Characterization of BioID tagging systems in budding yeast and exploring the interactome of the Ccr4-Not complex.

The modified E. coli biotin ligase BirA* was the first developed for proximity labeling of proteins (BioID). However, it has low activity at temperatures below 37°C, which reduces its effectiveness in organisms growing at lower temperatures, such as budding yeast. Multiple derivatives of the enzymes have been engineered, but a comparison of these variations of biotin ligases has not been reported in Saccharomyces cerevisiae. Here, we designed a suite of vectors to compare the activities of biotin ligase enzymes in yeast. We found that the newer TurboID versions were the most effective at labeling proteins, but they displayed low constitutive activity from biotin contained in the culture medium. We describe a simple strategy to express free BioID enzymes in cells that can be used as an appropriate control in BioID studies to account for the promiscuous labeling of proteins caused by random interactions between bait-BioID enzymes in cells. We also describe chemically-induced BioID systems exploiting the rapamycin-stabilized FRB-FKBP interaction. Finally, we used the TurboID version of the enzyme to explore the interactome of different subunits of the Ccr4-Not gene regulatory complex. We find that Ccr4-Not predominantly labeled cytoplasmic mRNA regulators, consistent with its function in mRNA decay and translation quality control in this cell compartment.

Deceased donor uterus retrieval: A novel technique and workflow.

Uterus transplantation has proven successful when performed with a living donor. Subsequently, interest in the novel field of reproductive transplantation is growing. The procedure is still considered experimental, with fewer than 25 cases performed worldwide, and the techniques of both uterus procurement and transplantation are still developing. We detail a new approach to deceased donor uterus procurement. In contrast to reported techniques and our own initial experience, in which the deceased donor uterus was procured post cross-clamp and after other organs were procured, our approach now is to perform the uterus procurement prior to the procurement of other organs in a multiorgan donor and hence prior to cross-clamp. We describe our practical experience in developing and implementing the logistical workflow for deceased donor uterus procurement in a deceased multiorgan donor setting.

Evaluation of late vegetative and reproductive stage soybeans for resistance to soybean aphid (Hemiptera: Aphididae).

The soybean aphid, Aphis glycines Matsumura, has become the most significant soybean [Glycine max (L.) Merrill] insect pest in the north central soybean production region of North America. The objectives of this research were to measure selected genotypes for resistance to the soybean aphid in the later vegetative and reproductive stages under field conditions, and confirm the presence of tolerance in KS4202. The results from 2007 to 2011 indicate that KS4202 can support aphid populations with minimal yield loss at levels where significant yield loss would be expected in most other genotypes. The common Nebraska cultivar, 'Asgrow 2703', appears to show signs of tolerance as well. None of the yield parameters were significantly different between the aphid infested and noninfested treatments. Based on our results, genotypes may compensate for aphid feeding in different ways. Asgrow 2703 appears to produce a similar number of seeds as its noninfested counterpart, although the seeds produced are slightly smaller. Field evaluation of tolerance in KS4202 indicated a yield loss of only 13% at 34,585-53,508 cumulative aphid-days, when 24-36% yield loss would have been expected.

Effects of thiamethoxam seed treatments on soybean aphid (Hemiptera: Aphididae) feeding behavior.

Since its discovery in North America in 2000, the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), has rapidly become an important pest of soybean [Glycine max (L.) Merrill], sometimes resulting in significant yield losses. Previous research has documented the toxicity of neonicotinoid seed treatments to soybean aphids, but control under field conditions has been inconsistent. Imidacloprid, a popular neonicotinoid insecticide, has been shown to exhibit antifeedant effects on aphids. Antifeedant activity has not been demonstrated for other neonicotinoids, including thiamethoxam. This research investigated the effects of a thiamethoxam seed treatment on soybean aphid feeding behavior by using electronic penetration graphs (EPG) to visualize stylet penetration behavior. Soybean aphid feeding behavior was assessed for 9 h on thiamethoxam-treated and untreated soybeans (V2 and V4 stages). Because results were inconclusive from initial experiments, a study was conducted to document the effects of thiamethoxam-treated soybeans on soybean aphid survival. The seed treatment was shown to negatively affect aphid survival at 4, 8, and 11 d after aphid introduction. A subsequent EPG study then was designed to document soybean aphid feeding behavior for 15 h, after an initial exposure of 9 h to thiamethoxam-treated soybeans. In this study, the exposed aphids exhibited significant differences in feeding behavior compared with those aphids feeding on untreated soybeans. Soybean aphids on thiamethoxam-treated soybeans spent significantly less time feeding in the sieve element phase, with a greater duration of nonprobing events. These studies suggest soybean aphids are unable to ingest phloem sap, which may be another important element in seed treatment protection.

