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The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in smalltrans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.

The serine/threonine kinase p38 is a ubiquitous, highly conserved, stress responsive, signal-transducing enzyme. It regulates the production of proinflammatory mediators and is the target of the cytokine synthesis inhibitory pyridinylimidazoles. We have expressed human p38 in Drosophila S2 cells and characterized preparations of mixed unphosphorylated/monophosphorylated (inactive) and homogeneously diphosphorylated (active) forms of the enzyme. We observed that only the active preparation of the enzyme has significant kinase activity when assayed using an ATF2-GST fusion protein as the substrate. We determined that the value of KM[ATP] in this reaction is 25 microM and that the pyridinylimidazole inhibitor of p38 kinase activity, SB203580, competes with ATP. We have found that a tritiated pyridinylimidazole, SB202190, has an equal affinity for both the active and inactive forms of the enzyme and that SB203580 competes with it equally well for binding to either form of the enzyme. However, ATP can compete with the tritiated inhibitor for binding to only the active form of the enzyme. Further, we demonstrate in vivo that at concentrations consistent with its IC50 as a cytokine inhibitor, SB203580 can inhibit stimulus-induced phosphorylation of p38 at the Thr-Gly-Tyr activation motif. Our observations suggest that pyridinylimidazoles may block the biological activity of p38 kinase by binding to the inactive form of p38 and reducing its rate of activation. Under these conditions, ATP would not effectively compete with the inhibitors in vivo.

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37°C and contained 1.15 ± 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret λmax at 396 nm with a shoulder at 460 nm and contained 0.28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 ± 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.

The destruction of articular cartilage in immune inflammatory arthritic disease involves the proteolytic degradation of its extracellular matrix. The role of activated matrix metalloproteinases (MMPs) in the chondrodestructive process was studied by identifying a selective cleavage product of aggrecan in murine arthritis models initiated by immunization with either type II collagen or proteoglycan. We conducted semiquantitative immunocytochemical studies of VDIPEN341 using a monospecific polyclonal antibody requiring the free COOH group of the COOH-terminal Asn for epitope detection. This antibody recognizes the aggrecan G1 domain fragment generated by MMP [i.e., stromelysin (SLN) or gelatinase A] cleavage of aggrecan between Asn341-Phe342 but does not recognize intact aggrecan. VDIPEN was undetectable in normal mouse cartilage but was observed in the articular cartilage (AC) of mice with collagen-induced arthritis 10 d after immunization, without histological damage and clinical symptoms. This aggrecan neoepitope was colocalized with high levels of glycosaminoglycans (GAGs) in pericellular matrices of AC chondrocytes but was not seen at the articular surface at this early time. Digestion of normal (VDIPEN negative) mouse paw cryosections with SLN also produced heavy pericellular VDIPEN labeling. Computer-based image analysis showed that the amount of VDIPEN expression increased dramatically by 20 d (70% of the SLN maximum) and was correlated with GAG depletion. Both infiltration of inflammatory cells into the synovial cavity and early AC erosion were also very prominent at this time. Analysis of adjacent sections showed that both induction of VDIPEN and GAG depletion were strikingly codistributed within sites of articular cartilage damage. Similar results occurred in proteoglycan-induced arthritis, a more progressive and chronic model of inflammatory arthritis. These studies demonstrate for the first time the MMP-dependent catabolism of aggrecan at sites of chondrodestruction during inflammatory arthritis.

