Growth factor release from amylopectin hydrogel based on copper coordination

This paper describes a biodegradable hydrogel matrix releasing basic fibroblast growth factor (bFGF) on the basis of protein metal coordination with the protein drug. The biodegradable hydrogel was prepared from amylopectin by its crosslinking with ethylene glycol diglycidyl ether, followed by introduction of diethylenetriaminepentaacetic acid (DTPA) residues for copper chelation. When bFGF was incorporated into the DTPA-introduced amylopectin hydrogel after chelation with Cu2+, an insignificant amount of bFGF was released from the hydrogel in buffered solution, in contrast to that without Cu2+ chelation. An increased ionic strength in the solution did not affect the bFGF release, indicating the occurrence of coordinate bonding of bFGF to the DTPA-introduced hydrogel through Cu2+ chelation. An implantation study with 125I-labeled amylopectin hydrogels demonstrated that they underwent degradation in the back subcutis of mice. Cu2+ chelation of hydrogels enabled bFGF to remain in the mouse back for a long time period, irrespective of DTPA introduction. However, DTPA residues were necessary to induce significant neovascularization by the Cu2+-chelating hydrogels incorporating bFGF. The DTPA-introduced amylopectin prevented Cu2+-induced deactivation of bFGF, again in marked contrast to DTPA-free amylopectin. It was concluded that biologically active bFGF could be incorporated to DTPA-introduced amylopectin through Cu2+ chelation in a stabilized state and was released as a result of hydrogel biodegradation, resulting in prolonged neovascularization.     

Pharmacokinetics of methotrexate-albumin conjugates in tumor-bearing rats

Linking chemotherapeutic drugs to a macromolecular carrier system may enhance tumor targeting, reduce toxicity and overcome drug resistance mechanisms. As an elementary model to evaluate the pharmacological properties of macromolecular drug carrier systems we chose rat serum albumin (RSA) for carrier and methotrexate (MTX) as antineoplastic drug. The conjugation procedure yielded conjugates with an approximate 1:1 molar loading rate (MTX(1)-RSA). In the first part of the study a residualizing [111In]DTPA protein label was used for mapping in vivo the catabolic sites of the native carrier protein and of the MTX(1)-RSA drug conjugate in Walker 256 carcinosarcoma bearing rats. The tumor accumulation was about 14% of the injected dose for the RSA and MTX(1)-RSA tracers after 24 h. Tracer entrapment by organs with an active mononuclear phagocyte system was low (liver below 7% and spleen below 1.5% of the injected dose after 24 h). The 1:1 conjugation of MTX to RSA did not decisively alter the pharmacokinetic properties nor the tumor or tissue distribution of the native carrier protein RSA. In the second part of the study the different properties of the MTX(1)-RSA conjugate were compared with MTX in vivo. About 2 mg MTX/kg body weight either of the drug conjugate or of the original drug were injected after being additionally spiked with radiolabeled tracers. Plasma concentrations were simultaneously determined by immunological and radioactive means. After 24 h about 12% MTX(1)-RSA was found in circulation compared to 0.03% MTX. Favorable tumor accumulation rates of about 14% were achieved for MTX(1)-RSA versus 0.04% for MTX. About 45-fold more of the injected dose of [3H]MTX accumulated in the liver as compared to the tumor (1.5 versus 0.03%) after 24 h. Conjugation of MTX to RSA reversed this ratio in favor of the tumor to 1:1.4 (13.6 versus 9.6%). In conclusion, the potential therapeutic benefit of the MTX(1)-RSA conjugate lies in its very long tumor exposure time and its improved tumor accumulation rate compared to conventional MTX. In addition the conjugation to albumin might enhance the therapeutic effects over those achieved by long-term continuous infusion of MTX, as MTX(1)-RSA enters the cells by a different uptake mechanism. This might also help to circumvent MTX resistance mechanisms, such as a reduction in folate receptor numbers or impaired MTX polyglutamylation.     

