Efficient control of Plasmodium yoelii infection in BALB/c and C57BL/6 mice with pre-existing Strongyloides ratti infection.

About 225 million malaria cases have been reported worldwide in 2009, and one-third of the world's population is infected with parasitic helminths. As helminths and Plasmodium are co-endemic, concurrent infections frequently occur. Helminths have been shown to modulate the host's immune response; therefore, pre-existing helminth infections may interfere with the efficient immune response to Plasmodium. To study the interaction between helminths and Plasmodium, we established a murine model of co-infection using the gastrointestinal nematode Strongyloides ratti and Plasmodium yoelii. We show that a pre-existing Strongyloides infection slightly enhanced peak parasitemia and weight loss in P. yoelii-infected BALB/c mice, while disease progression was not altered in co-infected C57BL/6 mice. The Plasmodium-induced IFN-γ production and final clearance of Plasmodium infection were not affected by S. ratti co-infection in both C57BL/6 and BALB/c mice. Interestingly, the T helper cell (Th) 2 response induced by S. ratti was significantly suppressed upon P. yoelii co-infection. This suppressed Th2 response, however, was still sufficient to allow expulsion of S. ratti parasitic adults. Taken together, we provide evidence that simultaneous presence of helminth and protist parasites does not interfere with efficient host defence in our co-infection model although changes in Th responses were observed.

Efficient control of Leishmania and Strongyloides despite partial suppression of nematode-induced Th2 response in co-infected mice.

Endemic regions for the pathogenic nematode Strongyloides and parasitic protist Leishmania overlap and therefore co-infections with both parasites frequently occur. As the Th2 and Th1 immune responses necessary to efficiently control Strongyloides and Leishmania infections are known to counterregulate each other, we analysed the outcome of co-infection in the murine system. Here, we show that Leishmania major-specific Th1 responses partially suppressed the nematode-induced Th2 response in co-infected mice. Despite this modulation, successful expulsion of gut dwelling Strongyloides was not suppressed in mice with pre-existing or subsequent Leishmania infection. A pre-existing Strongyloides infection, in contrast, did not interfere with efficient type-1 responses but even increased pro-inflammatory cytokine production. Also, control of L. major infections was not affected by pre-existing nematode infection. Taken together, we provide evidence that simultaneous presence of helminth and protist parasites did not interfere with efficient host defence in our co-infection model.

Strongyloides ratti infection induces transient nematode-specific Th2 response and reciprocal suppression of IFN-gamma production in mice.

Over one-third of the world population is infected with parasitic helminths, Strongyloides ssp. accounting for approximately 30-100 million infected people. In this study, we employ the experimental system of murine Strongyloides ratti infection to investigate the interaction of this pathogenic nematode with its mammalian host. We provide a comprehensive kinetic description of the immune response to S. ratti infection that was reflected by induction of antigen-specific IgM and IgG1, mast cell activation and a Th2-like cytokine response. T cells derived from infected mice displayed an increased IL-3, IL-4, IL-5, IL-13 and IL-10 response to CD3-engagement in comparison with T cells derived from naïve mice. The IFN-gamma response to CD3-engagement that was well detectable in T cells derived from naïve mice, however, was suppressed in T cells derived from infected mice. Both, the induction of the S. ratti-specific Th2 response and the suppression of pro-inflammatory cytokines were transient and observed in strict correlation to the course of infection and the number of infective larvae used. Finally, comparing artificial infections induced by subcutaneous injection of larvae to natural infections, we observed similar antigen-specific T cell responses although the natural infection led to a significantly lower worm burden.

Receptor specificity of the Escherichia coli T-even type phage Ox2. Mutational alterations in host range mutants.

