Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed on cancer cell membranes and activates the zymogen of MMP-2 (gelatinase A). We have recently isolated MT1-MMP complexed with tissue inhibitor of metalloproteinases 2 (TIMP-2) and demonstrated that MT1-MMP exhibits gelatinolytic activity by gelatin zymography (Imai, K., Ohuchi, E., Aoki, T., Nomura, H., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1996) Cancer Res. 56, 2707-2710). In the present study, we have further purified to homogeneity a deletion mutant of MT1-MMP lacking the transmembrane domain (DeltaMT1) and native MT1-MMP secreted from a human breast carcinoma cell line (MDA-MB-231 cells) and examined their substrate specificities. Both proteinases are active, without any treatment for activation, and digest type I (guinea pig), II (bovine), and III (human) collagens into characteristic 3/4 and 1/4 fragments. The cleavage sites of type I collagen are the Gly775-Ile776 bond for alpha1(I) chains and the Gly775-Leu776 and Gly781-Ile782 bonds for alpha2(I) chains. DeltaMT1 hydrolyzes type I collagen 6.5- or 4-fold more preferentially than type II or III collagen, whereas MMP-1 (tissue collagenase) digests type III collagen more efficiently than the other two collagens. Quantitative analyses of the activity of DeltaMT1 and MMP-1 indicate that DeltaMT1 is 5-7.1-fold less efficient at cleaving type I collagen. On the other hand, gelatinolytic activity of DeltaMT1 is 8-fold higher than that of MMP-1. DeltaMT1 also digests cartilage proteoglycan, fibronectin, vitronectin and laminin-1 as well as alpha1-proteinase inhibitor and alpha2-macroglobulin. The activity of DeltaMT1 on type I collagen is synergistically increased with co-incubation with MMP-2. These results indicate that MT1-MMP is an extracellular matrix-degrading enzyme sharing the substrate specificity with interstitial collagenases, and suggest that MT1-MMP plays a dual role in pathophysiological digestion of extracellular matrix through direct cleavage of the substrates and activation of proMMP-2.

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Kikuchi et al. Reply: In the preceding Comment [1], Gu and Su (GS) reported the finite temperature transfer matrix renormalization group (TMRG) method results for the distorted diamond chain (DDC) model. They pointed out that the double-peak behavior of T found in experiment cannot be reproduced by our parameter set J 1 :J 2 :J 3 1:1:25:0:45 [2], but well fitted by J 1 :J 2 :J 3z 1:1:9: ÿ 0:3 with J 3x =J 3z J 3y =J 3z 1:7.In response to GS's Comment, we have performed the additional density matrix renormalization group (DMRG) and the exact diagonalization calculations for the magnetization curve MH at T 0 of the DDC model with GS's parameter set. As can be seen from Fig. 1, the DMRG MH curve with GS's parameter set does not well explain the experimental results.The positional relations between Cu 2 ions corresponding to J 1 and J 3 are very similar to each other as can be seen in the schematic view of the crystal structure of Cu 3 CO 3 2 OH 2 in Fig. (1b) of our previous Letter [2]. The distance of two Cu 2 ions corresponding to J 1 is 327.5 pm with bond angle 113.7 and that to J 3 is 329.0 pm with bond angle 113.5 . Thus it is unlikely that J 1 is antiferromagnetic without the XXZ anisotropy while J 3 is ferromagnetic with strong XXZ anisotropy. Further, as far as we know, such a strong XXZ anisotropy has not been observed at all in the S 1=2 spin systems of Cu 2 ions.The double-peak behaviors of T and CT are not necessarily attributed to the frustration effect. The mechanism for the double-peak behaviors will be as follows. In the case of J 2 J 1 , jJ 3 j as lowering the temperature, spins coupled by J 2 are going to form singlet dimers at first. The remaining spins are nearly free because they are separated PRL 97,

