Acetohydroxy acid synthase (AHAS) and isomeroreductase (IR) catalyze subsequent reactions in the flux of metabolites towards isoleucine, valine, leucine, and pantothenate. A 4,705-bp DNA fragment from Corynebacterium glutamicum known to code for AHAS and IR was sequenced and analyzed by Northern (RNA blot) analysis. As in other bacteria, the AHAS of this gram-positive organism is encoded by two genes, ilvB and ilvN. Gene disruption verified that these genes encode the single AHAS activity in C. glutamicum. The start of ilvB was determined by amino-terminal sequencing of a fusion peptide. By Northern analysis of the ilvBNC cluster, three in vivo transcripts of 3.9, 2.3, and 1.1 kb were identified, corresponding to ilvBNC, ilvNC, and ilvC messages, respectively. The ilvC transcript (encoding IR) was by far the most abundant one. With a clone from which the ilvB upstream regions had been deleted, only the ilvNC and ilvC transcripts were synthesized, and with a clone from which the ilvN upstream regions had been deleted, only the smallest ilvC transcript was formed. It is therefore concluded that in the ilv operon of C. glutamicum, three promoters are active. The amounts of the ilvBNC and ilvNC transcripts increased in response to the addition of alpha-ketobutyrate to the growth medium. This was correlated to an increase in specific AHAS activity, whereas IR activity was not increased because of the relatively large amount of the ilvC transcript present under all conditions assayed. Therefore, the steady-state level of the ilvBNC and ilvNC messages contributes significantly to the total activity of the single AHAS. The ilvC transcript of this operon, however, is regulated independently and present in a large excess, which is in accord with the constant IR activities determined.

Cytotoxic T cells (CTLs) perceive the world through small peptides that are eight to ten amino acids long. These peptides (epitopes) are initially generated by the proteasome, a multi-subunit protease that is responsible for the majority of intra-cellular protein degradation. The proteasome generates the exact C-terminal of CTL epitopes, and the N-terminal with a possible extension. CTL responses may diminish if the epitopes are destroyed by the proteasomes. Therefore, the prediction of the proteasome cleavage sites is important to identify potential immunogenic regions in the proteomes of pathogenic microorganisms (or humans). We have recently shown that NetChop, a neural network-based prediction method, is the best method available at the moment to do such predictions; however, its performance is still lower than desired. Here, we use novel sequence encoding methods and show that the new version of NetChop predicts approximately 10% more of the cleavage sites correctly while lowering the number of false positives with close to 15%. With this more reliable prediction tool, we study two important questions concerning the function of the proteasome. First, we estimate the N-terminal extension of epitopes after proteasomal cleavage and find that the average extension is relatively short. However, more than 30% of the peptides have N-terminal extensions of three amino acids or more, and thus, N-terminal trimming might play an important role in the presentation of a substantial fraction of the epitopes. Second, we show that good TAP ligands have an increased chance of being cleaved by the proteasome, i.e., the specificity of TAP has evolved to fit the specificity of the proteasome. This evolutionary relationship allows for a more efficient antigen presentation.

NF-kappa B/Rel transcription factors control apoptosis, also known as programmed cell death. This control is crucial for oncogenesis, cancer chemo-resistance and for antagonizing tumour necrosis factor alpha (TNFalpha)-induced killing. With regard to TNFalpha, the anti-apoptotic activity of NF-kappa B involves suppression of the c-Jun N-terminal kinase (JNK) cascade. Using an unbiased screen, we have previously identified Gadd45 beta/Myd118, a member of the Gadd45 family of inducible factors, as a pivotal mediator of this suppressive activity of NF-kappa B. However, the mechanisms by which Gadd45 beta inhibits JNK signalling are not understood. Here, we identify MKK7/JNKK2--a specific and essential activator of JNK--as a target of Gadd45 beta, and in fact, of NF-kappa B itself. Gadd45 beta binds to MKK7 directly and blocks its catalytic activity, thereby providing a molecular link between the NF-kappa B and JNK pathways. Importantly, Gadd45 beta is required to antagonize TNFalpha-induced cytotoxicity, and peptides disrupting the Gadd45 beta/MKK7 interaction hinder the ability of Gadd45 beta, as well as of NF-kappa B, to suppress this cytotoxicity. These findings establish a basis for the NF-kappa B control of JNK activation and identify MKK7 as a potential target for anti-inflammatory and anti-cancer therapy.