The T-even type Escherichia coli phage Ox2 uses the outer membrane protein OmpA as a receptor. The protein is recognized with the ends of the virion's long tail fibers. The 266 residue protein 38 is located at this site and acts as an adhesin. Host-range mutants had previously been isolated from Ox2. Mutant Ox2h5 is able to infect cells possessing an altered OmpA protein, which renders the cell resistant to Ox2. Ox2h10 was selected from Ox2h5. This phage recognizes the OmpC protein in addition to the OmpA protein. Ox2h12, which stems from Ox2h10, binds to OmpC with high affinity, but has lost efficient binding to OmpA. The mutational alterations caused in genes 38 are: Asp231----Asn(h5) and His170----Arg(h10). The triple mutant Ox2h12 possesses an insertion of a Gly residue next to Gly121. The three mutants have additionally acquired mutations affecting their base plate, making them "trigger-happy". When protein 38 was compared with the same protein derived from other E. coli phages, it was found to contain two constant and one variable domains, the latter harboring four hypervariable regions flanked by a largely conserved glycine-rich sequence. The h5 and h10 mutations occurred within two hypervariable areas, while the additional Gly residue was present in one of the flanking conserved sequences. On the basis of these results, as well as those obtained from host-range mutants analyzed previously, a model for such adhesins is proposed. Receptor recognition is most likely performed via the hypervariable regions, which may form loops held together in close proximity by the oligoglycine sequences. The latter may achieve this by being part of highly compact omega loops.

DNA sequence of the tail fiber genes 37, encoding the receptor recognizing part of the fiber, of bacteriophages T2 and K3.

The DNA sequences of genes 37 of bacteriophages T2 and K3 are presented and compared with that of phage T4. The corresponding proteins constitute, as dimers, the part of the long tail fibers that recognizes the bacterial receptor. The CO2H termini of the polypeptides are located at the free ends of the fibers. Morphologically, the three phages are essentially identical, but they use different receptors. The genes from phages T4, T2 and K3 encode proteins consisting of 1026, 1341 and 1243 amino acid residues, respectively. DNA-DNA hybridizations had shown earlier that genes 37, in contrast to the gene for the major capsid protein, of a number of T-even type phages are highly polymorphic. The deduced amino acid sequences now show that this polymorphism extends to the protein primary structures. About 50 NH2-terminal residues are conserved and are probably required for binding to the adjacent protein 36. This area is followed by more or less irregularly spaced regions of non-homology, partial homology or complete homology. The heterogeneity is most prominent in a region encompassing about 600 CO2H-terminal residues of the T2 or K3 proteins. Nevertheless, the amino acid compositions of the three proteins are very similar and all are rich in glycine. It has been found that the receptor specificities of phages K3 and T2 are determined by protein 38, a polypeptide required for the efficient dimerization of protein 37 of phage T4. Proteins 38 of phages K3 and T2 are functionally interchangeable, those of T4 and T2 or K3 are not. Proteins 37 of phages K3 and T2 possess a conserved sequence of 160 CO2H-terminal residues. This area is missing in the T4 protein. This region may serve as a binding site for polypeptides 38 of phages K3 and T2. The overall picture of the protein primary structures of the three phages strongly suggests that the evolution of genes 37, which was most likely driven by selection for variations in receptor recognition specificities, has not been a steady process but has involved loss and gain of segments of DNA.

DNA sequence of genes 38 encoding a receptor-recognizing protein of bacteriophages T2, K3 and of K3 host range mutants.