Categorizing the resistance of soybean genotypes to the soybean aphid (Hemiptera: Aphididae).

We evaluated selected soybean, Glycine max (L.) Merr., genotypes during their reproductive stages for resistance to the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), under greenhouse conditions and documented the categories of aphid-resistant soybean. Two screening studies were performed to assess the level of resistance to the soybean aphid on six soybean genotypes during the reproductive stages of development. Significant differences in aphid damage ratings were detected among the soybean evaluated in the screening studies. Three genotypes (KS4202, K-1639-2, and K1621) were considered moderately resistant based on the assessed damage ratings. Two of these genotypes (K-1639-2 and KS4202), along with a commercial variety ('Asgrow 2703') were used in a follow-up greenhouse study to test for antibiosis and tolerance. For the antibiosis evaluation, KS4202 had significantly more nymphs than Asgrow 2703 and K-1639-2. In fact, KS4202 had a threefold difference in the number of nymphs compared with Asgrow 2703 (81.8 +/- 14.7 and 26.2 +/- 13.9 nymphs, respectively) and a fivefold difference compared with K-1639-2 (15.6 +/- 13.9). Although not significant, Asgrow 2703 had more nymphs than K-1639-2. The lower aphid numbers on infested K-1639-2 plants compared with aphid numbers on Asgrow 2703 and KS4202 plants indicates antibiosis for this genotype. No significant differences in average seed weight, number of seeds per pod, or plant damage were observed between infested and control KS4202 plants; however, significant differences in biomass, total seed weight, number of pods per plant, and number of seeds per plant were detected.

H9, H10, and H11 compose a cluster of Hessian fly-resistance genes in the distal gene-rich region of wheat chromosome 1AS.

H9, H10, and H11 are major dominant resistance genes in wheat, expressing antibiosis against Hessian fly [(Hf) Mayetiola destructor (Say)] larvae. Previously, H9 and H10 were assigned to chromosome 5A and H11 to 1A. The objectives of this study were to identify simple-sequence-repeat (SSR) markers for fine mapping of these genes and for marker-assisted selection in wheat breeding. Contrary to previous results, H9 and H10 did not show linkage with SSR markers on chromosome 5A. Instead, H9, H10, and H11 are linked with SSR markers on the short arm of chromosome 1A. Both H9 and H10 are tightly linked to flanking markers Xbarc263 and Xcfa2153 within a genetic distance of 0.3-0.5 cM. H11 is tightly linked to flanking markers Xcfa2153 and Xbarc263 at genetic distances of 0.3 cM and 1.7 cM. Deletion bin mapping assigned these markers and genes to the distal 14% of chromosome arm 1AS, where another Hf-resistance gene, Hdic (derived from emmer wheat), was also mapped previously. Marker polymorphism results indicated that a small terminal segment of chromosome 1AS containing H9 or H10 was transferred from the donor parent to the wheat lines Iris or Joy, and a small intercalary fragment carrying H11 was transferred from the resistant donor to the wheat line Karen. Our results suggest that H9, H10, H11, Hdic, and the previously identified H9- or H11-linked genes (H3, H5, H6, H12, H14, H15, H16, H17, H19, H28, and H29) may compose a cluster (or family) of Hf-resistance genes in the distal gene-rich region of wheat chromosome 1AS; and H10 most likely is the same gene as H9.

Categories of resistance to greenbug (Homoptera: Aphididae) biotype K in wheat lines containing Aegilops tauschii genes.