A rat chondrosarcoma cell line and primary bovine chondrocytes have been used to study cell-mediated aggrecan catabolism. Addition of 1 microM retinoic acid to chondrosarcoma cultures resulted in aggrecan proteolysis with the release of greater than 90% of the cell layer aggrecan into the medium within 4 days. NH2-terminal sequencing of chondroitin sulfate-substituted catabolic products gave a single major NH2-terminal sequence of ARGNVILTXK, initiating at Ala374. This showed that the proteinase, commonly referred to as "aggrecanase," which cleaves the Glu373-Ala374 bond of the interglobular domain of aggrecan (Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R. (1990) J. Biol. Chem. 266, 8683-8685), is active in this cell system. Aggrecan G1 domain, generated by cleavage of the interglobular domain, was also liberated during catabolism and this was characterized with three antipeptide antisera. Anti-CDAGWL was used as a general probe for G1 domain. Anti-FVDIPEN was used to specifically detect G1 domain with COOH terminus of Asn341, the form which is readily generated by cleavage of aggrecan by a wide range of matrix metalloproteinases. Anti-NITEGE antiserum was used to specifically detect G1 domain with COOH terminus of Gln373, the form which is the expected product of "aggrecanase"-mediated cleavage of aggrecan. Western blot analysis indicated that a single form of G1 domain of about 60 kDa was formed. G1 domain of this size reacted with both anti-CDAGWL and anti-NITEGE but not with anti-FVDIPEN. Similar experiments with primary bovine chondrocyte cultures, treated with either retinoic acid or interleukin 1, showed that two forms of catabolic G1 domain, of about 62 and 66 kDa, were formed. Both of these forms reacted on Western blots with anti-CDAGWL and also with anti-NITEGE. It is suggested that cell-mediated catabolism of the aggrecan interglobular domain in these culture systems, whether promoted by retinoic acid or interleukin 1, primarily involves cleavage of the Glu373-Ala374 bond by aggrecanase. The accumulation of G1 domain with a COOH-terminal of Glu373 shows that such aggrecanase-mediated cleavage can occur independent of the cleavage of the Asn341-Phe342 bond by matrix metalloproteinases.

FK-506, a potent immunosuppressive drug, acts during the commitment phase of T-lymphocyte activation to block a subset of calcium-associated events necessary for transcription of certain early lymphokine genes. The drug binds to an abundant, cytosolic 11.8-kDa protein termed the FK-506-binding protein (FKBP12). The FKBP12.FK-506 complex inhibits calcineurin, a calcium-dependent phosphatase that is a component of the signal transduction pathway leading to early lymphokine gene transcription. FKBP12 is one member of a growing gene family. Prior to this report, all other FKBP family members had been irrelevant to the mechanism of action of FK-506 because no other FKBP.FK-506 complexes were able to bind and inhibit calcineurin. Here, we report the purification and characterization of a novel FK-506-binding protein, FKBP12.6. Having 85% amino acid sequence identity to FKBP12, FKBP12.6 is, among the FKBPs, most closely related to FKBP12. When complexed with FK-506, FKBP12.6 binds to and inhibits calcineurin, making it only the second FKBP discovered thus far to do so. The ability to inhibit calcineurin establishes the potential relevance of FKBP12.6 to the immunosuppressive or toxic side effects of FK-506.

The report by Zimmerman et al. (J. Biol. Chem. 257, 1982, 6529-6536) that the active site of Escherichia coli leader peptidase (LPase) is located in the periplasm led us to explore the possibility that soluble, active short forms of LPase could be produced. Detergent free Δ2-75 mutant protein (LPase-sf) lacking the two N-terminal transmembrane spanning and the cytoplasmic domains was produced in high yield. The mass of the protein determined by electrospray ionisation mass spectrometry was 27,952 amu. The increase of 42 amu over that predicted by the expected amino acid sequence indicates that the N-terminus of LPase-sf is acetylated. This is consistent with the inability to obtain an N-terminal sequence. LPase-sf lacks the site of autolysis present in LPase, thus circumventing problems associated with enzyme autocatalytic instability. LPase-sf and wild type LPase displayed comparable activity versus two peptide substrates. The peptides, based upon the cleavage sites of procoat and the precursor of maltose binding protein, were processed at the expected sites. In addition, the activity of both LPase′s was not inhibited by classical inhibitors of the four classes of proteases. LPase-sf displayed similar activity in the presence and absence of detergent. Wild type LPase displayed specificity for alanine in the P1 subsite of the peptide WSASALX*KI and did not hydrolyse peptides with glycine, valine, or serine in that position. The availability of a detergent-free active form of LPase should facilitate mechanistic and structural studies.

The stimulation of human blood with a Ca2+ ionophore, A23187, leads to activation of polymorphonuclear leucocytes (PMN) with release of small amounts of catalyticaly active elastase, as demonstrated by the formation of a characteristic product, the N-terminal A alpha (1-21) peptide of the Aa subunit of fibrinogen. The identity of the peptide was initially established by radioimmunoassay (r.i.a.) with an antibody raised to A alpha (1-21). We now provide independent confirmation of the formation of A alpha (1-21) by fast-atom-bombardment-m.s. analysis of the fractions separated chromatographically after spiking of plasma samples with peptide labelled with [2H8]Phe at position 8. Identity of the peptides was established on the basis of their chromatographic retention time and by the distinct peaks in the mass spectra of these fractions. The relative intensities of the molecular ions of natural and labelled peptides were measured. On the basis of a comparison of the peaks of similar intensities, the concentration of the natural peptide at the time of spiking was close (79%) to the amount obtained by r.i.a. An additional peptide, des-alanyl-A alpha (2-21), was also seen. The total amount of material measured by r.i.a. could be accounted for by the sum of these two provides. The addition of label and assay by m.s. has provided an independent physical-chemical method for identifying A alpha (1-21) as a characteristic product of PMN elastase release in whole blood, but which is absent in freshly drawn blood.