Genes 38, which code for a receptor-recognizing protein present at the tip of the long tail fibers, have been sequenced from phages T2, the T-even-type phage K3 and its host range mutants K3hx, K3h1 and K3h1h. The genes from phages T2 and K3 code for proteins consisting of 262 and 260 amino acid residues, respectively. Fifty amino-terminal and 25 carboxy-terminal residues are highly conserved. The amino-terminal amino acids are most likely involved in binding to the neighboring protein 37. Between residues 116 and 226 of the T2 protein and residues 116 and 223 of the K3 protein, sequences exist that are similar to sequences present in Escherichia coli outer membrane proteins and which serve as phage receptors. Most likely, all of these regions in the latter proteins are exposed on the cell surface and are part of their phage receptor areas. In the phage proteins, these sequences are flanked by stretches rich in glycine, perhaps providing an increased flexibility for the polypeptide at these sites; some "wobble" may be required during the protein 38-receptor interaction. The mutational alterations in the host range mutants were found in gene 38. In the K3hx protein, a duplication of six base-pairs caused the wild-type sequence -Gly163-Lys-Leu-Ile- to be changed to -Gly163-Lys-Leu-Lys-Leu-Ile-. In the K3h1 protein, a glutamic acid residue at position 203 was substituted by a lysine. Both alterations occurred within areas similar to outer membrane proteins. Mutant K3h1h, derived from K3h1, exhibits an extended host range as compared to K3h1. No mutational alteration, in addition to that found in K3h1, was found in g38 nor was the part of gene 37 that encodes the carboxy-terminal moiety of the protein altered. K3h1h may represent a "trigger-happy" phage. The results of this and other work show that the phage-phage receptor systems under study represent a primitive immune system.

Receptor-recognizing proteins of T-even type bacteriophages. Constant and hypervariable regions and an unusual case of evolution.

Proteins 38 of bacteriophages T2, K3, Ox2 and M1 are located at the free ends of their long tail fibers and function as adhesins, i.e. they mediate binding to the bacterial receptors. The latter three phages use the Escherichia coli outer membrane protein OmpA as a receptor, while T2 uses the outer membrane proteins OmpF or Ttr. The DNA sequences of genes 38 of phages Ox2 and M1 have been determined and are compared with those known for T2 and K3. The genes encode 262(T2), 260(K3), 266(Ox2) and 262(M1) amino acid residues. Three domains are distinguishable in these proteins. There are two conserved regions encompassing about 120 NH2-terminal and about 25 CO2H-terminal residues, respectively. The area between these was found to be hypervariable, and it is shown that a very large number of amino acid substitutions, deletions and/or insertions have occurred. Glycine-rich stretches are present within and flanking these areas. Their positions are essentially conserved, indicating an important structural role in receptor recognition. The hypervariability, most likely caused by a constant struggle with bacterial phage-resistant mutants, is so drastic that one cannot discern that T2 uses different receptors from those of the other phages. The partially known sequence of gene 38 of phage T4 has been completed. The gene encodes a protein consisting of 183 amino acid residues. The amino acid composition and sequence of this protein is completely different from those of phages T2, K3, Ox2 and M1. Also, the protein is functionally unrelated to the other proteins 38: it is not present in phage T4 and, unlike the other proteins 38, is required for the efficient dimerization of protein 37. All phages under study are of the same morphology and the genomic organization of the tail fiber genes is identical, with genes 36, 37 and 38 most likely representing, in this order, a transcriptional unit. Sequence similarities between the CO2H-termini of genes 37 of the non-T4 phages and gene 38 of phage T4 were found; this part of gene 37 does not exist in T4. It is suggested that gene 38 of phage T4 originated from a segment of gene 37 of a T2-type phage. Gene 38 of phage T4 is not unique, DNA-DNA hybridization experiments indicated that two other T-even type phages, TuIa and TuIb, possess a T4-type gene 38.

Evidence that TraT interacts with OmpA of Escherichia coli.

The OmpA protein is one of the major outer membrane proteins of Escherichia coli. Among other functions the protein serves as a receptor for several phages and increases the efficiency of F-mediated conjugation when present in recipient cells. TraT is an F-factor-coded outer membrane lipoprotein involved in surface exclusion, the mechanism by which E. coli strains carrying F-factors become poor recipients in conjugation. To determine a possible interaction of TraT with OmpA, the influence of TraT on phage binding to cells was measured. Because TraT inhibits inactivation of OmpA-specific phages it is suggested that TraT interacts directly with OmpA. Sequence homology of TraT with proteins 38, the phage proteins recognizing outer membrane proteins, supports this finding. A model of protein interactions is discussed.

Topological analysis of the amino-terminal region of lactose permease using the Escherichia coli outer membrane protein, OmpA, as a marker.