The wheat lines (cultivars) 'Largo', 'TAM110', 'KS89WGRC4', and 'KSU97-85-3' conferring resistance to greenbug, Schizaphis graminum (Rondani), biotypes E, I, and K were evaluated to determine the categories of resistance in each line to greenbug biotype K. Our results indicated that Largo, TAM110, KS89WGRC4, and KSU97-85-3 expressed both antibiosis and tolerance to biotype K. Largo, KS89WGRC4, and KSU97-85-3, which express antixenosis to biotype I, did not demonstrate antixenosis to biotype K. The results indicate that the same wheat lines may possess different categories of resistance to different greenbug biotypes. A new cage procedure for measuring greenbug intrinsic rate of increase (r(m)) was developed, by using both drinking straw and petri dish cages, to improve the efficiency and accuracy of r(m)-based antibiosis measurements.

A group of related cDNAs encoding secreted proteins from Hessian fly [Mayetiola destructor (Say)] salivary glands.

A group of cDNAs has been isolated and characterized from Hessian fly [Mayetiola destructor (Say)] salivary glands. Members in this group appear to encode proteins with secretion signal peptides at the N-terminals. The mature putative proteins are small, basic proteins with calculated molecular weights that ranged from 8.5 to 10 kDa, and isoelectric points from 9.92 to 10.90. Sequence analysis indicated a strong selection for mutations that generate amino acid changes within the coding region. Northern blot analysis revealed that these genes are expressed only in the first instar larvae, a critical stage that determines if the interaction between a specific Hessian fly biotype and a specific wheat cultivar is compatible. Genomic analysis demonstrated that multiple copies of similar genes are clustered within a short region on chromosome 2A. This is the same arm in which two avirulence genes have been mapped.

Analysis of TAF90 mutants displaying allele-specific and broad defects in transcription.

Yeast TAF90p is a component of at least two transcription regulatory complexes, the general transcription factor TFIID and the Spt-Ada-Gcn5 histone acetyltransferase complex (SAGA). Broad transcription defects have been observed in mutants of other TAF(II)s shared by TFIID and SAGA but not in the only two TAF90 mutants isolated to date. Given that the numbers of mutants analyzed thus far are small, we isolated and characterized 11 temperature-sensitive mutants of TAF90 and analyzed their effects on transcription and integrity of the TFIID and SAGA complexes. We found that the mutants displayed a variety of allele-specific defects in their ability to support transcription and maintain the structure of the TFIID and SAGA complexes. Sequencing of the alleles revealed that all have mutations corresponding to the C terminus of the protein, with most clustering within the conserved WD40 repeats; thus, the C terminus of TAF90p is required for its incorporation into TFIID and function in SAGA. Significantly, inactivation of one allele, taf90-20, caused the dramatic reduction in the levels of total mRNA and most specific transcripts analyzed. Analysis of the structure and/or activity of both TAF90p-containing complexes revealed that this allele is the most disruptive of all. Our analysis defines the requirement for the WD40 repeats in preserving TFIID and SAGA function, demonstrates that the defects associated with distinct mutations in TAF90 vary considerably, and indicates that TAF90 can be classified as a gene required for the transcription of a large number of genes.

Genetic analysis of TAF68/61 reveals links to cell cycle regulators.

In yeast, inactivation of certain TBP-associated factors (TAF(II)s) results in arrest at specific stages of the cell cycle. In some cases, cell cycle arrest is not observed because overlapping defects in other cellular processes precludes the manifestation of an arrest phenotype. In the latter situation, genetic analysis has the potential to reveal the involvement of TAF(II)s in cell cycle regulation. In this report, a temperature-sensitive mutant of TAF68/61 was used to screen for high-copy dosage suppressors of its growth defect. Ten genes were isolated: TAF suppressor genes, TSGs 1-10. Remarkably, most TSGs have either a genetic or a direct link to control of the G(2)/M transition. Moreover, eight of the 10 TSGs can suppress a CDC28 mutant specifically defective for mitosis (cdc28-1N) but not an allele defective for passage through start. The identification of these genes as suppressors of cdc28-1N has identified four unreported suppressors of this allele. Moreover, synthetic lethality is observed between taf68-9 and cdc28-1N. The isolation of multiple genes involved in the control of a specific phase of the cell cycle argue that the arrest phenotypes of certain TAF(II) mutants reflect their role in specifically regulating cell cycle functions.

Promoter targeting of chromatin-modifying complexes.