Target cell lysis is regulated by natural killer (NK) cell receptors that recognize class I MHC molecules. Here we report the crystal structure of the human immunoglobulin-like NK cell receptor KIR2DL2 in complex with its class I ligand HLA-Cw3 and peptide. KIR binds in a nearly orthogonal orientation across the alpha1 and alpha2 helices of Cw3 and directly contacts positions 7 and 8 of the peptide. No significant conformational changes in KIR occur on complex formation. The receptor footprint on HLA overlaps with but is distinct from that of the T-cell receptor. Charge complementarity dominates the KIR/HLA interface and mutations that disrupt interface salt bridges substantially diminish binding. Most contacts in the complex are between KIR and conserved HLA-C residues, but a hydrogen bond between Lys 44 of KIR2DL2 and Asn 80 of Cw3 confers the allotype specificity. KIR contact requires position 8 of the peptide to be a residue smaller than valine. A second KIR/HLA interface produced an ordered receptor-ligand aggregation in the crystal which may resemble receptor clustering during immune synapse formation.

Ubiquitin chains are formed through the action of a set of enzymes that covalently link ubiquitin either through peptide bonds or through isopeptide bonds between their C terminus and any of four lysine residues. These naturally occurring polyproteins allow one to study the mechanical stability of a protein, when force is applied through different linkages. Here we used single-molecule force spectroscopy techniques to examine the mechanical stability of N-C-linked and Lys48-C-linked ubiquitin chains. We combined these experiments with steered molecular dynamics (SMD) simulations and found that the mechanical stability and unfolding pathway of ubiquitin strongly depend on the linkage through which the mechanical force is applied to the protein. Hence, a protein that is otherwise very stable may be easily unfolded by a relatively weak mechanical force applied through the right linkage. This may be a widespread mechanism in biological systems.

BACKGROUND

Excess body-mass index (BMI) has been associated with adverse outcomes in prostate cancer, and hyperinsulinaemia is a candidate mediator, but prospective data are sparse. We assessed the effect of prediagnostic BMI and plasma C-peptide concentration (reflecting insulin secretion) on prostate cancer-specific mortality after diagnosis.

METHODS

This study involved men diagnosed with prostate cancer during the 24 years of follow-up in the Physicians' Health Study. BMI measurements were available at baseline in 1982 and eight years later in 1990 for 2546 men who developed prostate cancer. Baseline C-peptide concentration was available in 827 men. We used Cox proportional hazards regression models controlling for age, smoking, time between BMI measurement and prostate cancer diagnosis, and competing causes of death to assess the risk of prostate cancer-specific mortality according to BMI and C-peptide concentration.

RESULTS

Of the 2546 men diagnosed with prostate cancer during the follow-up period, 989 (38.8%) were overweight (BMI 25.0-29.9 kg/m(2)) and 87 (3.4%) were obese (BMI>>/=30 kg/m(2)). 281 men (11%) died from prostate cancer during this follow-up period. Compared with men of a healthy weight (BMI <25 kg/m(2)) at baseline, overweight men and obese men had a significantly higher risk of prostate cancer mortality (proportional hazard ratio [HR] 1.47 [95% CI 1.16-1.88] for overweight men and 2.66 [1.62-4.39] for obese men; p(trend)<0.0001). The trend remained significant after controlling for clinical stage and Gleason grade and was stronger for prostate cancer diagnosed during the PSA screening era (1991-2007) compared with during the pre-PSA screening era (1982-1990) or when using BMI measurements obtained in 1990 compared with those obtained in 1982. Of the 827 men with data available for baseline C-peptide concentration, 117 (14%) died from prostate cancer. Men with C-peptide concentrations in the highest quartile (high) versus the lowest quartile (low) had a higher risk of prostate cancer mortality (HR 2.38 [95% CI 1.31-4.30]; p(trend)=0.008). Compared with men with a BMI less than 25 kg/m(2) and low C-peptide concentrations, those with a BMI of 25 kg/m(2) or more and high C-peptide concentrations had a four-times higher risk of mortality (4.12 [1.97-8.61]; p(interaction)=0.001) independent of clinical predictors.