LacY-ompA fusions, encoding the N-terminal 50, 71 or 143 residues of lactose permease, were constructed. The observed orientation of the OmpA part of each hybrid protein with respect to the plasma membrane supports current models of the N-terminus of Lac permease. Hybrids possessing the entire mature OmpA were very stable; those with only a part thereof were much less stable. Due to their in vivo stability and accessibility to antibody it is proposed that such hybrids may represent potential models to investigate the assembly pathway of lactose permease.

Localization and functional analysis of the cytosolic and extracellular CuZn superoxide dismutases in the human parasitic nematode Onchocerca volvulus.

This study describes the histological localization of two CuZn superoxide dismutases (SOD1 and SOD2) in the parasitic nematode Onchocerca volvulus, and a functional characterization of the 'extracellular' form of this enzyme (SOD2) which provides evidence that it is involved in the defense against environmental superoxide anion radicals. These essential enzymes are detected in larval and adult stages of the parasite, determined at the mRNA and protein levels by in situ hybridization and immunolocalization studies. These proteins are distributed throughout the worm, at various concentrations with particularly high levels produced in the hypodermis. In vitro maintenance of parasites indicated that SOD2 was secreted outside the parasite into the medium. Baculovirus constructs designed to test the ability of the SOD2 hydrophobic N-terminal region to function in processing and secretion confirmed the ability of this polypeptide sequence to direct the secretion of a marker protein, as well as of the mature SOD2 enzyme. Analyses of the native, mature SOD2 enzyme molecular mass, and the primary and quaternary structure, indicate that unlike other extracellular SODs, the SOD2 is active as a non-glycosylated dimer, rather than as a tetrameric glycoprotein. The detection of SOD2 outside of the parasite maintained in vitro, and the confirmation that the SOD2 is a secreted enzyme, indicate that this enzyme plays a role in the interactive biology of parasitic nematodes with their hosts.

Identification of a stress-responsive Onchocerca volvulus glutathione S-transferase (Ov-GST-3) by RT-PCR differential display.

The effects of oxidative insult on gene transcript levels in the filarial nematode Onchocerca volvulus were investigated using differential display RT-PCR. Oxidative stress was applied with the reagents paraquat, plumbagin and xanthine-xanthine oxidase. In all three cases, a cDNA fragment encoding a novel glutathione S-transferase (GST) resembling members of the theta-class was identified as upregulated (PQ29, PG112, XOD26). The subsequently isolated full-length cDNA harbors a 753-bp open reading frame encoding a GST with 268 amino acid residues and a predicted molecular mass of 31 kDa. This stress-responsive GST (Ov-GST-3) possesses only 14 and 21% sequence identity with the other O. volvulus GSTs (Ov-GST-1 and Ov-GST-2, respectively). Interestingly, Ov-GST-3 shares higher sequence identity with GSTs that are upregulated due to environmental stress. In order to confirm the specific upregulation of the Ov-GST-3 transcripts identified by differential display and to analyze the mRNA levels of the other Ov-GSTs (Ov-GST-1 and Ov-GST-2) under elevated stress conditions, a semi-quantitative polymerase chain reaction-enzyme-linked immunosorbent assay was performed. The Ov-GST-3 gene transcript level increased dramatically in response to xanthine-xanthine oxidase and to a lesser extent with paraquat and plumbagin. In contrast, Ov-GST-1 and Ov-GST-2 did not show any significant alterations in their steady-state mRNA levels in response to oxidative stress when examining the same mRNA samples. The present study clearly demonstrates that Ov-GST-3 is a critical enzyme in the defense against oxidative stress.

DNA sequence heterogeneity in the genes of T-even type Escherichia coli phages encoding the receptor recognizing protein of the long tail fibers.

