0.18 %ID/g at 4 h after injection). Compared with (64)Cu-DOTA-RGD, this tracer showed improved in vivo kinetics, with significantly reduced liver uptake (0.99 +/- 0.08 %ID/g vs. 1.73 +/- 0.39 %ID/g at 30 min after injection and 0.58 +/- 0.07 %ID/g vs. 2.57 +/- 0.49 %ID/g at 4 h after injection). The pegylated RGD peptide showed higher renal accumulation at early time points (3.51 +/- 0.24 %ID/g vs. 2.18 +/- 0.23 %ID/g at 30 min after infection) but more rapid clearance (1.82 +/- 0.29 %ID/g vs. 2.01 +/- 0.25 %ID/g at 1 h after injection) than 64Cu-DOTA-RGD. The integrin receptor specificity of this radiotracer was demonstrated by blocking of tumor uptake by coinjection with nonradiolabeled c(RGDyK). The high tumor-to-organ ratios for the pegylated RGD peptide tracer (at 1 h after injection: tumor-to-blood ratio, 20; tumor-to-muscle ratio, 12; tumor-to-liver ratio, 2.7; and tumor-to-kidney ratio, 1.2) were confirmed by microPET and autoradiographic imaging in a subcutaneous U87MG tumor model. This tracer was also able to detect an orthotopic brain tumor in a model in which U87MG cells were implanted into the mouse forebrain. Although the magnitude of tumor uptake in the orthotopic xenograft was lower than that in the subcutaneous xenograft, the orthotopic tumor was still visualized with clear contrast from normal brain tissue.

CONCLUSIONS

This study demonstrated the suitability of a PEG moiety for improving the in vivo kinetics of a 64Cu-RGD peptide tracer without compromising the tumor-targeting ability and specificity of the peptide. Systematic investigations of the effects of the size and geometry of PEG on tumor targeting and in vivo kinetics will lead to the development of radiotracers suitable for clinical applications such as visualizing and quantifying alphav-integrin expression by PET. In addition, the same ligand labeled with therapeutic radionuclides may be applicable for integrin-targeted internal radiotherapy.

Bone marrow cells are multipotent cells. When bone marrow cells were cultured with type I collagen matrix gels, they showed high alkaline phosphatase activity, collagen synthesis, and formed mineralized tissues. Furthermore, cells expressed osteocalcin and bone sialoprotein genes, which are osteoblast-specific genes. These findings indicate that type I collagen matrix gels induce osteoblastic differentiation of bone marrow cells. Type I collagen interacts with the alpha 2 beta 1 integrin receptor on the cell membrane and mediates extracellular signals into cells. DGEA peptide is a cell-binding domain of type I collagen molecule. When collagen-integrin interaction was interrupted by the addition of Asp-Gly-Glu-Ala (DGEA) peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. Furthermore, anti-alpha 2 integrin antibody, which interacts with alpha subunit of integrin and blocks the binding of integrin with collagen, suppressed the expression of osteoblastic phenotypes. These findings imply that collagen-alpha 2 beta 1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.

Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe.

A number of mitochondrial proteins have been identified in Leishmania sp. and Trypanosoma brucei that may be involved in U-insertion/deletion RNA editing. Only a few of these have yet been characterized sufficiently to be able to assign functional names for the proteins in both species, and most have been denoted by a variety of species-specific and laboratory-specific operational names, leading to a terminology confusion both within and outside of this field. In this review, we summarize the present status of our knowledge of the orthologous and unique putative editing proteins in both species and the functional motifs identified by sequence analysis and by experimentation. An online Supplemental sequence database (http://164.67.60.200/proteins/protsmini1.asp) is also provided as a research resource.

P2X receptor subunits have intracellular N and C termini, two membrane-spanning domains, and an extracellular loop of about 280 amino acids. We expressed the rat P2X(2) receptor in human embryonic kidney cells, and used alanine-scanning mutagenesis on 30 residues with polar side chains conserved among the seven rat P2X receptor subunits. This identified a region proximal to the first transmembrane domain which contained 2 lysine residues that were critical for the action of ATP (Lys(69) and Lys(71)). We substituted cysteines in this region (Asp(57) to Asp(71)) and found that for S65C and I67C ATP-evoked currents were inhibited by methanethiosulfonates. At I67C, the inhibition by negatively charged ethylsulfonate and pentylsulfonate derivatives could be overcome by increasing the ATP concentration, consistent with a reduced affinity of ATP binding. The inhibitory action of the methanethiosulfonates was prevented by pre-exposure to ATP, suggesting occlusion of the binding site. Finally, introduction of negative charges into the receptor by mutagenesis at this position (I67E and I67D) also gave receptors in which the ATP concentration-response curve was right-shifted. The results suggest that residues close to Ile(67) contribute to the ATP-binding site.