Lectin-mediated drug targeting: preparation, binding characteristics, and antiproliferative activity of wheat germ agglutinin conjugated doxorubicin on Caco-2 cells

PURPOSE: To investigate the usefulness of wheat germ agglutinin as a targeting carrier protein for an acid-labile chemotherapeutic prodrug directed against colon carcinoma cells in vitro. METHODS: Cis-aconityl-linked doxorubicin-wheat germ agglutinin was prepared by a two step procedure and the conjugate-binding capacity of target- and non-target cells was assayed by flow cytometry. The antiproliferative activity of the prodrug on Caco-2 and MOLT-4 cells was determined by the XTT- and BrdU-test and compared with that of the parent drug and the lectin alone. RESULTS: At pH 4.0, about 50% of the conjugated doxorubicin were released within 24 h from the water soluble prodrug exhibiting a conjugation number of 24 (mol doxorubicin/mol WGA). The prodrug-binding capacity of colon carcinoma cells exceeded that of human colonocytes and lymphoblastic MOLT-4 cells 4.5-fold. Additionally, the antiproliferative effect of the conjugate on Caco-2 cells was 39% as opposed to 5% in case of MOLT-4 cells. As the unmodified carrier protein inhibited or stimulated Caco-2 cell growth in a concentration-dependent manner, the cytostatic activity of the conjugate was determined at WGA concentrations without an effect on cell-proliferation. Considering 50% release of conjugated drug at the most, the prodrug yielded 160% of the cytostatic activity of free doxorubicin. CONCLUSIONS: WGA-prodrug targeting offers new perspectives for site-specific, cytoinvading drug delivery in colon cancer chemotherapy.     

New modular delivery system for diagnostic and therapeutic pre-targeting using tautomer-specific monoclonal antibody EM-6-47 and 3-substituted adenines

We have developed a new modular affinity system for the 2-step delivery of functional molecules to target cells. The system is based on the tautomer-specific monoclonal antibody (MAb) EM-6-47, which binds to 3- and 3,8-substituted adenines with high affinity (Ka > 10(9) l/mol) without cross-reacting with naturally occurring purine derivatives. This MAb serves as the hapten-specific fusion partner to produce bispecific MAbs (bs-MAbs) recognizing a target cell antigen and a low-m.w. hapten as carrier molecule for, e.g., radionuclides. Either the C-8 or the N-3 position of adenines can be used for conjugation with effector molecules; the remaining position may be substituted with different moieties to modulate the pharmacokinetics of the haptens. Different 3- and 3,8-substituted adenines conjugated to the chelates DOTA and DTPA or to the drug daunomycin were synthesized. Adenine-chelate derivatives were efficiently labeled with (111)In and 90Y, while high-affinity binding of 3-substituted adenines to MAb EM-6-47 remained almost unaffected by the conjugation to radiochelates. To confirm the validity of the delivery system, a prototype bs-MAb, EM-168-47, was generated by somatic cell fusion of MAb EM-6-47 and MAb EM-168-2, the latter recognizing a surface antigen on canine hematopoietic cells. Two-step targeting assays in vitro verified the bs-MAb-mediated, dose-dependent delivery of (111)In-labeled adenine-chelate derivatives to myeloid cells. This system represents a powerful tool for new pre-targeting approaches relying on bs-MAbs and low-m.w. haptens. Suitable cellular antigens can be targeted by fusing the appropriate MAbs with hapten-specific MAb EM-6-47, and tailor-made 3-substituted adenines may be labeled with diagnostic or therapeutic radionuclides, cytotoxic drugs or other functional molecules.     