A series of substituted 2-aminopyridines was prepared and evaluated as inhibitors of human nitric oxide synthases (NOS). 4,6-Disubstitution enhanced both potency and specificity for the inducible NOS with the most potent compound having an IC50 of 28 nM.

The objective of this study was to compare the specificity and potency of recombinant human SLN-1 (rhSLN) and human leukocyte elastase (HLE) as proteoglycan (PG)-degrading enzymes after intraarticular injection into rabbits. Another objective was to evaluate the elicitation of a rhSLN-induced hyaluronan-binding region (HABR) fragment from rabbit aggrecan in joints using a polyclonal antiserum (anti-FVDIPEN) against the synthetic peptide, Phe-Val-Asp-Ile-Pro-Glu-Asn (FVDIPEN). The intraarticular injection of either activated rhSLN or HLE resulted in enzyme-specific quantitative release of PG fragments into synovial fluid. Based on the criteria used herein, HLE appears to be a more potent PG-degrading enzyme than SLN. Intraarticular injection of rhSLN also resulted in time- and dose-dependent release of a new HABR fragment of aggrecan (HABR-FMDIPEN) into both articular cartilage and synovial fluid. HABR-FVDIPEN is likely to be a good marker of matrix metalloprolcinase (MMP)-induced degradation of aggrecan.

The peptide leukotrienes LTC4, D4 and E4 collectively account for the biological activity known as slow-reacting substance of anaphylaxis (SRS-A). These metabolites of arachidonic acid are thought to play a role in lung pathophysiology. A role in asthma is postulated as a result of their potent bronchoconstrictor activity both in vitro (Dahlen et al. 1980; Drazen et al. 1980; Hanna et al. 1981; Piper et al. 1981; Jones et al. 1982; Peters et al. 1984) and in vivo (Manning et al. 1990; Holroyde et al. 1981; Griffin et al. 1983; Weiss et al. 1982; Barnes et al. 1984). Increased production of leukotrienes has been demonstrated following antigen challenge of the airways of allergic patients in vivo (Creticos et al. 1984; Ishihara et al. 1985; Isono et al. 1985) and in vitro (Dalhen et al. 1983).

Human neutrophil elastase was inactivated by methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane. The modified enzyme was crystallized from 40 mM ammonium phosphate, pH 7.0 in the hexagonal space group P6(3) with unit cell parameters a = 74.53 A, b = 74.53 A, c = 70.88 A, alpha = beta = 90 degrees, gamma = 120 degrees. These crystals were resistant to radiation damage and diffracted beyond 1.84-A resolution. The asymmetric unit contained one 25,000-dalton monomer of human neutrophil elastase. Crystals were also grown from the enzyme modified with the analogous iodinated inactivator, p-iodoanilinosuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane. These crystals proved to be isomorphous with those of methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane-modified human neutrophil elastase, and served as a single-site, heavy atom derivative for solving the tertiary structure of the enzyme.

1. 1. Collagenase and gelatinase of human PMN leukocytes were separated by serial chromatography. 2. 2. The enzymes were shown to be similar in latency, activatability, chromatographic and electrophoretic behavior and the response to inhibitors. 3. 3. They recognize the same peptide linkage for cleavage, only each with a distinct difference in the effect caused by the secondary binding sites of the substrate molecules.

