The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix

We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti–MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the α2 integrin subunit but not by antibodies against the α1 or α3 subunits. We propose that interaction of the α2β1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.     

Serine racemase modulates intracellular D-serine levels through an alpha,beta-elimination activity

Mammalian brain contains high levels of d-serine, an endogenous co-agonist of N-methyl D-aspartate type of glutamate receptors. D-Serine is synthesized by serine racemase, a brain enriched enzyme converting L- to D-serine. Degradation of D-serine is achieved by D-amino acid oxidase, but this enzyme is not present in forebrain areas that are highly enriched in D-serine. We now report that serine racemase catalyzes the degradation of cellular D-serine itself, through the alpha,beta-elimination of water. The enzyme also catalyzes water alpha,beta-elimination with L-serine and L-threonine. alpha,beta-Elimination with these substrates is observed both in vitro and in vivo. To investigate further the role of alpha,beta-elimination in regulating cellular D-serine, we generated a serine racemase mutant displaying selective impairment of alpha,beta-elimination activity (Q155D). Levels of D-serine synthesized by the Q155D mutant are several-fold higher than the wild-type both in vitro and in vivo. This suggests that the alpha,beta-elimination reaction limits the achievable D-serine concentration in vivo. Additional mutants in vicinal residues (H152S, P153S, and N154F) similarly altered the partition between the alpha,beta-elimination and racemization reactions. alpha,beta-Elimination also competes with the reverse serine racemase reaction in vivo. Although the formation of L- from D-serine is readily detected in Q155D mutant-expressing cells incubated with physiological D-serine concentrations, reversal with wild-type serine racemase-expressing cells required much higher D-serine concentration. We propose that alpha,beta-elimination provides a novel mechanism for regulating intracellular D-serine levels, especially in brain areas that do not possess D-amino acid oxidase activity. Extracellular D-serine is more stable toward alpha,beta-elimination, likely due to physical separation from serine racemase and its elimination activity.     

Bovine brain cathepsin D: inhibition by pepstatin and binding to concanavalin A.

1. Cathepsin D from bovine brain has been purified 1100-fold in 46% recovery. Three isozymes are present with pI (+/- 0.05) = 6.10, 6.30 and 6.40. 2. The isozymes are single polypeptide chains with apparent Mr = 42,000 and are similar with respect to substrate binding and cleavage; the pH-optimum is 3.5 with virtually no activity at neutral pH. 3. Pepstatin inhibits the enzyme and kinetic data are consistent with a "tight binding" mechanism. 4. The dissociation constant for the concanavalin A-enzyme complex is Kd = 19 nM at pH 5.0. 5. Under conditions where 90% of the enzyme is bound to soluble concanavalin A, full enzymatic activity is observed.     

Characteristic features of temperature evolution in ultracold plasmas

The theoretical interpretation of recent experiments on the time evolution of the temperature in freely expanding, ultracold plasma clouds released from a magneto-optical trap is discussed. The most interesting result of those experiments was the asymptotic behavior T _e ∝ t ^{−(1,2±0.1)}, instead of the behavior proportional to t ⁻², which was expected for a rarefied monatomic gas in the inertial expansion stage. It is shown that such a substantially slower temperature fall can be well explained by the specific properties of the equation of state of ultracold plasma with a large Coulomb coupling parameter; whereas the heat release in inelastic processes (in particular, three-body recombination) turns out to be relatively unimportant in the first approximation. This conclusion is confirmed by approximate analytic estimates from the model of virializing the energies of charged particles and also by the results of ab initio computer simulations; moreover, the computations demonstrate that the law of decrease in the electron temperature is established very rapidly, when the virialization criterion begins to be satisfied only to within a factor of order unity.     