Nurse case management to improve glycemic control in diabetic patients in a health maintenance organization. A randomized, controlled trial

A monomeric protein, the hemoglobin alpha chain, was used to compare four protocols for conjugation with diethylene triamine pentaacetic (DTPA) anhydride. Carbamylation and succinylation were also performed. The isoelectric point (pI) was 7.7 for the native protein versus only 5.5 to 7.3 for the five carbamylated derivatives and 4.0 to 7.0 for the six succinylated derivatives. With carbamylation or succinylation, increasing the molar ratio (agent/protein) was associated with a gradual downward pI shift producing trains of bands. This phenomenon did not occur with DTPA conjugation, whose results varied with the method used; only one derivate (pI 6.7) was produced by all four methods, and multiple fine bands with pH values in the vicinity of 3.6 were seen. For the protein, the pI shift varied with the number of groups inserted on the primary amine residues. Also, the shift was larger if the inserted groups carried electrically-charged moieties.     

Dietary guidelines for Americans: an historical perspective

The laminin peptide fragments GYIGSR-NH2 and CDPGYIGSR-NH2 are known to bind to a 67-kDa laminin receptor. This receptors is understood to be expressed at higher than normal levels in malignant tumor cells, particularly those of breast and colon carcinomas. Peptides DTPA-GYIGSR-NH2 (1), DTPA-(GYIGSR-NH2)2 (2), DTPA-CDPGYIGSR-NH2 (3), DTPA-(CDPGYIGSR-NH2)2 (4), and negative control DTPA-GAGAGA-NH2 (5) were prepared by solid-phase peptide synthesis. All five DTPA-conjugated peptides were subsequently radiolabeled with 111In and their tissue distribution evaluated in mice bearing C3H tumors. 111In-3 and 111In-4 showed the highest specific tumor localization. These preliminary data support further study of radiolabeled petide fragments for the potential detection of malignant tumors of the breast and other organs.     

Salmonella typhi O:9,12 polysaccharide-protein conjugates: characterization and immunoreactivity with pooled and individual normal human sera, sera from patients with paratyphoid A and B and typhoid fever, and animal sera

Polysaccharide of O:9,12 specificity purified from Salmonella typhi was conjugated to tetanus toxoid or bovine serum albumin in order to obtain defined antigenic material that would contain O chain free of other S. typhi antigens and that would be suitable for characterizing host humoral response to only S. typhi O-chain antigens. These artificial conjugates were strongly reactive in immunodots with 18 pooled and 3 individual serum samples from patients with typhoid fever and with rabbit anti-Salmonella O antiserum (group D, factors 1, 9, and 12). They reacted weakly with one serum sample from one human with paratyphoid A. These results suggest that the periodate oxidation and the reductive amination used in the conjugation conserved the immunogenicity of the O chain and allowed its absorption to nitrocellulose. They also suggest that the bovine serum albumin conjugate could be used in the diagnosis of S. typhi infections as normal sera may react with the protein molecule of the tetanus toxoid conjugate.     

Pretreatment plasminogen activator inhibitor-1 (PAI-1) levels and the outcome of thrombolysis with streptokinase in patients with acute myocardial infarction

We have evaluated the potential usefulness of radiolabelled [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide as radiopharmaceuticals for somatostatin receptor-targeted scintigraphy and radiotherapy. In vitro somatostatin receptor binding and in vivo metabolism in rats of the compounds were investigated in comparison with [111In-DTPA0] octreotide. Comparing different peptide-chelator constructs, [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide were found to have a higher affinity than [DTPA0]octreotide for subtype 2 somatostatin receptors (sst2) in mouse AtT20 pituitary tumour cell membranes (all IC50 values obtained were in the low nanomolar range). In vivo studies in CA20948 tumor-bearing Lewis rats revealed a significantly higher uptake of both 111In-labelled [DOTA0,Tyr3]octreotide and [DTPA0,Tyr3]octreotide in sst2-expressing tissues than after injection of [111In-DTPA0]octreotide, showing that substitution of Tyr for Phe at position 3 in octreotide results in an increased affinity for its receptor and in a higher target tissue uptake. Uptake of 111In-labelled [DTPA0]octreotide, [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide in pituitary, pancreas, adrenals and tumour was decreased to less than 7% of control by pre-treatment with 0.5 mg unlabelled octreotide/rat, indicating specific binding to sst2. Comparing different radionuclides, [90Y-DOTA0,Tyr3]octreotide had the highest uptake in sst2-positive organs, followed by the [111In-DOTA0,Tyr3]octreotide, whereas [DOTA0,125I-Try3]octreotide uptake was low compared to that of the other radiopharmaceuticals, when measured 24 hr after injection. Renal uptake of 111In-labelled [DTPA0]octreotide, [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide was reduced over 50% by an i.v. injection of 400 mg/kg D-lysine, whereas radioactivity in blood, pancreas and adrenals was not affected.     