CONCLUSIONS

Excess bodyweight and a high plasma concentration of C-peptide both predispose men with a subsequent diagnosis of prostate cancer to an increased likelihood of dying of their disease. Patients with both factors have the worst outcome. Further studies are now needed to confirm these findings.

Caspases are a large family of evolutionarily conserved proteases found from Caenorhabditis elegans to humans. Although the first caspase was identified as a processing enzyme for interleukin-1beta, genetic and biochemical data have converged to reveal that many caspases are key mediators of apoptosis, the intrinsic cell suicide program essential for development and tissue homeostasis. Each caspase is a cysteine aspartase; it employs a nucleophilic cysteine in its active site to cleave aspartic acid peptide bonds within proteins. Caspases are synthesized as inactive precursors termed procaspases; proteolytic processing of procaspase generates the tetrameric active caspase enzyme, composed of two repeating heterotypic subunits. Based on kinetic data, substrate specificity, and procaspase structure, caspases have been conceptually divided into initiators and effectors. Initiator caspases activate effector caspases in response to specific cell death signals, and effector caspases cleave various cellular proteins to trigger apoptosis. Adapter protein-mediated oligomerization of procaspases is now recognized as a universal mechanism of initiator caspase activation and underlies the control of both cell surface death receptor and mitochondrial cytochrome c-Apaf-1 apoptosis pathways. Caspase substrates have bene identified that induce each of the classic features of apoptosis, including membrane blebbing, cell body shrinkage, and DNA fragmentation. Mice deficient for caspase genes have highlighted tissue- and signal-specific pathways for apoptosis and demonstrated an independent function for caspase-1 and -11 in cytokine processing. Dysregulation of caspases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders, and increasing evidence shows that altering caspase activity can confer therapeutic benefits.

Growth hormone factor 1 (GHF-1) is a pituitary-specific transcription factor that plays a critical role in cell type-specific expression of the growth hormone (GH) gene. Here, we describe the isolation of bovine and rat GHF-1 cDNA clones. These cDNAs encode proteins whose molecular mass, 33K, is identical to purified GHF-1 and whose sequence agrees with a partial GHF-1 peptide sequence. The predicted GHF-1 sequence contains a region, near its C-terminus, that exhibits considerable similarity to a homeobox consensus sequence. DNAase I footprinting with bacterially expressed fusion protein containing a fragment of GHF-1 encompassing the homeobox indicates that this region of the protein functions as its DNA binding domain. Expression of GHF-1 is restricted to cells of the somatotropic lineage in the pituitary. This remarkable specificity of GHF-1 expression correlates with the selective transcription of its target, the GH gene. Other mammalian homeobox-containing proteins may function similarly as transcription factors controlling cell type-specific expression in other locations.