Genes (g) 36 and 37 code for the proteins of the distal half of the long tail fibers of phage T4, gene product (gp) 35 links the distal half to the proximal half of this fiber. The receptor, lipopolysaccharide, most likely is recognized by gp37. Using as probe a restriction fragment consisting of most of g36 and g37 of phage T4 the genes corresponding to g35, g36, and g37 of phages T2 and K3 (using the E. coli outer membrane proteins OmpF and OmpA, respectively, as receptors) have been cloned into plasmid pUC8. Partial DNA sequences of g37 of phage K3 have been determined. One area, corresponding to residues 157 to 210 of the 1026 residue gp37 of phage T4, codes for an identical sequence in phage K3. Another area corresponds to residues 767 to 832 of the phage T4 sequence. Amino acid residues 786 to 832 of the T4 sequence are almost identical in both phage proteins while the remainder is rather different. DNAs of T2, T4, T6, another T-even type phage using protein Tsx as a receptor, and 10 different T-even type phages using the OmpA protein as a receptor have been hybridized with restriction fragments covering various parts of the g37 area of phage K3. With probably only one exception all of the 13 phages tested possess unique genes 37 and within the majority of these, sequences highly homologous to parts of g37 of K3 are present in a mosaic type fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of DNA, encoding parts of bacteriophage tail fiber genes, in the chromosome of Escherichia coli K-12.

The classical T-even bacteriophages recognize host cells with their long tail fibers. Gene products 35, 36, and 37 constitute the distal moiety of these fibers. The free ends of the tail fibers, which are formed by the CO2H terminus of gene product 37, possess the host range determinants. It was found that 4 out of 10 different strains of Escherichia coli K-12 contained regions of chromosomal DNA which hybridized with a probe consisting of genes 35, 36, and 37 of the T-even phage K3. From one strain this homologous DNA, which was associated with an EcoRI fragment of about 5 kilobases, was cloned into plasmid pUC8. Two independently recovered hybrid plasmids had undergone a peculiar rearrangement which resulted in the loss of about 3 kilobases of cloned DNA and a duplication of both the vector and the remaining chromosomal DNA. The mechanisms causing this duplication-deletion may be related to that of transposases. The cloned DNA was capable of recombination with phage T4 gene 36 and a phage T2 gene 37 amber mutant. DNA sequencing revealed the existence of regions of identity between the cloned DNA and genes 36 and 37 of phage T2. In addition, after growth of a derivative of phage K3 on a strain harboring T2 DNA, it was found that this phage contained the same parts of the T2 tail fiber genes which had been recovered from the bacterial chromosome. There appears to be little doubt that the phage had picked up this DNA from the host. The possibility is considered that a repertoire of parts of genes 36 and 37 of various T-even-type phages is present in their hosts, allowing the former to change their host ranges.

Purification of gelatin-specific neutral protease from human skin by conventional and high-performance liquid chromatography.

Human skin, maintained in serum-free organ culture, secretes a neutral metalloendopeptidase which is remarkably specific for gelatin. Because the product peptides from the action of collagenase on collagen become denatured into random coil polypeptides of 25000 and 75000 daltons at physiological temperature, it is thought that this "gelatinase" is the second, and possibly the only other enzyme in the pathway of extracellular collagen degradation. New types of high-performance liquid chromatography (HPLC) columns have enabled us to improve the yields of active gelatinase from skin culture medium. Raw medium, which has been dialyzed and lyophilized, is fractionated with ammonium sulfate, and applied to Pharmacia Blue Sepharose in a batch step. The 0.4 M sodium chloride eluate is then subjected to gel filtration on Sephacryl S-200, followed by gradient elution from Amicon Green Sepharose. The fractions with gelatinolytic activity are applied to a Bio-Rad TSK-Phenyl-5PW HPLC column for mild hydrophobic chromatography with a gradient of decreasing ammonium sulfate concentration. In the final step, the enzyme is applied to a Pharmacia Mono-Q FPLC column and eluted with a gradient of sodium chloride. At this point, the enzyme appears as two bands, corresponding to enzymatic activity zymograms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The nucleotide sequences of the tail fiber gene 36 of bacteriophage T2 and of genes 36 of the T-even type Escherichia coli phages K3 and Ox2.

