An efficient method for computing steady state solutions with Gillespie's direct method.

Gillespie's direct method is a stochastic simulation algorithm that may be used to calculate the steady state solution of a chemically reacting system. Recently the all possible states method was introduced as a way of accelerating the convergence of the simulations. We demonstrate that while the all possible states (APS) method does reduce the number of required trajectories, it is actually much slower than the original algorithm for most problems. We introduce the elapsed time method, which reformulates the process of recording the species populations. The resulting algorithm yields the same results as the original method, but is more efficient, particularly for large models. In implementing the elapsed time method, we present robust methods for recording statistics and empirical probability distributions. We demonstrate how to use the histogram distance to estimate the error in steady state solutions.

Structure of the human MMP-19 gene.

The recently identified MMP-19 belongs to the multi-protein family of zinc-binding matrix metalloproteinases (MMP). In order to analyze its genomic organization and to identify transcription factor binding sites that may be involved in the regulation of human MMP-19 expression, the gene coding for MMP-19 has been cloned and sequenced. The MMP-19 gene spans over 7.6kb and is composed of nine exons and eight introns. Furthermore, a 1.9kb fragment of 5'-flanking DNA was isolated and the transcription start point mapped. Nucleotide sequence analysis of its 5'-flanking region revealed several potential transcription factor binding sites typical of MMP promoters. Thus, a TATA-box, a consensus AP-1 binding element, and a putative PEA3 site were identified. The 1.9kb MMP-19 promoter fragment and several deletion constructs thereof were able to drive transcription of the luciferase reporter gene in transiently transfected CHO cells. Finally, it has been shown by an electrophoretic mobility shift assay that the AP-1 consensus sequence is able to bind a HeLa nuclear extract derived AP-1 factor.

Matrix metalloproteinase-19 in capillary endothelial cells: expression in acutely, but not in chronically, inflamed synovium.

Matrix metalloproteinase-19 (MMP-19), originally isolated as an autoantigen from the synovium of a patient suffering from rheumatoid arthritis (RA), is expressed in smooth muscle cells of the tunica media of large blood vessels of an RA patient, but not in the endothelial cell layer. By contrast, in acutely inflamed tissue, synovial capillaries strongly express MMP-19 in the cytoplasm, as shown by immunofluorescence of cryostat sections. In MMP-19-producing capillaries the beta3 integrin chain was found at the endothelial cell surface, as was the vascular endothelial cell growth factor receptor-2 (KDR). The specific tissue inhibitor of metalloproteinases TIMP-1 was absent or faintly stained in MMP-19-expressing capillaries, whereas TIMP-1, but not TIMP-2, was strongly expressed in large vessels and in MMP-19-negative capillaries of RA synovia. In the spontaneously transformed human umbilical vein endothelial cell line ECV304 neither MMP-19 transcripts nor protein could be detected. By contrast, primary cultures of human endothelial cells of either dermal or adipose tissue origin produced MMP-19 mRNA and protein. The results strongly suggest the regulated induction of matrix metalloproteinase-19 in capillary endothelial cells during acute inflammation and hint at a role of MMP-19 in angiogenesis.

Isolation and identification of cyclic imide and deamidation products in heat stressed pramlintide injection drug product.

This report summarizes the identification of six cyclic imide [Asu] and two deamidation products from a sample of pramlintide final drug product that had been stressed at 40 degrees C for 45 days. The pramlintide degradation products were isolated by cation exchange high-performance liquid chromatography (HPLC) followed by reversed-phase HPLC. The isolated components were characterized by mass spectrometry (MS), tandem MS (MS/MS) and when necessary, by enzymatic (thermolysin) digestion followed by liquid chromatography/mass spectrometry (LC/MS) and sequence analysis. The isolated products were identified as [Asu14]-pramlintide, [Asu21]-pramlintide, [Asu22]-pramlintide, [Asu35]-pramlintide, [1-21]-succinimide-pramlintide, and [1-22]-succinimide-pramlintide. Also identified were [Asp35]-pramlintide, the deamidation product of pramlintide at Asn35, and [Tyr37-OH]-pramlintide, the deamidation product of the pramlintide amidated C-terminal Tyr. Together these data support those presented earlier (C. Hekman et al., Isolation and identification of peptide degradation products of heat stressed pramlintide injection drug product. Pharm Res 1998;15:650-9) indicating that the primary mechanism of degradation for pramlintide in this pH 4.0 formulation is deamidation, with six of the eight possible deamidation sites observed to undergo deamidation. Gln-10 and Asn-31 are the only two residues subject to deamidation for which none is observed. The data indicate that the cyclic imide products account for approximately 20% of the total thermal degradation while the deamidation products account for 64%. The remaining degradation is due to peptide backbone hydrolysis.

Matrix metalloproteinase MMP-19 (RASI-1) is expressed on the surface of activated peripheral blood mononuclear cells and is detected as an autoantigen in rheumatoid arthritis.

In order to characterize the autoimmune response participating in the pathogenesis of rheumatoid arthritis (RA) a cDNA expression library constructed from mRNAs which had been isolated from the inflamed synovium of an RA patients was screened with autologous IgG autoantibodies. This led to the identification of gene rasi-1 which encodes a protein showing sequence identity with the zinc-binding matrix metalloproteinase MMP-19. MMP-19 is detected on the surface of activated PBMCs, TH1 lymphocytes, and Jurkat T lymphoma cells. It exhibits gelatinolytic activity and is recognized by autoantibodies in 26% and, respectively, 33% of sera collected from RA patients and systemic lupus erythematosus (SLE) patients. The novel autoantigen MMP-19 thus could play a role in the pathological processes participating in RA-associated joint tissue destruction.