Inhibition of the accumulation of amyloid beta-peptide (Abeta) and the formation of beta-amyloid fibrils (fAbeta) from Abeta, as well as the destabilization of preformed fAbeta in the central nervous system, would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We reported previously that nordihydroguaiaretic acid (NDGA) and wine-related polyphenols inhibit fAbeta formation from Abeta(1-40) and Abeta(1-42) and destabilize preformed fAbeta(1-40) and fAbeta(1-42) dose-dependently in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of curcumin (Cur) and rosmarinic acid (RA) on the formation, extension, and destabilization of fAbeta(1-40) and fAbeta(1-42) at pH 7.5 at 37 degrees C in vitro. We next compared the anti-amyloidogenic activities of Cur and RA with NDGA. Cur and RA dose-dependently inhibited fAbeta formation from Abeta(1-40) and Abeta(1-42), as well as their extension. In addition, they dose-dependently destabilized preformed fAbetas. The overall activities of Cur, RA, and NDGA were similar. The effective concentrations (EC(50)) of Cur, RA, and NDGA for the formation, extension, and destabilization of fAbetas were in the order of 0.1-1 microM. Although the mechanism by which Cur and RA inhibit fAbeta formation from Abeta and destabilize preformed fAbeta in vitro remains unclear, they could be a key molecule for the development of therapeutics for AD.

Many surface proteins of Gram-positive bacteria are anchored to the cell wall envelope by a transpeptidation mechanism, requiring a C-terminal sorting signal with a conserved LPXTG motif. Sortase, a membrane protein of Staphylococcus aureus, cleaves polypeptides between the threonine and the glycine of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl-group of threonine and the amino-group of peptidoglycan cross-bridges. S. aureus mutants lacking the srtA gene fail to anchor and display some surface proteins and are impaired in the ability to cause animal infections. Sortase acts on surface proteins that are initiated into the secretion (Sec) pathway and have their signal peptide removed by signal peptidase. The S. aureus genome encodes two sets of sortase and secretion genes. It is conceivable that S. aureus has evolved more than one pathway for the transport of 20 surface proteins to the cell wall envelope.

The neuropeptide control of gonadotropin secretion at the level of the anterior pituitary gland is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), which was originally isolated from mammals and subsequently from non-mammals. To date, however, an inhibitory peptide of gonadotropin release is unknown in vertebrates. Here we show, in a bird, that the hypothalamus also contains a novel peptide which inhibits gonadotropin release. Acetic acid extracts of quail brains were passed through C-18 reversed-phase cartridges, and then the retained material was subjected to the reversed-phase and cation-exchange high-performance liquid chromatography (HPLC). The peptide was isolated from avian brain and shown to have the sequence Ser-Ile-Lys-Pro-Ser-Ala-Tyr-Leu-Pro-Leu-Arg-Phe-NH(2). Cell bodies and terminals containing this peptide were localized immunohistochemically in the paraventricular nucleus and median eminence, respectively. This peptide inhibited, in a dose-related way, gonadotropin release from cultured quail anterior pituitaries. This is the first hypothalamic peptide inhibiting gonadotropin release reported in a vertebrate. We therefore term it gonadotropin-inhibitory hormone (GnIH).

Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages.

The retroviral transmembrane envelope protein p15E is immunosuppressive in that it inhibits immune responses of lymphocytes, monocytes, and macrophages. A region of p15E has been conserved among murine and feline retroviruses; a homologous region is also found in the transmembrane envelope proteins of the human retroviruses HTLV-I and HTLV-II and in a putative envelope protein encoded by an endogenous C-type human retroviral DNA. A peptide (CKS-17) was synthesized to correspond to this region of homology and was examined for its effects on lymphocyte proliferation. CKS-17 inhibited the proliferation of an interleukin-2-dependent murine cytotoxic T-cell line as well as alloantigen-stimulated proliferation of murine and human lymphocytes. Four other peptides, representing different regions of virus proteins, were inactive. These results suggest that the immunosuppressive portion of retroviral transmembrane envelope proteins may reside, at least in part, in a-conserved sequence represented by the CKS-17 peptide.