A cationic amino acid copolymer (CP530) with a molar ratio of lysine, leucine, tryptophan and phenylalanine of 11:2:1:1 and a Mr of about 2300 was prepared and its inhibitory effects on the complement cascade was compared with those of polylysine with a Mr of about 3000. The effects by these two cationic peptides appeared to be at the early stage of complement activation. CP530 and polylysine inhibited the binding of C1q to insoluble IgG aggregates with a concentration required for 50% inhibition of 0.7 and 0.9 mM, respectively. Both compounds were also potent inhibitors of immune hemolysis (a concentration causing 50% inhibition, 0.5 and 3.5 microM respectively) as well as assembly of EAC cell intermediates required for formation of C3 and C5 convertases (a concentration of 50% inhibition of 1.0 microM for CP530 and 3.8 microM for polylysine). However, CP530 was shown to be distinctly more effective against the activation of C1r . Cls complex induced by insoluble IgG aggregate-bound C1q, requiring 0.15 mM for 50% inhibition compared to greater than 10 mM for polylysine. The 50% inhibition value for soluble IgG aggregate-induced activation of C1 in whole serum was 0.7 mM for CP530 and 5.0 mM for polylysine. The greater inhibition of C1 activation by CP530 than that exerted by polylysine could be attributable to the presence of non-lysyl residues which provide the structural basis for specificity and potency.

The similarities in the structure and properties of C1q and collagen prompted us to examine the susceptibility of C1q to human polymorphonuclear leukocyte collagenase. Incubation of C1q with a collagenase preparation resulted in no change in (1) the binding of C1q to immunoglobulin aggregates, (2) the hemolytic function of C1q as measured by reconstitution of C1q-depleted serum in immune hemolysis, or (3) the structural properties of C1q as revealed by gel electrophorettic patterns of the whole molecule or its polypeptide chains. In contrast, rapid inactivation and degradation of C1q was caused by leukocyte elastase. The collagenase preparation was, however, capable of cleaving reduced and carboxamidomethylated C1q into discrete fragments. This activity was attributed to a gelatinase present in the enzyme preparation since (1) the cleavage reaction was inhibited by denatured collagen but not by native collagen and (2) a collagenase fraction free of gelatinolytic activity could not degrade reduced and carboxamidomethylated C1q, while a gelatinase fraction devoid of collagenase activity retained the capacity to effect reduced and carboxamidomethylated C1q. Both collagenase and gelatinase activities were activated from the latent form by trypsin, and inhibited by EDTA. Therefore, it appears that native C1q lacks the structural features present in collagen which are recognized by leukocyte collagenase for hydrolytic action even though the denatured molecule still contains that region capable of being cleaved by gelatinase.

Human neutrophil elastase (HNE) has been implicated as a major contributor to tissue destruction in various disease states, including emphysema. The structure of HNE, at neutral pH, in complex with methoxysuccinyl-Ala-Ala-Pro-Ala chloromethyl ketone (MSACK), has been solved and refined to an R factor of 16.4% at 1.84-A resolution. Results are consistent with the currently accepted mechanism of peptide chloromethyl ketone inhibition of serine proteases, in that MSACK cross-links the catalytic residues His-57 and Ser-195. The structure of the HNE-MSACK complex is compared with that of porcine pancreatic elastase in complex with L-647,957, a beta-lactam inhibitor of both elastases. The distribution of positively charged residues on HNE is highly asymmetric and may play a role in its specific association with the underlying negatively charged proteoglycan matrix of the neutrophil granules in which the enzyme is stored.

The binding constants of a number of ligands were measured for pancreatic elastase (PE) and anhydro-elastase (AE) in order to assess the contribution of Ser-195 to substrate and inhibitor binding by PE. AE was purified by affinity chromatography on a column containing immobilized turkey ovomucoid inhibitor. The AE had 0.1 +/- 0.1% of the activity of the native enzyme and contained 0.8 +/- 0.06 residue of dehydroalanine per molecule. A difference electron-density map, derived from an X-ray crystallographic analysis of AE, showed that the modified residue was Ser-195. The complexing of 3-carboxypropionyl-Ala-Ala-Ala-p-nitroanilide (SAN) to the active site of AE was also demonstrated by X-ray-diffraction analysis of an AE crystal soaked overnight with substrate. The nitroanilide moiety was not observed in the difference map. AE was shown to bind turkey ovomucoid inhibitor with a dissociation constant (Kd) of 0.3 +/- 0.06 microM compared with 0.10 microM for PE. The Kd of the AE-SAN complex (0.2 mM) was comparable with the Michaelis constant for SAN with PE (1.0 mM). A number of inhibitors, such as elastatinal, which forms a hemiketal adduct with PE, while others such as the beta-lactams, which function as acylators of the active-site serine residue, bound AE with a lower affinity than to PE. The binding of a peptidylchloromethane (acetyl-Ala-Ala-Pro-Ala-CH2Cl) to AE occurs without evidence for alkylation of histidine. The binding constants for benzoisothiazolinone and 3,4-dichloroisocoumarin to PE differed from their binding constants to AE by less than a factor of 4.0-fold. The contribution of the hydroxy group of Ser-195 to the binding of these inhibitors to PE in their non-covalent complexes is relatively small, even though they inactivate PE by an acylation mechanism. These results suggest that the hydroxy group on Ser-195 in PE is of secondary importance in the energetics of ligand binding, in contrast with its essential role in the catalytic properties of the enzyme.