The action of multi-subunit complexes that are able to overcome the repressive effects of chromatin is an important step in the regulation of eukaryotic gene expression. Identification of complexes that modify the structure of chromatin to help factors access the underlying DNA has enhanced our understanding of how some genes are controlled. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) represent one group of complexes that regulate the level of acetylation on the N-terminal tails of core histone proteins. The SWI/SNF complex is the prototype of a second group of complexes, which use the energy of ATP-hydrolysis to alter histone-DNA contacts, leading to changes in chromatin conformation. Genetic studies in yeast have revealed that some of these multi-subunit complexes interact in vivo to control transcription of a subset of genes. It has become apparent that some gene promoters require modifications by both types of complexes. An important question regarding these two types of complexes is how they are recruited to the promoters of genes that are dependent on their activity for their expression. This review will tie together many studies on promoter recruitment of both HATs and SWI/SNF. Emphasis will be placed on recent data that demonstrates functional interplay between these two types of chromatin-remodeling activities. In addition, this review summarizes recent data demonstrating the ability of repressors and corepressors to recruit histone deacetylase complexes. Interestingly, many subunits of chromatin-modifying complexes in humans have been implicated in the development of cancer. Thus, studying how these complexes work can help us better understand human diseases.

Ssn6-Tup1 regulates RNR3 by positioning nucleosomes and affecting the chromatin structure at the upstream repression sequence.

The DNA damage inducible gene ribonucleotide reductase (RNR3) is regulated by a transcriptional repression mechanism by the recruitment of the Ssn6-Tup1 corepressor complex to its promoter by the sequence-specific DNA-binding protein Crt1. Ssn6-Tup1 is reported to represses transcription by interfering with transcription factors, recruiting histone deacetylases, and positioning nucleosomes at the promoter of its target genes. Two of the three mechanisms involve effects on chromatin structure, and therefore, we have delineated the nucleosomal structure of RNR3 in the repressed and derepressed state using multiple nuclease mapping strategies. A regular array of positioned nucleosomes is detected over the repressed RNR3 promoter that extends into the coding sequence. Treating cells with DNA damaging agents or deleting CRT1, SSN6, or TUP1 derepresses RNR3 transcription, and causes a dramatic disruption of nucleosome positioning over its promoter. Furthermore, derepression of RNR3 correlated with changes in nuclease sensitivity within the upstream repression sequence (URS) region. Specifically, the loss of a MNase-hypersensitive site, and the appearance of strong DNase I hypersensitivity, was observed over the URS. Interestingly, we find that the binding of Crt1 to the promoter in the absence of Ssn6 or Tup1 is insufficient for nucleosome positioning or regulating chromatin structure at the URS; thus, these two functions are strictly dependent upon Ssn6-Tup1. We propose that RNR3 is regulated by changes in nucleosome positioning and chromatin structure that are mediated by Ssn6, Tup1, and Crt1.

Categories of resistance to greenbug (Homoptera: Aphididae) biotype I in Aegilops tauschii germplasm.

Categories of resistance to greenbug, Schizaphisgraminum (Rondani), biotype I, were determined in goatgrass, Aegilops tauschii (Coss.) Schmal., accession 1675 (resistant donor parent), 'Wichita' wheat, Triticum aestivum L., (susceptible parent), and an Ae. tauschii-derived resistant line, '97-85-3'. Antibiosis was assessed using the intrinsic rate of increase (rm) of greenbugs confined to each of the three genotypes. Neither parent nor the resistant progeny expressed antibiosis. Mean rm values for greenbug I on Wichita (0.0956), and Ae. tauschii (0.10543) were not significantly different. Mean rm values for Wichita and 97-85-3 were also not significantly different. Antixenosis was determined by allowing aphids a choice to feed on plants of each of the three genotypes. Ae. tauschii 1675 exhibited antixenosis, but this resistance was not inherited and expressed in '97-85-3'. In experiments comparing Wichita and Ae. tauschii 1675, greenbug I population distributions were not significantly different on Wichita at 24 h, but were shifted toward Wichita at 48 h. In the second antixenosis experiment, there were no significant differences in greenbug I population distributions on 97-85-3 or Wichita at 24 or 48 h. When all three lines were compared, there were no significant differences in greenbug biotype I populations at 24 or 48 h after infestation. Comparisons of proportional dry plant weight loss (DWT) and SPAD meter readings were used to determine tolerance to greenbug I feeding. Ae. tauschii 1675 and 97-85-3 were highly tolerant compared with Wichita. Infested and uninfested Ae. tauschii 1675 DWT was nonsignificant, and infested Wichita plants weighed significantly less than uninfested plants. When Wichita and 97-85-3 were contrasted, DWT of infested and uninfested Wichita plants were significantly different, but those of 97-85-3 were not. Mean percent leaf chlorophyll losses for the three genotypes, as measured by the SPAD chlorophyll meter, were as follows: Wichita = 65%; Ae. tauschii 1675 = 25%; and 97-85-3 = 39%. Percent leaf chlorophyll losses caused by greenbug feeding was significantly different in comparisons between Wichita and Ae. tauschii 1675, and comparisons between Wichita and 97-85-3, although feeding damage was not significantly different in comparisons between Ae. tauschii 1675 and 97-85-3. These data provided further evidence of the expression of tolerance to greenbug feeding in Ae. tauschii 1675 and 97-85-3.