The synthesis of virulence factors and other exoproteins in Staphylococcus aureus is controlled by the global regulator, agr. Expression of secreted proteins is up-regulated in the postexponential growth phase, whereas expression of surface proteins is down-regulated by agr. The agr locus consists of two divergent operons, transcribed from neighboring but non-overlapping promoters, P2 and P3. The P2 operon sequence, reported here, contains 4 open reading frames, agrA, C, D, and B, of which A and C appear to encode proteins of a classical 2-component signal transduction pathway. The P3 operon specifies a 0.5 kb transcript, RNA III, which is the actual effector of the agr response, and, incidentally, encodes the agr-regulated peptide delta-hemolysin. Transcriptional fusions have shown that both P2 and P3 are agr sensitive (function in an agr+ but not in an agr- background) and deletion analysis has shown that all four of the P2 ORFs are involved; agrA and agrC seem to be absolutely required for the transcriptional activation of the agr locus, whereas agrB and agrD seem to be partially required. Since transcription of P2 requires P2 operon products, the P2 operon is autocatalytic, and is thus admirably suited to the need for rapid production of exoproteins at a time when overall growth is coming to a halt.

BACKGROUND

B-type natriuretic peptides have been shown to predict cardiovascular disease in apparently healthy individuals but their predictive ability for mortality and future cardiovascular events compared with C-reactive protein (CRP) and urinary albumin/creatinine ratio is unknown.

OBJECTIVE

To assess the prognostic value of the N-amino terminal fragment of the prohormone brain natriuretic peptide (NT-proBNP) vs CRP and urinary albumin/creatinine ratio in an older adult population.

METHODS

A population-based prospective study of 764 participants aged 50 to 89 years from a community in Copenhagen, Denmark, in which 658 participants provided blood and urinary samples and were examined between September 1, 1998, and January 24, 2000. Of these participants, 626 without heart or renal failure were enrolled. A subgroup of 537 had no history of cardiovascular disease at baseline. During 5 years of follow-up (to December 31, 2003), 94 participants died and 65 developed a first major cardiovascular event.

METHODS

Risk of mortality and first major cardiovascular event by baseline levels of NT-proBNP, CRP, and urinary albumin/creatinine ratio levels.

RESULTS

After adjustment for the cardiovascular risk factors of age, sex, smoking, diabetes mellitus, hypertension or ischemic heart disease, total cholesterol, and serum creatinine, the hazard ratio (HR) of mortality for values above the 80th percentile of NT-proBNP was 1.96 (95% confidence interval [CI], 1.21-3.19); for CRP, 1.46 (95% CI, 0.89-2.24); and for urinary albumin/creatinine ratio, 1.88 (95% CI, 1.18-2.98). Additional adjustment for left ventricular systolic dysfunction did not markedly attenuate the predictive value of NT-proBNP (HR, 1.82; 95% CI, 1.11-2.98). The absolute unadjusted increase in mortality risk for participants with values above the 80th percentile vs equal to or below the 80th percentile was 24.5% for NT-proBNP, 7.8% for CRP, and 19.5% for urinary albumin/creatinine ratio. The NT-proBNP levels were associated with first major cardiovascular events (nonfatal myocardial infarction, fatal coronary heart disease, unstable angina, heart failure, stroke, and transient ischemic attack) with an adjusted HR of 3.24 (95% CI, 1.80-5.79) vs 1.02 (95% CI, 0.56-1.85) for CRP and 2.32 (95% CI, 1.33-4.05) for urinary albumin/creatinine ratio when comparing participants with values above the 80th percentile with those with values equal to or below the 80th percentile.

CONCLUSIONS

Measurements of NT-proBNP provide prognostic information of mortality and first major cardiovascular events beyond traditional risk factors. NT-proBNP was a stronger risk biomarker for cardiovascular disease and death than CRP was in nonhospitalized individuals aged 50 to 89 years.

We have isolated the bovine and human genes for Müllerian inhibiting substance (MIS), a testicular glycoprotein that causes regression of the Müllerian duct during development of the male embryo. The mRNA sequence of bovine MIS, determined from an analysis of cDNA and genomic clones, codes for a protein of 575 amino acids containing a 24 amino acid leader peptide. The human gene has five exons that code for a protein of 560 amino acids. A comparison of the bovine and human MIS proteins reveals a highly conserved C-terminal domain that shows marked homology with human transforming growth factor-beta and the beta chain of porcine inhibin. Animal cells transfected with the human gene secrete biologically active MIS, which causes regression of the rat Müllerian duct in vitro.