Indium-111-DTPA-folate as a potential folate-receptor-targeted radiopharmaceutical

Indium-111-labeled diethylenetriamine pentaacetic acid (DTPA)-folate was evaluated as a radiopharmaceutical for targeting tumor-associated folate receptors. METHODS: Athymic mice were subcutaneously inoculated with approximately 1.8 x 10(6) folate receptor-positive KB (human nasopharyngeal carcinoma) cells, yielding 0.2- to 0.6-g tumors in 15 days, at which time (111)In-DTPA-folate, (111)In-DTPA or (111)In-citrate was administered by intravenous injection. RESULTS: The (111)In-DTPA-folate conjugate afforded marked tumor-specific (111)In deposition in vivo using this mouse model. The involvement of the folate receptor in mediating tumor uptake of (111)In-DTPA-folate was demonstrated by the blocking of tumor uptake by coadministration of free folic acid (intravenous). The (111)In-DTPA-folate also shows folate receptor-mediated uptake and retention in the kidneys, presumably reflecting radiotracer binding to folate receptors of the proximal tubules. In control experiments, the (111)In-citrate radiopharmaceutical precursor was also shown to afford significant tumor uptake of (111)In, but with much poorer tumor-to-background tissue contrast than that obtained with (111)In-DTPA-folate. Unconjugated (111)In-DTPA showed no tumor affinity. CONCLUSION: Indium-111-DTPA-folate appears suitable as a radiopharmaceutical for targeting tumor-associated folate receptors.     

Stereoselective conjugation of prostaglandin A2 and prostaglandin J2 with glutathione, catalyzed by the human glutathione S-transferases A1-1, A2-2, M1a-1a, and P1-1

Prostaglandins containing an alpha,beta-unsaturated keto group, such as prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2), inhibit cell proliferation. These cyclopentenone prostaglandins may be conjugated with GSH chemically or enzymatically via glutathione S-transferases, and this has been suggested to result in inhibition of the antiproliferative mode of action. In the present study, the role of the major human GSTs in the conjugation of PGA2 and PGJ2 with GSH was investigated with purified enzymes, i.e., the Alpha-class enzymes GST A1-1 and GST A2-2, the Mu-class enzyme GST M1a-1a, and the Pi-class enzyme GST P1-1. The GSH conjugates were separated from the parent compound by HPLC and identified by fast atom bombardment mass spectrometry and 1H-NMR. Two GSH conjugates were found for both PGA2 and PGJ2, the R- and S-GSH conjugates of both prostaglandins. Incubation experiments with PGA2 and PGJ2 (70-600 microM) clearly showed the role of individual GSTs in the conjugation of PGA2 and PGJ2. Compared to the chemical reaction, enzyme activities towards PGA2 were up to 5.4 times as high (GSTA1-1) at the lowest concentration (70 microM), while at the highest concentration (600 microM) enzyme activities were up to 3.0 times as high (GST P1-1). For PGJ2, enzyme activities were up to 4.3 (GSTM1a-1a, 70 microM) and up to 3.1 (GSTM1a-1a, 600 microM) times as high. As expected, similar amounts of the R- and S-conjugates of both prostaglandins were found in the chemical reaction. Striking stereoselectivities in conjugating activities were observed for GST A1-1 and GST P1-1. GST A1-1 favors the formation of the R-GSH conjugates of both prostaglandins. GST P1-1 showed a clear selectivity with regard to the formation of the S-GSH conjugate of PGA2. However, this selectivity was not found for the formation of the S-GSH conjugate of PGJ2. GSTM1a-1a showed no stereoselectivity with regard to the GSH conjugation of both PGA2 and PGJ2. GSTA2-2 only showed some minor formation of the R-GSH conjugate of PGJ2. The possible implications of the observed stereoselectivity on the effects of PGA2 and PGJ2 are discussed.     