Genes 36 have been cloned from phage T2 and the T-even type phages K3 and Ox2. The products of these genes are part of the long tail fibers of the phages, they form the proximal moiety of the distal half fiber. The genes have been sequenced, the nucleotide sequence of gene 36 of phage T4 is known (Oliver, D.B. & Crowther, R.A. (1981) J.Mol.Biol. 153, 545-568). Comparison of the deduced amino acid sequences of the four proteins revealed a surprising pattern. These sequences can be divided into two highly conserved and one very variable region. The former consist of about 60 NH2-terminal and 70 CO2H-terminal residues flanking the variable middle region of about 100 residues. Thus, an identical and unique morphology can be formed by a number of different primary structures. It is proposed that the conserved areas are involved in binding of the proteins to the neighboring products of genes 35 and 37 and that this function has put constraints on the variability of the primary protein structure. The overall amino acid composition of the proteins is rather similar; the codon usage is that known for phage T4. The intercistronic region between genes 35 and 36 consisting of 62 base pairs and containing a presumed terminator for g35 transcription and the 'late type' promoter for transcription of genes 36, 37, and 38, is almost completely identical in the four phages.

Export incompatibility of N-terminal basic residues in a mature polypeptide of Escherichia coli can be alleviated by optimising the signal peptide.

Export of the outer membrane protein, OmpA, across the cytoplasmic membrane of Escherichia coli was severely inhibited by the presence of two, three, four or six additional basic residues at the N-terminus of the mature polypeptide, but not by three similarly positioned acidic residues. Because a few bacterial proteins do possess basic residues close to the leader peptidase cleavage site and because the type of inhibition described here could pose problems in the construction of hybrid secretory proteins, we also studied means of alleviating this form of export incompatibility. Inhibition was abolished when basic residues were preceded by acidic ones. Also, the processing rates of the mutants with two or six basic residues could be partially restored by increasing the length of the hydrophobic core of the signal peptide. Taking this as a precedent, it is suggested that the structure of the signal peptide is an important feature for maintenance of a reasonable rate of translocation of those exported proteins which possess basic residue(s) at the N-terminus of the mature polypeptide.

An artificial hydrophobic sequence functions as either an anchor or a signal sequence at only one of two positions within the Escherichia coli outer membrane protein OmpA.

The 325-residue outer membrane protein, OmpA, of Escherichia coli, like most other outer membrane proteins with known sequence, contains no long stretch of hydrophobic amino acids. A synthetic oligonucleotide, encoding the sequence Leu-Ala-Leu-Val, was inserted four times between the codons for amino acid residues 153 and 154 and two, three, or four times between the codons for residues 228 and 229, resulting in the OmpA153-4, OmpA-228-2, -3, and -4 proteins, respectively. In the first case, the lipophilic sequence anchored the protein in the plasma membrane. In the OmpA228 proteins, 16 but not 12 or 8 lipophilic residues most likely also acted as an anchor. By removal of the NH2-terminal signal peptide, the function of the insert in OmpA153-4 was converted to that of a signal-anchor sequence. Possibly due to differences in amino acid sequences surrounding the insert, no signal function was observed with the insert in OmpA228-4. Production of the OmpA153-4 protein, with or without the NH2-terminal signal sequence, resulted in a block of export of chromosomally encoded OmpA. Clearly, long hydrophobic regions are not permitted within proteins destined for the bacterial outer membrane, and these proteins, therefore, have had to evolve another mechanism of membrane assembly.

Internal deletions in the gene for an Escherichia coli outer membrane protein define an area possibly important for recognition of the outer membrane by this polypeptide.