Genes that function in translocation of secretory protein precursors into the ER have been identified by a genetic selection for mutant yeast cells that fail to translocate a signal peptide-cytosolic enzyme hybrid protein. The new mutants, sec62 and sec63, are thermosensitive for growth and accumulate a variety of soluble secretory and vacuolar precursors whose electrophoretic mobilities coincide with those of the corresponding in vitro translated polypeptides. Proteolytic sensitivity of precursor molecules in extracts of mutant cells confirms that polypeptide translocation is blocked. Some form of interaction among the SECCell Biol. 105:633-645), SECCC and show a marked enhancement of the translocation defect. The translocation defects of two mutants (sec62 and sec63) have been reproduced in vitro. sec63 microsomes display low and thermolabile translocation activity for prepro-alpha-factor (pp alpha F) synthesized with a cytosol fraction from wild type yeast. These gene products may constitute part of the polypeptide recognition or translocation apparatus of the ER membrane. Pulse-chase analysis of the translocation-defective mutants demonstrates that insertion of pp alpha F into the ER can proceed posttranslationally.

In bacteria, SsrA RNA recognizes ribosomes stalled on defective messages and acts as a tRNA and mRNA to mediate the addition of a short peptide tag to the C-terminus of the partially synthesized nascent polypeptide chain. The SsrA-tagged protein is then degraded by C-terminal-specific proteases. SmpB, a unique RNA-binding protein that is conserved throughout the bacterial kingdom, is shown here to be an essential component of the SsrA quality-control system. Deletion of the smpB gene in Escherichia coli results in the same phenotypes observed in ssrA-defective cells, including a variety of phage development defects and the failure to tag proteins translated from defective mRNAs. Purified SmpB binds specifically and with high affinity to SsrA RNA and is required for stable association of SsrA with ribosomes in vivo. Formation of an SmpB-SsrA complex appears to be critical in mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain. SsrA RNA is present at wild-type levels in the smpB mutant arguing against a model of SsrA action that involves direct competition for transcription factors.

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We and others have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting signaling molecules within caveolae membranes. In this regard, it has been shown that a 20-amino acid membrane-proximal region of the cytosolic NH2-terminal domain of caveolin is sufficient to mediate the interaction of caveolin with signaling proteins, namely G-proteins, Src-like kinases, eNOS, and H-Ras. This caveolin-derived protein domain has been termed the caveolin-scaffolding domain. Binding of the caveolin-scaffolding domain functionally suppresses the activity of G-protein alpha subunits, eNOS, and Src-like kinases, suggesting that caveolin binding may also play a negative regulatory role in signal transduction. Here, we report the direct interaction of caveolin with a growth factor receptor, EGF-R, a known caveolae-associated receptor tyrosine kinase. Two consensus caveolin binding motifs have been previously defined using phage display technology. One of these motifs is present within the conserved kinase domains of most known receptor tyrosine kinases (termed region IX). We now show that this caveolin binding motif within the kinase domain of the EGF-R can mediate the interaction of the EGF-R with the scaffolding domains of caveolins 1 and 3 but not with caveolin 2. In addition, the scaffolding domains of caveolins 1 and 3 both functionally inhibit the autophosphorylation of the EGF-R kinase in vitro. Importantly, this caveolin-mediated inhibition of the EGF-R kinase could be prevented by the addition of an EGF-R-derived peptide that (i) contains a well conserved caveolin binding motif and (ii) is located within the kinase domain of the EGF-R and most known receptor tyrosine kinases. Similar results were obtained with protein kinase C, a serine/threonine kinase, suggesting that caveolin may function as a general kinase inhibitor. The implications of our results are discussed within the context of caveolae-mediated signal transduction. In this regard, caveolae-coupled signaling might explain how linear signaling pathways can branch and interconnect extensively, forming a signaling module or network.