Labelled polycyanoacrylate nanoparticles for human in vivo use

Isobutyl and isohexyl cyanoacrylate nanoparticles are used as drug carriers, particularly for some anti-cancer drugs. Body distribution as well as pharmacokinetics have been well studied in animal and partially in man. Labelling of the monomer itself or of the carried drug with beta-emitters allowed such studies. In man, however, organ distribution and uptake could easily be done and followed by means of scintigraphy (imaging) techniques if one could achieve nanoparticle labelling with gamma-emitting isotopes. We have developed labelling methods able to supply such carriers using gamma-emitters like radioactive iodine (125I or 131I), indium or technetium. We used DTPA as a spacer in order to fix the last two isotopes. This would mean that any other gamma-emitting cation can theoretically be tried pending on its ability to be chelated by DTPA. The preparations were obtained with high labelling yields, usually > 80% and were relatively stable in human plasma over the whole period of investigation. 111In and 99mTc labelled forms have been administered to rabbit and then to man with 60-75% accumulation in the reticulo-endothelial system.     

Ubiquitin is conjugated to the cytoskeletal protein alpha-spectrin in mature erythrocytes

Ubiquitination of red blood cell (RBC) proteins was investigated by encapsulation of 125I-ubiquitin into human erythrocytes using a procedure of hypotonic dialysis, isotonic resealing, and reannealing. Incubation (37 degrees C, up to 2 h) of 125I-ubiquitin-loaded cells resulted in the recovery of 125I-ubiquitin with the cytosolic proteins (9.22 +/- 0.4 micrograms/ml RBC) and conjugated to membrane proteins (2.18 +/- 0.05 micrograms/ml RBC). This conjugation was time-dependent, and the predominant membrane protein band that became labeled showed an apparent molecular mass of 240 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Western blotting experiments with three different anti-ubiquitin antibodies revealed that this protein is also ubiquitinated in vivo. Cell-free experiments have shown that fraction II (a DEAE-bound protein fraction eluted by 0.5 M KCl) prepared from both mature erythrocytes and reticulocytes is able to conjugate ubiquitin to this protein. Ubiquitin conjugation was ATP-dependent (Km 0.09 mM), time-dependent, and fraction II-dependent (8 +/- 0.5 pmol of 125I-ubiquitin/h/mg of fraction II). Isolation of the major RBC membrane protein that is ubiquitinated was obtained by using biotinylated ubiquitin. Membrane proteins, once ubiquitinated with this derivative, were extracted and purified by affinity chromatography on immobilized avidin. The major components retained by the column were two peptides of molecular masses 220 and 240 kDa. Both proteins are recognized by a monoclonal anti-spectrin antibody, but only the 240-kDa component is detected by streptavidin peroxidase conjugate. That indeed the ubiquitinated membrane protein of 240-kDa is alpha-spectrin was confirmed by immunoaffinity chromatography using 125I-ubiquitin and a monoclonal anti-spectrin antibody. Antigen-antibody complexes were purified by protein A chromatography and analyzed by SDS-PAGE and autoradiography. Again two bands of 240 and 220 kDa were eluted (alpha- and beta-spectrin), but only one band corresponding to the electrophoretic mobility of alpha-spectrin was detected by autoradiography. Thus, alpha-spectrin is a substrate for the ATP-dependent ubiquitination system, suggesting that the cytoskeleton is covalently modified by ubiquitination both in reticulocytes and mature RBC.     