Transfer RNAs specific for Gln, Lys, and Glu from all organisms (except Mycoplasma) and organelles have a 2-thiouridine derivative (xm(5)s(2)U) as wobble nucleoside. These tRNAs read the A- and G-ending codons in the split codon boxes His/Gln, Asn/Lys, and Asp/Glu. In eukaryotic cytoplasmic tRNAs the conserved constituent (xm(5)-) in position 5 of uridine is 5-methoxycarbonylmethyl (mcm(5)). A protein (Tuc1p) from yeast resembling the bacterial protein TtcA, which is required for the synthesis of 2-thiocytidine in position 32 of the tRNA, was shown instead to be required for the synthesis of 2-thiouridine in the wobble position (position 34). Apparently, an ancient member of the TtcA family has evolved to thiolate U34 in tRNAs of organisms from the domains Eukarya and Archaea. Deletion of the TUC1 gene together with a deletion of the ELP3 gene, which results in the lack of the mcm(5) side chain, removes all modifications from the wobble uridine derivatives of the cytoplasmic tRNAs specific for Gln, Lys, and Glu, and is lethal to the cell. Since excess of the unmodified form of these three tRNAs rescued the double mutant elp3 tuc1, the primary function of mcm(5)s(2)U34 seems to be to improve the efficiency to read the cognate codons rather than to prevent mis-sense errors. Surprisingly, overexpression of the mcm(5)s(2)U-lacking tRNA(Lys) alone was sufficient to restore viability of the double mutant.

OmpT from Escherichia coli belongs to a family of highly homologous outer membrane proteases, known as omptins, which are implicated in the virulence of several pathogenic Gram-negative bacteria. Here we present the crystal structure of OmpT, which shows a 10-stranded antiparallel beta-barrel that protrudes far from the lipid bilayer into the extracellular space. We identified a putative binding site for lipopolysaccharide, a molecule that is essential for OmpT activity. The proteolytic site is located in a groove at the extracellular top of the vase-shaped beta-barrel. Based on the constellation of active site residues, we propose a novel proteolytic mechanism, involving a His-Asp dyad and an Asp-Asp couple that activate a putative nucleophilic water molecule. The active site is fully conserved within the omptin family. Therefore, the structure described here provides a sound basis for the design of drugs against omptin-mediated bacterial pathogenesis. Coordinates are in the Protein Data Bank (accession No. 1I78)

Neurotransmitter:sodium symporters (NSS)(1) mediate sodium-dependent reuptake of neurotransmitters from the synaptic cleft and are targets for many psychoactive drugs. The crystal structure of the prokaryotic NSS protein, LeuT, was recently solved at high resolution; however, the mechanistic details of regulation of the permeation pathway in this class of proteins remain unknown. Here we combine computational modeling and experimental probing in the dopamine transporter (DAT) to demonstrate the functional importance of a conserved intracellular interaction network. Our data suggest that a salt bridge between Arg-60 in the N terminus close to the cytoplasmic end of transmembrane segment (TM) 1 and Asp-436 at the cytoplasmic end of TM8 is stabilized by a cation-pi interaction between Arg-60 and Tyr-335 at the cytoplasmic end of TM6. Computational probing illustrates how the interactions may determine the flexibility of the permeation pathway, and mutagenesis within the network and results from assays of transport, as well as the state-dependent accessibility of a substituted cysteine in TM3, support the role of this network in regulating access between the substrate binding site and the intracellular milieu. The mechanism that emerges from these findings may be unique to the NSS family, where the local disruption of ionic interactions modulates the transition of the transporter between the outward- and inward-facing conformations.

We have genotyped 292 affected sibling pairs (ASPs) with Alzheimer's disease (AD) according to NINCDS-ADRDA diagnostic criteria and with onset ages of>>/=65 years using 237 microsatellite markers separated by an average distance of 16.3 cM. Data were analysed by SPLINK and MAPMAKER/SIBS on the whole sample of 292 ASPs and subsets of 162 ASPs where both members possessed an apolipoprotein E (APOE)straightepsilon4 allele and 63 pairs where neither possessed anstraightepsilon4 allele. Sixteen peaks with a multipoint lod score (MLS) >1 either in the whole sample, the straightepsilon4-positive or -negative subgroups were observed on chromosomes 1 (two peaks), 2, 5, 6, 9 (two peaks), 10 (two peaks), 12, 13, 14, 19, 21 and X (two peaks). Simulation studies revealed that these findings exceeded those expected by chance, although many are likely to be false positives. The highest lod scores on chromosomes 1 (MLS 2.67), 9 (MLS 2.38), 10 (MLS 2.27) and 19 (MLS 1.79) fulfilLander and Kruglyak's definition of 'suggestive' in that they would be expected to occur by chance once or less per genome scan. Several other peaks were only marginally less significant than this, in particular those on chromosomes 14 (MLS 2.16), 5 (MLS 2.00), 12, close to alpha2-macroglobulin (MLS 1.91), and 21, close to amyloid precursor protein (MLS 1.77). This is the largest genome scan to date in AD and shows for the first time that this is a genetically complex disorder involving several, perhaps many, genes in addition to APOE. Moreover, our data will be of interest to those hoping to identify positional candidate genes using information emerging from neurobiological studies of AD.