Nachweis, dass das lokaleShwartzman-Phanomen in Kaninchen durch intradermale oder i.v. Gabe von Pepstatin, einem sauren Proteinase-Hemmer, oder von Sojabohnen-Trypsin-Hemmer kurz vor der auslosenden Injektion von bakteriellem Endotoxin unterdruckt werden kann.

Mixtures of 14C-labelled glucose plus pyruvate were incubated either with rat diaphragm or slices of rat liver. Incorporation of glucose carbon into glycogen was compared with its incorporation into glucose 6-phosphate relative to the incorporation of pyruvate carbon into these metabolic products. There was no preferential incorporation of glucose carbon relative to pyruvate carbon into glycogen compared with glucose 6-phosphate in the liver slices, but there was in diaphragm. On the assumption that glucose 6-phosphate is a necessary intermediate in the conversion of pyruvate carbon into glycogen, this is evidence for the existence in muscle, but not in liver, of more than one pool of glucose 6-phosphate or of a pathway from glucose to glycogen without glucose 6-phosphate as an intermediate. Galactose carbon, relative to pyruvate carbon, was preferentially incorporated into liver glycogen, so that a substrate converted in liver into glycogen without glucose 6-phosphate as an intermediate could be detected by this approach.

A simple radioisotopic assay for the proteolytic enzyme activity with methyl- 14 C-glycinated bovine hemoglobin as the substrate has been developed. The radioactive hemoglobin was prepared by the reaction of hemoglobin with a water-soluble carbodiimide and methyl [ 14 C]glycinate in 5 M guanidine hydrochloride.

[1- 14 C]fructose and [6- 14 C]fructose were incubated with liver slices and injected intraportally into rats. Distributions of 14 C in the carbons of glucose, the glucose of glucose-6-P, and the glucose unit of glycogen from the slices and from the livers of the rats were determined. With both [1- 14 C]fructose and [6- 14 C]fructose the distribution of 14 C in the glucose unit of glycogen was the same as in glucose-6- P , in accord with glucose-6- P being an obligate intermediate in the conversion of fructose to glycogen. With [6- 14 C]fructose the distribution in glucose was also similar to that of glucose-6- P , in accord with a metabolism involving glucose-6- P . With [1- 14 C]fructose there was a difference in distribution between glucose and glucose-6- P , but the difference was less than the difference reported by Muntz (1) and its significance is not certain. Except for these results with [1- 14 C]fructose, the data are not in accord with the existence of a pathway for the conversion of fructose to glucose without glucose-6- P as intermediate.

A definite contribution of the glucuronic acid pathway to total glucose metabolism by rat adipose tissue in vitro in the presence of growth hormone preparations has not been found. Randomization of 14C of glucose-5-14C into glycerol and glucose-1-14C and -2-14C into glycogen on incubation of rat epididymal tissue indicates that the pathway contributes at most only a few per cent to the over-all metabolism of glucose. Results of incubations with glucose-1-14C and -6-14 are in accord with metabolism solely via the pentose cycle, Embden-Meyerhof pathway, and Krebs cycle. Thus, essentially all of the 14C utilized on incubation of glucose-1-14C and -6-14C is isolated in products formed via these pathways. Nonequilibration of dihydroxyacetone phosphate with glyceraldehyde 3-phosphate occurs, and this results in a preferential oxidation to CO2 of carbon 6 as compared to carbon 1 via the Krebs cycle. This coupled with an increase in the fraction of carbon 3 of pyruvate converted to CO2 and a decreased pentose cycle contribution in the presence compared to the absence of the preparations can account for the enhanced yields of 14CO2 from glucose-6-14C as compared to -1-14C that have been reported on growth hormone addition.

The contribution of the pentose and Krebs cycles to glucose metabolism has been determined in the isolated goosefish islet, in vitro, using glucose-2-C-14. Hyperglycemia caused increased net glucose metabolism by both the pentose and Krebs cycles, while glucagon and tolbutamide had no significant effect on net glucose metabolism by either pathway. The rate of reduced pyridine nucleotide formation was calculated and was increased with hyperglycemia but not with tolbutamide or glucagon.