Urinary metabolites of p-hydroxyamphetamine in man, rat and guinea-pig

1. The metabolism of (+/-)-p-hydroxy[14C]amphetamine has been studied in the rat, guinea-pig and man. 2. Most of the administered 14C was excreted in the urine within the first 24 h (64-92%), and was present mainly as free and conjugated p-hydroxy[14C]-amphetamine. In the female rat and female guinea-pig the conjugate was a glucuronide, but in man, who received a much smaller dose, the conjugate was a sulphate ester. A sex difference in conjugation was found in the rat, the female partly conjugating the drug but not the male. 3. Small quantities (1-6% of dose) of p-hydroxynorephedrine, a putative false neurotransmitter, were found in the urine of the three species. 4. Some oxidative degradation of the side chain of p-hydroxyamphetamine occurred in rat and guinea-pig since small amounts of p-hydroxybenzoic acid (1-3%) were detected in the urine.     

The use of the optical disector to estimate the number of neurons, glial and endothelial cells in the spinal cord of the mouse--with a comparative note on the rat spinal cord

Poly(ethylene glycol) (PEG)-bound chelated metal ions partition preferentially into the top, PEG-rich, phase of a PEG-salt or PEG-dextran aqueous two-phase system. Extraction by this soluble affinity ligand of proteins is due to a selective interaction of the chelated metal ion with accessible histidine residues on the protein surface. Using Cu-iminodiacetate-PEG (Cu-IDA-PEG) the surface of lactate dehydrogenase (LDH) isoenzymes from different species was probed for the presence of metal chelate binding sites. It was demonstrated that the homotetramers (LDH-1)(H4) from rabbit, bovine and pig displayed weak binding to chelated copper whereas the M4-type isoenzymes (LDH-5) bound strongly to this ligand. The binding of the different heterotetramers increases as the number of M-type subunits increases. In contrast, the human isoenzymes are bound to chelated copper in a reversed sequence. The comparison of the affinity partitioning effect of Cu-IDA-PEG in PEG-salt and PEG-dextran systems revealed that the discriminatory effect of copper is promoted by high salt concentrations. Resolution of isoenzymes by multiple extraction using counter-current distribution provides valuable data on the partitioning of enzymes relative to that of the bulk proteins. The efficacy of metal chelate affinity partitioning for the purification of LDH from tissue samples by batchwise extraction was also demonstrated.     

Tobramycin-EDTA conjugate: a noninnocent affinity-cleaving reagent

The design, synthesis, and ribozyme inhibitory activity of a novel EDTA-aminoglycoside conjugate are reported. This affinity cleaving reagent is a noninnocent RNA binder: its RNA affinity, judged by its ability to inhibit the hammerhead ribozyme HH16, is different than the parent natural product and is markedly dependent on the oxidation state of the chelated metal ion.     

Characterization of a second member of the sentrin family of ubiquitin-like proteins

Sentrin is a novel ubiquitin-like protein that can be conjugated to other proteins in a manner analogous to ubiquitination. Two additional cDNA sequences that encode proteins highly homologous to sentrin have been reported to GenBankTM. It is not known whether these sentrin-like proteins could also function as protein modifiers. In this report, a second member of the sentrin family was characterized in detail. Sentrin-2 is a 95-amino acid polypeptide that is 46% identical and 66% homologous to sentrin-1. Northern blot analysis showed that the sentrin-2 message was expressed in all tissues, but was barely detectable in the liver and placenta. The ability of sentrin-2 to conjugate to other proteins was tested by expressing hemagglutinin epitope-tagged sentrin-2 in COS cells. Western blot analysis showed that sentrin-2 could be transferred to other proteins in a pattern similar to that of sentrin-1 conjugation and had similar C-terminal processing. We further showed that both sentrin-1 and sentrin-2 could covalently modify RanGAP1, a Ran GTPase-activating protein critically involved in nuclear transport. Immunocytochemical analysis showed that sentrin-2 derivatives were highly enriched in the nucleus. Taken together, our results demonstrate that sentrin-2 is another protein modifier for the sentrinization pathway.     