Integrin alpha(v)beta(3) plays a critical role in tumor-induced angiogenesis and metastasis and has become a promising diagnostic indicator and therapeutic target for various solid tumors. Radiolabeled RGD peptides that are integrin specific can be used for noninvasive imaging of integrin expression level as well as for integrin-targeted radionuclide therapy.

METHODS

In this study we developed a tetrameric RGD peptide tracer (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) for PET imaging of integrin alpha(v)beta(3) expression in female athymic nude mice bearing the subcutaneous UG87MG glioma xenografts.

RESULTS

The RGD tetramer showed significantly higher integrin binding affinity than the corresponding monomeric and dimeric RGD analogs, most likely due to a polyvalency effect. The radiolabeled peptide showed rapid blood clearance (0.61 +/- 0.01 %ID/g at 30 min and 0.21 +/- 0.01 %ID/g at 4 h after injection, respectively [%ID/g is percentage injected dose per gram]) and predominantly renal excretion. Tumor uptake was rapid and high, and the tumor washout was slow (9.93 +/- 1.05 %ID/g at 30 min after injection and 4.56 +/- 0.51 %ID/g at 24 h after injection). The metabolic stability of (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) was determined in mouse blood, urine, and liver and kidney homogenates at different times after tracer injection. The average fractions of intact tracer in these organs at 1 h were approximately 70%, 58%, 51%, and 26%, respectively. Noninvasive microPET studies showed significant tumor uptake and good contrast in the subcutaneous tumor-bearing mice, which agreed well with the biodistribution results. Integrin alpha(v)beta(3) specificity was demonstrated by successful blocking of tumor uptake of (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) in the presence of excess c(RGDyK) at 1 h after injection. The highest absorbed radiation doses determined for the human reference adult were received by the urinary bladder wall (0.262 mGy/MBq), kidneys (0.0296 mGy/MBq), and liver (0.0242 mGy/MBq). The average effective dose resulting from a single (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) injection was estimated to be 0.0164 mSv/MBq.

CONCLUSIONS

The high integrin and avidity and favorable biokinetics make (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) a promising agent for peptide receptor radionuclide imaging and therapy of integrin-positive tumors.

The aim of this investigation was to determine whether murine models of inflammatory, neuropathic and cancer pain are each characterized by a unique set of neurochemical changes in the spinal cord and sensory neurons. All models were generated in Cce and hyperalgesia and allodynia behaviorally characterized. A variety of neurochemical markers that have been implicated in the generation and maintenance of chronic pain were then examined in spinal cord and primary afferent neurons.Three days after injection of complete Freund's adjuvant into the hindpaw (a model of persistent inflammatory pain) increases in substance P, calcitonin gene-related peptide, protein kinase C gamma, and substance P receptor were observed in the spinal cord. Following sciatic nerve transection or L5 spinal nerve ligation (a model of persistent neuropathic pain) significant decreases in substance P and calcitonin gene-related peptide and increases in galanin and neuropeptide Y were observed in both primary afferent neurons and the spinal cord. In contrast, in a model of cancer pain induced by injection of osteolytic sarcoma cells into the femur, there were no detectable changes in any of these markers in either primary afferent neurons or the spinal cord. However, in this cancer-pain model, changes including massive astrocyte hypertrophy without neuronal loss, increase in the neuronal expression of c-Fos, and increase in the number of dynorphin-immunoreactive neurons were observed in the spinal cord, ipsilateral to the limb with cancer. These results indicate that a unique set of neurochemical changes occur with inflammatory, neuropathic and cancer pain in Cce and further suggest that cancer induces a unique persistent pain state. Determining whether these neurochemical changes are involved in the generation and maintenance of each type of persistent pain may provide insight into the mechanisms that underlie each of these pain states.