Summary Objective To analyze the roles of two classes of proteinases, ‘aggrecanase', and matrix metalloproteinases (MMPs), in chondrodestruction during murine collagen-induced arthritis (CIA). Methods Generation of the ‘aggrecanase' neo-epitope (NITEGE 373 ), and the MMP neo-epitope (VDIPEN 341 ) within aggrecan was studied by immunoperoxidase microscopy using specific anti-peptide antibodies in normal and stromelysin-1 (SLN-1) deficient knockout mice with CIA. Results High levels of NITEGE 373 and VDIPEN 341 neo-epitopes were observed in foci within CIA paw articular cartilage exhibiting depletion of glycosaminoglycans, in advance of significant cartilage erosion. The highest concentrations of NITEGE 373 and VDIPEN 341 labeling were observed and often co-distributed in the chondrocyte pericellular matrix, suggesting that stimulated chondrocytes can synthesize and/or activate both enzymes. Other regions of the cartilage frequently exhibited either NITEGE 373 or VDIPEN 341 labeling, but not both neo-epitopes simultaneously, suggesting that ‘aggrecanase' and MMP cleavages of aggrecan may be generated independently. No detectable differences were observed in expression or distribution of either neo-epitope in SLN-1 knockout versus wild-type mice. In addition, in vitro digestion of joint sections with SLN-1 did not alter the expression of cartilage NITEGE 373 , while markedly increasing VDIPEN 341 labeling. Peripheral nerves and brains of naive mice also exhibited intense anti-NITEGE 373 labeling. Conclusions These data indicate that NITEGE 373 and VDIPEN 341 aggrecan neo-epitopes are sensitive and specific markers of early joint pathology, and are consistent with the hypothesis that SLN-1 does not have ‘aggrecanase' activity, and that ‘aggrecanase' is distinct from the MMPs which cleave aggrecan at the MMP site.

l-Lactate-3-14C, dl-lactate-2-14C, or pyruvate-2-14C were injected into nine human subjects, and 1 hour later glucose from their blood was isolated and degraded. The distributions of 14C in the glucoses are in accord with metabolism via the Krebs cycle where the carbons of lactate and pyruvate are extensively randomized, and the Embden-Meyerhof pathway with conversion to glucose by condensation of triose phosphates in isotopic nonequilibrium. The results differ from those previously reported for man, but are essentially in accord with observations in animals. New degradations of glucoses isolated in the experiments in man previously reported, yielded distributions similar to those of the present study.

The continuing discovery and development of beta-lactams as antibiotics has had an unparalleled impact on the overall health and well-being of society. Recently, appropriately substituted cephalosporins were shown to be potent inhibitors of elastase, suggesting a novel therapeutic role for the beta-lactams in the control of emphysema and other degenerative diseases. We have now solved and partially refined at atomic resolution the structure of a complex of porcine pancreatic elastase with the time-dependent irreversible inhibitor 3-acetoxymethyl-7-alpha-chloro-3-cephem-4-carboxylate-1,1-dioxide tert-butyl ester (I), the most potent of the beta-lactam elastase inhibitors yet reported. (Porcine pancreatic elastase is a close relative of the desired drug target, human polymorphonuclear leukocyte elastase.) A mechanism of action is presented, based on the structure and on biochemical evidence (T.-Y.L. et al., in preparation), which clarifies the operational similarities and differences between beta-lactam elastase inhibitors and antibiotics. Features of the reaction include the expulsion of a leaving group at the cephalosporin 3' position and the formation of two covalent bonds with the active site of porcine pancreatic elastase at residues Ser 195 and His 57.

Pyruvate-1-14C, pyruvate-2-14C and pyruvate-3-14C, and unlabeled pyruvate in the presence of 14CO2, were incubated in a bicarbonate buffer with rat epididymal adipose tissue. Incorporation of 14C into CO2, glycerol and fatty acids of triglycerides, lactate and glycogen were measured and the glycerol and lactate were degraded to yield the relative activities of 14C in each of their carbons. The results are in accord with metabolism via the Embden-Meyerhof Pathway and Krebs Cycle. The dicarboxylic acid shuttle, involving conversion of pyruvate to phosphoenolpyruvate via CO2 fixation with oxaloacetate as an intermediate, and equilibration with symmetrical dicarboxylic acids of the Krebs Cycle, occurs in adipose tissue and plays a major role in the distribution of the carbons of pyruvate.