A protein conjugation system essential for autophagy

Autophagy is a process for the bulk degradation of proteins, in which cytoplasmic components of the cell are enclosed by double-membrane structures known as autophagosomes for delivery to lysosomes or vacuoles for degradation. This process is crucial for survival during starvation and cell differentiation. No molecules have been identified that are involved in autophagy in higher eukaryotes. We have isolated 14 autophagy-defective (apg) mutants of the yeast Saccharomyces cerevisiae and examined the autophagic process at the molecular level. We show here that a unique covalent-modification system is essential for autophagy to occur. The carboxy-terminal glycine residue of Apg12, a 186-amino-acid protein, is conjugated to a lysine at residue 149 of Apg5, a 294-amino-acid protein. Of the apg mutants, we found that apg7 and apg10 were unable to form an Apg5/Apg12 conjugate. By cloning APG7, we discovered that Apg7 is a ubiquitin-E1-like enzyme. This conjugation can be reconstituted in vitro and depends on ATP. To our knowledge, this is the first report of a protein unrelated to ubiquitin that uses a ubiquitination-like conjugation system. Furthermore, Apg5 and Apg12 have mammalian homologues, suggesting that this new modification system is conserved from yeast to mammalian cells.     

Safety of revaccination with pneumococcal polysaccharide vaccine

Macromolecular conjugates of Gd3+-diethylenetriaminepentaacetic acid with dextran were synthesized from dextran 40 (about 40 kg/mol). Diethylenetriaminepentaacetic acid (DTPA) was coupled to aminated dextran by means of a watersoluble carbodiimide and macromolecular conjugates containing DTPA ratios as high as 1.25 mmol/g of polymer were obtained. First, it was found that the polymer had a favourable influence on relaxivity, as at 20 MHz, the r1 longitudinal relaxivity of the Gd3+-complexed macromolecular conjugates was 2 to 4 times as great as that of free GdDTPA2-, depending on the DTPA content. Second, r1 greatly increased with the increase in the conjugate DTPA content, from 7.4 to 15.9 mM-1s-1 for an increase in the DTPA content from 0.36 to 0.96 mmol/g. Further increase in the ligand content had no more effect on relaxivity.     

A novel protein modification pathway related to the ubiquitin system

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.     

Eosinophil accumulation induced by human interleukin-8 in the guinea-pig in vivo

Interleukin-8 (IL-8) is a neutrophil chemoattractant cytokine. Initially IL-8 appeared to exhibit specificity for neutrophils over other cells of the immune system. However, several recent studies have shown that this mediator can also activate other leucocyte types in vitro. In this study we have used an in vivo model of local [111In]leucocyte accumulation in the guinea-pig and an in vitro assay of leucocyte activation (changes in cytosolic-free Ca2+) to investigate the eosinophil chemoattractant activity of IL-8. The intradermal injection of recombinant human (rh)IL-8 induced a dose-dependent accumulation of intravenously administered [111In]eosinophils into the skin sites over 4 hr. Time-course experiments revealed that this cell infiltration was delayed in onset, occurring between 1 and 2 hr after injection of IL-8. The delay may indicate that IL-8 operates via an indirect mechanism. In contrast, eosinophil accumulation induced by the complement fragment C5a occurred within the first hour following injection. Other human cytokines, IL-1, IL-3, IL-5, tumour necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), were not eosinophil chemoattractants in this in vivo test system. Direct activation of eosinophils by IL-8 was demonstrated in vitro by a transient elevation in cytoplasmic-free Ca2+ levels where it was less potent than either rhC5a or leukotriene B4 (LTB4). Experiments using [111In]neutrophils in vivo indicated that rhIL-8 and rhC5a were similar in potency in inducing local neutrophil infiltration into guinea-pig skin. The demonstration of the eosinophil chemoattractant activity of IL-8 in vivo raises the possibility that this cytokine, or a structurally related molecule, contributes towards eosinophil infiltration in a number of inflammatory conditions such as asthma, helminthic infections and adult respiratory distress syndrome.