The genome of influenza A virus is comprised of eight viral RNA (vRNA) segments. Although the products of all eight vRNA segments must be present for viral replication, little is known about the mechanism(s) responsible for incorporation of these segments into virions. Two models have been proposed for the generation of infectious virions containing eight vRNA segments. The randomincorporation model assumes a common structural feature in all the vRNAs, enabling any combination of vRNAs to be incorporated randomly into virions. The selective-incorporation model predicts the presence of specific structures in each vRNA segment, leading to the incorporation of a set of eight vRNA segments into virions. Here we demonstrate that eight different vRNA segments must be present for efficient virion formation and that sequences within the coding region of (and thus unique to) the neuraminidase vRNA possess a signal that drives incorporation of this segment into virions. These findings indicate a unique contribution from individual vRNA segments and thus suggest a selective (rather than random) mechanism of vRNA recruitment into virions. The neuraminidase vRNA incorporation signal and others yet to be identified should provide attractive targets for the attenuation of influenza viruses in vaccine production and the design of new antiviral drugs.T he influenza A virus genome consists of eight segments of single-stranded RNA with negative polarity (complementary to mRNA). Each viral RNA (vRNA) segment resides within a complex [the viral ribonucleoprotein complex (vRNP)] of nucleoprotein and three polymerase subunits designated PA, PB1, and PB2. After transport of M1 and NS2 proteins into the nucleus, vRNPs formed in this compartment are exported to the cytoplasm, where presumably they interact with viral membraneassociated proteins including hemagglutinin (HA), neuraminidase (NA), and M1 to ensure the correct assembly of virions and their release from the host cell. Although all eight vRNPs must be present for efficient viral replication, little is known about the mechanism(s) responsible for incorporation of vRNA segments into virions and their stable maintenance during repeated cycles of replication.Two models have been proposed for the generation of infectious virions containing eight vRNA segments. The randomincorporation model assumes a common structural feature in all the vRNPs, enabling them to be incorporated randomly into virions. Support for this model comes from the observation that an influenza A virion can possess more than eight vRNPs (1). The selective-incorporation model predicts the presence of specific structures in each vRNA segment, leading to their individual incorporation into virions. This hypothesis was suggested by data showing that the presence of excess amounts of internally deleted segments encoding polymerase proteins resulted in a corresponding reduction of full-length segments in virions (2, 3).Air and coworkers (4-6) produced an influenza A virus with a large internal deletion in the NA vRNA s...

Hematopoietic prostaglandin (PG) D synthase is the key enzyme for production of the D and J series of prostanoids in the immune system and mast cells. We isolated a cDNA for the rat enzyme, crystallized the recombinant enzyme, and determined the three-dimensional structure of the enzyme complexed with glutathione at 2.3 A resolution. The enzyme is the first member of the sigma class glutathione S-transferase (GST) from vertebrates and possesses a prominent cleft as the active site, which is never seen among other members of the GST family. The unique 3-D architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism, responsible for the specific isomerization from PGH2 to PGD2.

At the final step in viral replication, the viral genome must be incorporated into progeny virions, yet the genomic regions required for this process are largely unknown in RNA viruses, including influenza virus. Recently, it was reported that both ends of the neuraminidase (

The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3 coding region are critical for efficient NS vRNA incorporation and that deletion of the 3 segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3 NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3 end) form a structure that is required for efficient influenza A virus vRNA packaging.The genetic information that characterizes all life systems must be transmitted to each generation. For viruses, this is achieved by amplification of the genome within cells and its subsequent packaging into progeny virions. The influenza A virus genome is fragmented into eight single-strand negativesense RNA segments that encode at least 11 proteins (3, 10). These viral RNAs (vRNAs) form the ribonucleoprotein (RNP) complex with heterotrimeric viral polymerase subunit proteins (PB2, PB1, and PA) and nucleoprotein (NP) (10). After introduction into cells via receptor-mediated endocytosis, the viral RNPs are transported into the nucleolus, where viral mRNAs are transcribed and translated into viral proteins (10). The vRNAs are replicated and, together with the viral proteins, form the RNP complexes (10), which are then exported to the cytoplasm in a CRM-1-dependent manner (4,14,15,18,21,26). The final step is packaging of the RNP complexes into progeny virions at the plasma membrane (10).Influenza A virus RNA contains noncoding regions at both ends of its coding region (10). These noncoding regions are composed of sequences that are conserved among eight vRNAs (U13 and U12) as well as sequences specific for each segment that are located inside the terminal noncoding region (10).These noncoding regions regulate replication of the viral genome and transcription (22). Luytjes et al. (13) generated an artificial viral RNA comprising the chloramphenicol acetyltransferase (CAT) gene flanked by the noncoding regions of an NS segment. They showed that this RNA was incorporated into influenza virus virions, using the CAT activity of virusinfected cells as an indicator of segment incorporation. However, the CAT activity gradually decreased upon passaging. Recently, we showed that both ends of th...

Brevican is a member of the lectican family of chondroitin sulfate proteoglycans that is predominantly expressed in the central nervous system. The susceptibility of brevican to digestion by matrix metalloproteinases (MMP-1, -2, -3, -7, -8, -9, -10, and -13 and membrane type 1 and 3 MMPs) and aggrecanase-1 (ADAMTS4) was examined. MMP-1, -2, -3, -7, -8, -10, and -13 degraded brevican into a few fragments with similar molecular masses, whereas the degradation products of aggrecanase-1 had apparently different sizes. NH 2 -terminal sequence analyses of the digestion fragments revealed that cleavages of the brevican core protein by these metalloproteinases occurred commonly within the central non-homologous domain. MMP-1, -2, -3, -7, -8, -10, and -13 preferentially attacked the Ala