Acute elevations in free fatty acids (FFAs) stimulate insulin secretion, but prolonged lipid exposure impairs beta-cell function in both in vitro studies and in vivo animal studies. In humans data are limited to short-term (< or =48 h) lipid infusion studies and have led to conflicting results. We examined insulin secretion and action during a 4-day lipid infusion in healthy normal glucose tolerant subjects with (FH+ group, n = 13) and without (control subjects, n = 8) a family history of type 2 diabetes. Volunteers were admitted twice to the clinical research center and received, in random order, a lipid or saline infusion. On days 1 and 2, insulin and C-peptide concentration were measured as part of a metabolic profile after standardized mixed meals. Insulin secretion in response to glucose was assessed with a +125 mg/dl hyperglycemic clamp on day 3. On day 4, glucose turnover was measured with a euglycemic insulin clamp with [3-3H]glucose. Day-long plasma FFA concentrations with lipid infusion were increased within the physiological range, to levels seen in type 2 diabetes (approximately 500-800 micromol/l). Lipid infusion had strikingly opposite effects on insulin secretion in the two groups. After mixed meals, day-long plasma C-peptide levels increased with lipid infusion in control subjects but decreased in the FH+ group (+28 vs. -30%, respectively, P < 0.01). During the hyperglycemic clamp, lipid infusion enhanced the insulin secretion rate (ISR) in control subjects but decreased it in the FH+ group (first phase: +75 vs. -60%, P < 0.001; second phase: +25 vs. -35%, P < 0.04). When the ISR was adjusted for insulin resistance (ISRRd = ISR / [1/Rd], where Rd is the rate of insulin-stimulated glucose disposal), the inadequate beta-cell response in the FH+ group was even more evident. Although ISRRd was not different between the two groups before lipid infusion, in the FH+ group, lipid infusion reduced first- and second-phase ISR(Rd) to 25 and 42% of that in control subjects, respectively (both P < 0.001 vs. control subjects). Lipid infusion in the FH+ group (but not in control subjects) also caused severe hepatic insulin resistance with an increase in basal endogenous glucose production (EGP), despite an elevation in fasting insulin levels, and impaired suppression of EGP to insulin. In summary, in individuals who are genetically predisposed to type 2 diabetes, a sustained physiological increase in plasma FFA impairs insulin secretion in response to mixed meals and to intravenous glucose, suggesting that in subjects at high risk of developing type 2 diabetes, beta-cell lipotoxicity may play an important role in the progression from normal glucose tolerance to overt hyperglycemia.

Various tissues and cells in culture contain a specific inhibitor of DNase I (EC 3.1.4.5). In this paper evidence is presented that this inhibitor is actin, one of the major structural proteins of muscle and nonmuscle cells. (a) The inhibitor is a major cellular component constituting 5-10% of the soluble protein. (b) It migrates with actin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, having a characteristic molecular weight of 42,000. (c) It has an amino-acid composition closely similar to that of actin. (d) The peptide maps of the two proteins are nearly identical. (e) Skeletal muscle actin inhibits the enzymatic activity of DNase I. (f) DNase I-agarose affinity chromatography quantitatively retains purified skeletal muscle actin, and actin, specifically, from high-speed supernatants of whole cell extracts. (g) An antibody to purified inhibitor protein from calf thymus, used in indirect immunofluorescence on cells grown in culture, stains a two-dimensional network of fibers similar to that seen with an actin-specific antibody.The observation that actin can be isolated by DNase-agarose affinity chromatography provides a useful tool for the biochemical study of actin under different physiological conditions.

PDZ domains are abundant protein interaction modules that often recognize short amino acid motifs at the C-termini of target proteins. They regulate multiple biological processes such as transport, ion channel signaling, and other signal transduction systems. This review discusses the structural characterization of PDZ domains and the use of recently emerging technologies such as proteomic arrays and peptide libraries to study the binding properties of PDZ-mediated interactions. Regulatory mechanisms responsible for PDZ-mediated interactions, such as phosphorylation in the PDZ ligands or PDZ domains, are also discussed. A better understanding of PDZ protein-protein interaction networks and regulatory mechanisms will improve our knowledge of many cellular and biological processes.