Effects of microwaves on the puffing pattern of <Emphasis Type="Italic">D. melanogaster</Emphasis>

The influence of electromagnetic field exposure on puffing pattern of salivary gland polythene chromosomes, viability and fertility of Drosophila melanogaster of the wild type Canton-S line was studied. Experimental conditions: Electromagnetic field characteristics: frequency — 36.64 GHz, power density — 0.4 W/m², exposure time −10 seconds. Electromagnetic field exposure was conducted on the egg stage. Results: in larvae developed from the exposed eggs 3 of 8 chromosomal puffs tested (71CE, 82EF, and 83E) had significantly smaller dimensions than these in control at the prepupal stage. Viability of Drosophila estimated by the number of adult flies hatched from exposed eggs decreased, while the number of dominant lethal mutations increased. Conclusion: the exposure to a low-level microwave irradiation suppressed puffing activity at ecdysone-inducible loci of Drosophila polythene chromosomes, increased frequency of dominant lethal mutations and decreased Drosophila viability but did not influence Drosophila fertility.     

Nuclear Compartmentalization of Serine Racemase Regulates d-Serine Production: IMPLICATIONS FOR N-METHYL-d-ASPARTATE (NMDA) RECEPTOR ACTIVATION*

d-Serine is a physiological co-agonist that activates N-methyl d-aspartate receptors (NMDARs) and is essential for neurotransmission, synaptic plasticity, and behavior. d-Serine may also trigger NMDAR-mediated neurotoxicity, and its dysregulation may play a role in neurodegeneration. d-Serine is synthesized by the enzyme serine racemase (SR), which directly converts l-serine to d-serine. However, many aspects concerning the regulation of d-serine production under physiological and pathological conditions remain to be elucidated. Here, we investigate possible mechanisms regulating the synthesis of d-serine by SR in paradigms relevant to neurotoxicity. We report that SR undergoes nucleocytoplasmic shuttling and that this process is dysregulated by several insults leading to neuronal death, typically by apoptotic stimuli. Cell death induction promotes nuclear accumulation of SR, in parallel with the nuclear translocation of GAPDH and Siah proteins at an early stage of the cell death process. Mutations in putative SR nuclear export signals (NESs) elicit SR nuclear accumulation and its depletion from the cytosol. Following apoptotic insult, SR associates with nuclear GAPDH along with other nuclear components, and this is accompanied by complete inactivation of the enzyme. As a result, extracellular d-serine concentration is reduced, even though extracellular glutamate concentration increases severalfold. Our observations imply that nuclear translocation of SR provides a fail-safe mechanism to prevent or limit secondary NMDAR-mediated toxicity in nearby synapses.     

An Ultra-specific Avian Antibody to Phosphorylated Tau Protein Reveals a Unique Mechanism for Phosphoepitope Recognition

Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K_D of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a “bowl-like” conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.     

Pro-collagenase-1 (matrix metalloproteinase-1) binds the alpha(2)beta(1) integrin upon release from keratinocytes migrating on type I collagen

In injured skin, collagenase-1 (matrix metalloproteinase-1 (MMP-1)) is induced in migrating keratinocytes. This site-specific expression is regulated by binding of the alpha(2)beta(1) integrin with dermal type I collagen, and the catalytic activity of MMP-1 is required for keratinocyte migration. Because of this functional association among substrate/ligand, receptor, and proteinase, we assessed whether the integrin also directs the compartmentalization of MMP-1 to its matrix target. Indeed, pro-MMP-1 co-localized to sites of alpha(2)beta(1) contacts in migrating keratinocytes. Furthermore, pro-MMP-1 co-immunoprecipitated with alpha(2)beta(1) from keratinocytes, and alpha(2)beta(1) co-immunoprecipitated with pro-MMP-1. No other MMPs bound alpha(2)beta(1), and no other integrins interacted with MMP-1. Pro-MMP-1 also provided a substrate for alpha(2)beta(1)-dependent adhesion of platelets. Complex formation on keratinocytes was most efficient on native type I collagen and reduced or ablated on denatured or cleaved collagen. Competition studies suggested that the alpha(2) I domain interacts with the linker and hemopexin domains of pro-MMP-1, not with the pro-domain. These data indicate that the interaction of pro-MMP-1 with alpha(2)beta(1) confines this proteinase to points of cell contact with collagen and that the ternary complex of integrin, enzyme, and substrate function together to drive and regulate keratinocyte migration.