A series of overlapping deletions has been constructed in the ompA gene which encodes the 325-residue Escherichia coli outer membrane protein OmpA. Immunoelectron microscopy showed that the OmpA fragments were either located in the periplasmic space or were associated with the outer membrane. Apparently an area between residues 154 and 180 is required for this association; all proteins missing this area were found to be periplasmic. The nature of this association remained unknown; no membrane-protected tryptic fragments could be identified for any of these polypeptides. Hybrid genes were constructed encoding parts of the periplasmic maltose binding protein and an area of the ompA gene coding for residues 154-274. The corresponding proteins were not localized to the outer membrane but remained attached to the outer face of the plasma membrane, possibly because the normal mechanism of release from this membrane was impaired. In the OmpA protein the conspicuous sequence Ala180-Pro-Ala-Pro-Ala-Pro-Ala-Pro187 exists. Frameshift mutants were constructed to eliminate this sequence. There was no effect on the incorporation of the mutant proteins into the outer membrane. Thus, this "hinge" region is not involved in sorting. A proposal suggesting the existence of a sorting signal common to several outer membrane proteins (Benson, S. A., Bremer, E., and Silhavy, T. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3830-3834) was subsequently rejected (Bosch, D., Leunissen, J., Verbakel, J., de Jong, M., van Erp, H., and Tommassen, J. (1986) J. Mol. Biol. 189, 449-455; Freudl, R., Schwarz, H., Klose, M., Movva, N. R., and Henning, U. (1985) EMBO J. 4, 3593-3598). Although it is not known whether or not the outer membrane association observed represents a step in the normal sorting mechanism, it is concluded that it remains an open question whether or not a sorting signal, as proposed originally, exists in outer membrane proteins.

Identification of stress-responsive genes in Caenorhabditis elegans using RT-PCR differential display.

In order to identify genes that are differentially expressed as a consequence of oxidative stress due to paraquat we used the differential display technique to compare mRNA expression patterns in Caenorhabditis elegans . A C.elegans mixed stage worm population and a homogeneous larval population were treated with 100 mM paraquat, in parallel with controls. Induction of four cDNA fragments, designated L-1, M-47, M-96 and M-132, was confirmed by Northern blot analysis with RNA from stressed and unstressed worm populations. A 40-fold increase in the steady-state mRNA level in the larval population was observed for the L-1/M-47 gene, which encodes the detoxification enzyme glutathione S-transferase. A potential stress-responsive transcription factor (M-132) with C2H2-type zinc finger motifs and an N-terminal leucine zipper domain was identified. The M-96 gene encodes a novel stress-responsive protein. Since paraquat is known to generate superoxide radicals in vivo , the response of the C.elegans superoxide dismutase (SOD) genes to paraquat was also investigated in this study. The steady-state mRNA levels of the manganese-type and the copper/zinc-type SODs increased 2-fold in the larval population in response to paraquat, whereas mixed stage populations did not show any apparent increase in the levels of these SOD mRNAs.

Cleavage specificity of type IV collagenase (gelatinase) from human skin. Use of synthetic peptides as model substrates.

Type IV collagenase (gelatinase) has a marked substrate specificity for denatured collagen (gelatin). Cleavage site specificity of type IV collagenase from human skin was determined using small collagenous peptides with varied sequences around Gly-Leu or Gly-Ile. Type IV collagenase showed essentially the same order of preference for the peptide substrates as did interstitial collagenase. Both required a peptide with a minimum of six amino acid residues to demonstrate significant gelatinolytic activity and were able to cleave uncharged molecules more rapidly than charged molecules. the repeating Gly-X-Y-Gly sequence of collagen is not an absolute requirement for either enzyme since both digested AcPro-Leu-Gly-Ile-Leu-Ala-Ala-OC2H5 at 70% of the rate of the best substrate peptide, AcPro-Leu-Gly-Leu-Leu-Gly-OC2H5. Km and kcat (Vmax) values were determined for several of the peptides and for the native substrate. Turnover numbers with type IV collagenase were similar to those with interstitial collagenase (Weingarten, H., Martin, R., and Feder, J. (1985) Biochemistry 24, 6730-6734). However, the Km for all peptides investigated was approximately 10-fold lower for type IV collagenase than for interstitial collagenase. Because type IV collagenase does not cleave helical interstitial collagens, the data support the conclusion that secondary structure determines whether the peptide bond can be hydrolyzed at any potential cleavage site.