The proteasome regulates the UV-induced activation of the AP-1-like transcription factor Gcn4.

The proteasome is well known for its regulation of the cell cycle and degradation of mis-folded proteins, yet many of its functions are still unknown. We show that RPN11, a gene encoding a subunit of the regulatory cap of the proteasome, is required for UV-stimulated activation of Gcn4p target genes, but is dispensable for their activation by the general control pathway. We provide evidence that RPN11 functions downstream of RAS2, and show that mutation of two additional proteasome subunits results in identical phenotypes. Our analysis defines a novel function of the proteasome: regulation of the RAS- and AP-1 transcription factor-dependent UV resistance pathway.

Derepression of DNA damage-regulated genes requires yeast TAF(II)s.

The general transcription factor TFIID and its individual subunits (TAF(II)s) have been the focus of many studies, yet their functions in vivo are not well established. Here we characterize the requirement of yeast TAF(II)s for the derepression of the ribonucleotide reductase (RNR) genes. Promoter mapping studies revealed that the upstream repressing sequences, the damage-responsive elements (DREs), rendered these genes dependent upon TAF(II)s. DREs are the binding sites for the sequence-specific DNA binding-protein Crt1 that represses transcription by recruiting the Ssn6-Tup1 co-repressor complex to the promoter. We demonstrate that deletion of SSN6, TUP1 or CRT1 alleviated the TAF(II) dependence of the RNR genes, indicating that TAF(II) dependence requires the co-repressor complex. Furthermore, we provide evidence that Crt1 specifies the TAF(II) dependence of these genes. Our studies show that TFIID interacts with the repression domain of Crt1, suggesting that the derepression mechanism involves an antagonism between TFIID and the co-repressor complex. Our results indicate that yeast TAF(II)s have other functions in addition to core promoter selectivity, and describe a novel activity: the derepression of promoters.

Identification of a yeast transcription factor IID subunit, TSG2/TAF48.

The RNA polymerase II general transcription factor TFIID is a complex containing the TATA-binding protein (TBP) and associated factors (TAFs). We have used a mutant allele of the gene encoding yeast TAF(II)68/61p to analyze its function in vivo. We provide biochemical and genetic evidence that the C-terminal alpha-helix of TAF(II)68/61p is required for its direct interaction with TBP, the stable incorporation of TBP into the TFIID complex, the integrity of the TFIID complex, and the transcription of most genes in vivo. This is the first evidence that a yeast TAF(II) other than TAF(II)145/130 interacts with TBP, and the implications of this on the interpretation of data obtained studying TAF(II) mutants in vivo are discussed. We have identified a high copy suppressor of the TAF68/61 mutation, TSG2, that has sequence similarity to a region of the SAGA subunit Ada1. We demonstrate that it directly interacts with TAF(II)68/61p in vitro, is a component of TFIID, is required for the stability of the complex in vivo, and is necessary for the transcription of many yeast genes. On the basis of these functions, we propose that Tsg2/TAF(II)48p is the histone 2A-like dimerization partner for the histone 2B-like TAF(II)68/61p in the yeast TFIID complex.

Anaphylactoid reaction to muromonab-CD3 in a pediatric renal transplant recipient.