Isolation and identification of peptide degradation products of heat stressed pramlintide injection drug product.

PURPOSE: This report summarizes the identification of nine deamidation and four hydrolysis products from a sample of pramlintide injection final drug product that was subjected to stress at 40 degrees C for 45 days. METHODS: The pramlintide degradation products were isolated by strong cation exchange HPLC followed by reversed-phase HPLC. Subsequent to isolation, the molecular weight of each component was determined by liquid chromatography-mass spectrometry (LC/MS). Further characterization was accomplished by amino acid sequence analysis and/ or enzymatic (thermolysin) digestion followed by LC/MS and sequence analysis. RESULTS: The isolated products were identified as [iso-Asp21]-pramlintide, [iso-Asp3]-pramlintide, and [iso-Asp22]-pramlintide, the deamidation products of pramlintide with rearrangement at Asn21, Asn3, and Asn22, respectively. Also found were [Asp/iso-Asp14]-pramlintide, and [Asp/iso-Asp35]-pramlintide, the deamidation products at Asn14, and Asn35, and [Asp21]-pramlintide together with [Asp22]-pramlintide. For the deamidations at the 14th and 35th residues, it could not be determined whether the substance corresponded to the Asp or the iso-Asp product. The [Asp21] and [Asp22] products could not be separated from each other chromatographically but were both identified in a single fraction. Two minor degradation products were also identified as deamidated species. However, the sites of deamidation remain unknown. Also identified were [1-18]-pramlintide, [1-19]-pramlintide, [19-37]-pramlintide, and [20-37]-pramlintide, the products of hydrolytic peptide backbone cleavage at amino acids His18/Ser19 and Ser19/Ser20, respectively. One other product was isolated and tentatively identified as a cyclic imide intermediate preceeding deamidation. CONCLUSIONS: The primary mode of thermally induced degradation for this peptide is deamidation. A second degradation mechanism is peptide backbone hydrolysis.

The matrix metalloproteinase RASI-1 is expressed in synovial blood vessels of a rheumatoid arthritis patient.

RASI-1 is a novel matrix metalloproteinase which we isolated from an expression cDNA library representing the mRNA of an inflamed synovium obtained from a patient with rheumatoid arthritis (RA). To investigate the involvement of RASI-1 in the pathology of RA, we examined synovial specimens from RA patients with antibodies directed against an unique RASI-1-derived peptide. In comparison to interstitial collagenase, gelatinase A and B, and stromelysin 1, the RASI-1 expression in the RA-synovium is located mainly in the tunica media of blood vessel walls and its synovial localization is not as ubiquitous as that of other MMPs. The tissue inhibitor of metalloproteinases (TIMP-1), although also widely expressed in the synovium, exhibits strong colocalization with RASI-1 in blood vessel walls. While RASI-1 is expressed in blood vessels of the inflamed synovium of an RA patient, its expression was not found in control synovial specimens from patients with luxation and arthrosis. However, RASI-1 expression can also be found in non-inflamed blood vessels of uterine ligaments and skin. RASI-1, although its function and substrates are unknown, could be involved in processes such as neovascularization and angiogenesis or lymphocyte extravasation and thus may participate in joint tissue destruction during RA.

In vivo efficacy of novel synthetic enediynes 1.

We have investigated the biodistribution, toxicity, and antitumor activity of a new type of synthetic compound containing an enediyne functional group capable of benzenoid diradical generation. The design of this cytotoxic molecule was based on the structures of naturally occurring enediyne antibiotics. Compared to the natural compounds, the synthetic enediyne displayed cytotoxicities approaching the natural analogs. Using a tritiated analog, biodistribution studies revealed relatively high uptake levels in kidney, lung, heart, and spleen with moderate levels in all other organs. Antitumor activity was apparent, with significant tumor regression observed in athymic nude mice with established M21 melanomas. Significant tumor antiproliferative effects were observed against L-1210 mouse leukemia, A549 lung carcinomas and PC3 prostate carcinomas in athymic nude mice, and against EMT-6 mouse mammary adenocarcinomas in Balb/cByJ mice. These results suggest that synthetic enediynes may be useful therapeutic compounds since their design reduces systemic toxicity compared to the natural products, without compromising antitumor activity. The relatively low sensitivity of many established cell lines to synthetic enediynes suggests a discrepancy between cell culture and in vivo tumor cytotoxicities. Adaptation of some cell lines for in vivo proliferation may affect their sensitivity to synthetic enediynes.

Regulation of apoptosis in leukemic cells by analogs of dynemicin A.

The naturally occurring enediyne antibiotics, which include calicheamicin, esperamicin, neocarzinostatin, kedarcidin and dynemicin, are a unique class of reactive compounds which can undergo aromatization to produce cytotoxic biradicals and can result in phosphodiester bond breakage of DNA. Synthetic enediynes designed with low molecular complexity are also highly cytotoxic, specifically to human leukemic cells, by a mechanism involving the induction of apoptosis. We have used a variety of biological assays to evaluate the cytotoxic properties of synthetic dynemicin analogs which contain a spectrum of structural modifications and reactivities. It was found that the induction of apoptosis and nuclear degradation by the synthetic compounds did not require an ability to bind or cleave DNA. Prevention of apoptosis was observed in analogs which were electronically stabilized to inhibit aromatic rearrangement and generation of diradicals. The preventive capability of the stabilized analogs was observed against a wide variety of toxic agents including topoisomerase I and II inhibitors, anti-mitotic and DNA anti-metabolite drugs, as well as alkylating agents. The structural determinants involved in inhibiting the induction of apoptosis are described. The significance of these results with respect to relevant mechanism of tumor regression are discussed.