Muromonab-CD3 (OKT3), a murine IgG2a antibody directed against the T3 (CD3) complex on mature lymphocytes, triggers adverse immune reactions. Anaphylactic reactions have occurred in patients exposed to OKT3 and are mediated by anti-OKT3 IgE antibodies. The reactions are not antibody mediated and can occur within seconds of administration of a mast cell secretogogue. A renal transplant recipient became hypotensive and hypoxic immediately after receiving her first dose of OKT3 and required advanced life support. Serum antibody tests were negative for anti-OKT3 IgG, IgE, and antimouse protein antibodies. To our knowledge, this is the first published report of a patient with an anaphylactoid reaction to the initial infusion of OKT3.

A subset of TAF(II)s are integral components of the SAGA complex required for nucleosome acetylation and transcriptional stimulation.

A number of transcriptional coactivator proteins have been identified as histone acetyltransferase (HAT) proteins, providing a direct molecular basis for the coupling of histone acetylation and transcriptional activation. The yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex requires the coactivator protein Gcn5 for HAT activity. Identification of protein subunits by mass spectrometry and immunoblotting revealed that the TATA binding protein-associated factors (TAF(II)s) TAF(II)90, -68/61, -60, -25/23, and -20/17 are integral components of this complex. In addition, TAF(II)68 was required for both SAGA-dependent nucleosomal HAT activity and transcriptional activation from chromatin templates in vitro. These results illustrate a role for certain TAF(II) proteins in the regulation of gene expression at the level of chromatin modification that is distinct from the TFIID complex and TAF(II)145.

Cyclosporine-induced white and grey matter central nervous system lesions in a pediatric renal transplant patient.

Major neurologic complications secondary to cyclosporine are well documented and are known to include confusion, cortical blindness, seizure, spasticity, paresis, ataxia and coma. Most previous reports attribute these to white matter central nervous system (CNS) lesions or white/grey matter border lesions. Many predisposing factors have been identified, including: elevated levels of cyclosporine, hypomagnesemia, hypocholesterolemia, aluminium toxicity, high dose steroids, hypertension and infection. However CNS events attributed to cyclosporine have been reported without any of these risk factors. We report a case of a child developing multiple white and grey matter thalamic and cortical lesions along with acute neurologic deterioration, and then review cyclosporine mediated CNS injury, including the roles of P-glycoprotein and cyclophilin.

Changes in arterial expression of fibrinolytic system proteins in atherogenesis.

Plasminogen activators (PAs) and their inhibitor, plasminogen activator inhibitor type-1 (PAI-1), have been implicated in modulation of luminal fibrinolysis and mural proteolysis contributing to atherogenesis. Expression of PAs/PAI-1 (normalized to extracted tissue protein) was delineated by assays of conditioned media and of extracts from walls of human arterial segments in culture. Arterial specimens (n = 39 from 26 subjects) were divided into four groups: normal (n = 14), fatty streak (n = 6), moderate atherosclerosis (mural thickening with < 70% lumen obstruction, n = 5), and severe atherosclerosis (mural thickening with > 70% lumen obstruction, n = 14). Paired samples from the same individual comprising a normal arterial segment and an atherosclerotic segment were evaluated also. A fourfold molar excess in PAI-1:t-PA was seen in conditioned media from samples with any evidence of atherosclerosis compared with normal specimens (normal 21 +/- 4, diseased 82 +/- 21, P < or = .05). Compared with normal pairs, the tissue content of PAI-1 (ng) was increased in fatty streak lesions (n = 3, normal 35 +/- 12, fatty streak 50 +/- 8, P < or = .05); stable to decreased in moderate atherosclerosis (n = 3, normal 34 +/- 3, moderate 22 +/- 7, P = .16); and increased in severe atherosclerosis (n = 6, normal 48 +/- 9, severe 85 +/- 19, P < or = .05). The tissue content of PAs (ng), though not increased in fatty streak lesions, was elevated in moderately and severely atherosclerotic segments (normal 0.7 +/- 0.2, moderate 1.6 +/- 0.1; normal 0.8 +/- 0.3, severe 2.1 +/- 0.3, P < or = .05 for each comparison). Atherogenesis is associated with decreased luminal fibrinolytic capacity that may exacerbate thrombosis. Decreased mural proteolysis in early atherogenesis may exacerbate matrix accumulation. Increased mural proteolysis later is associated with, and may potentiate, smooth muscle cell migration and proliferation.