Biomedical subjects

P Mathias

Publications and source records attributed to P Mathias.

At least 19 recordsLinked to original sources

PubMed2001

DNA strand breaks, oxidative damage, and 1-OH pyrene in roofers with coal-tar pitch dust and/or asphalt fume exposure.

OBJECTIVE: To determine the potential for asphalt fume exposure to increase DNA damage, we conducted a cross-sectional study of roofers involved in the application of roofing asphalt. METHODS: DNA strand breaks and the ratio of 8-hydroxydeoxyguanosine (8-OHdG) to 2-deoxyguanosine (dG) were measured in peripheral blood leukocytes of roofers. In addition, urinary excretion of 8-OHdG and 8-epi-prostaglandin F2alpha (8-epi-PGF) was also measured. The study population consisted of 26 roofers exposed to roofing asphalt and 15 construction workers not exposed to asphalt during the past 5 years. A subset of asphalt roofers (n = 19) was exposed to coal-tar pitch dust (coal tar) during removal of existing roofs prior to applying hot asphalt. Personal air monitoring was performed for one work-week to measure exposure to total particulates, benzene-soluble fraction of total particulates, and polycyclic aromatic compounds (PACs). Urinary 1-OH-pyrene levels were measured as an internal biomarker of PAC exposure. RESULTS: Full-shift breathing zone measurements for total particulates, benzene-solubles and PACs were significantly higher for coal-tar exposed workers than for roofers not exposed to coal tar. Similarly, urinary 1-OH-pyrene levels were higher in coal-tar exposed roofers than roofers not exposed to coal tar. Total particulates or benzene-soluble fractions were not associated with urinary 1-OH-pyrene, but PAC exposure was highly correlated with urinary 1-OH-pyrene. When stratified by 1-OH-pyrene excretion, DNA strand breaks increased in a dose-dependent manner, and leukocyte 8-OHdG/dG decreased in a dose-dependent manner. Significant changes in DNA damage appeared to be linked to PACs from coal-tar exposure, although asphalt fume alone was associated with a small but significant increase in urinary 1-OH-pyrene and DNA strand breaks. CONCLUSIONS: Results are consistent with previous reports that asphalt or coal-tar exposure can cause DNA damage. Urinary 8-epi-PGF remained relatively constant during the week for virtually all subjects, regardless of exposure indicating that neither asphalt nor coal-tar exposure induces an overt oxidative stress. A small, but statistically significant increase in 8OHdG was evident in end-of-week urine samples compared with start-of-week urine samples in roofers exposed to coal-tar. The increase in urinary 8OHdG coupled with the decrease in leukocyte 8-OHdG/dG, suggests that coal-tar exposure induces protective or repair mechanisms that result in reduced levels of steady-state oxidative-DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2000

50-Hertz magnetic field and calcium transients in Jurkat cells: results of a research and public information dissemination (RAPID) program study.

An effect on intracellular calcium continues to be proposed as a biochemical pathway for the mediation of biologic effects of electrical-power-frequency magnetic fields (MF). However, reproducible results among laboratories are difficult to attain and the characteristics of magnetic field effects on intracellular free calcium ([Ca(2+)](i)) are not well understood. We attempted to repeat the studies of Lindström et al. [Intracellular Calcium Oscillations in a T-Cell Line by a Weak 50 Hz Magnetic Field. J Cell Physiol 156:395-398 (1993)] by investigating the effect of a 1.5-G 50-Hz MF on [Ca(2+)](i) in the Jurkat lymphocyte T-cell line. Changes in [Ca(2+)](i) were determined using microscopic imaging of fura-2 loaded Jurkat cells on poly-l-lysine-coated glass coverslips. The MF was generated by a single coil constructed with bifilar wire and located in the same plane as the cells. Cells were randomly exposed for 8 min to MF, sham field (SF), or no field (NF) conditions. The exposure condition remained coded until data analysis was complete. Each exposure period was preceded by an 8-min data collection to establish a baseline for [Ca(2+)](i). After each exposure condition, cells were exposed to anti-CD3 antibody that induced a rapid increase in [Ca(2+)](i) in responsive cells; this provided a positive control. [Ca(2+)](i) was analyzed for individual cells as spatially-averaged background-corrected 340/380 nm ratios, and a [Ca(2+)](i) transient was considered significant for positive deviations from baseline of 3 [multiple] an estimate of noise in the baseline. Typically, 25-50 cells/field were viewed and approximately 50% had no [Ca(2+)](i) transients in the baseline period and also responded to positive control. Only cells responding to positive control and lacking changes in [Ca(2+)](i) during the baseline period were considered qualified for assessment during the exposure period. The incidences of [Ca(2+)](i) transients during the exposure period for two experiments (40 [multiple] objective) were 16.5, 14.6, and 14.2% for MF, SF, and NF, respectively, and were not statistically significantly different. Previous studies by Lindström et al. [Intracellular Calcium Oscillations in a T-Cell Line after Exposure to Extremely-Low-Frequency Magnetic Fields with Variable Frequencies and Flux Densities. Bioelectromagnetics 16:41-47 (1995)] showed a high response rate (92%) for exposure to 1. 5-G 50-Hz MF when individual cells were preselected for investigation. We found no such effect when examining many cells simultaneously in a random and blind fashion. These results do not preclude an effect of MF on [Ca(2+)](i), but suggest that responsive cells, if they exist, were not identified using the approaches that we used in this study.

Calcium↗

PubMed1999

Oxidative stress and DNA damage in Fischer rats following acute exposure to trichloroethylene or perchloroethylene.

Oxidative DNA damage is emerging as an biomarker of effect in studies assessing the health risks of occupational chemicals. Trichloroethylene (TCE) and perchloroethylene (PERC) are used in the dry cleaning industry and their metabolism can produce reactive oxygen compounds. The present study examined the potential for TCE and PERC to induce oxidative DNA damage in rats that was detectable as increased urinary excretion of 8-hydroxydeoxyguanosine (8OHdG). Thiobarbaturic acid reactive substances (TBARS) and 8-epiprostaglandin F2alpha (8epiPGF) were also measured as biomarkers of increased oxidative stress. Male Fischer rats were administered a single i.p. injection of 0, 100, 500, or 1000 mg/kg of PERC or TCE. Control rats received only vehicle (1:4 v/v of Alkamuls/water). A positive control group received 100 mg/kg 2-nitropropane (2NP). Rats were sacrificed 24 h after dosing. In rats receiving 2NP or TCE but not PERC, TBARS and the 8OHdG/dG ratios were significantly elevated in liver. Lymphocyte 8OHdG/dG was not affected significantly by 2NP, TCE or PERC. In rats receiving 2NP, urinary excretion of 8OHdG and 8epiPGF2 were significantly increased. In rats receiving TCE or PERC, significant increases in 8epiPGF2 or 8OHdG were not evident. Results indicate that a single high dose of TCE, but not PERC, can induce an increase in oxidative DNA damage in rat liver. However, the usefulness of 8OHdG as a biomarker of TCE-induced oxidative DNA damage is questionable.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1999

Structure of adenovirus complexed with its internalization receptor, alphavbeta5 integrin.

The three-dimensional structure of soluble recombinant integrin alphavbeta5 bound to human adenovirus types 2 and 12 (Ad2 and -12) has been determined at approximately 21-A resolution by cryoelectron microscopy (cryo-EM). The alphavbeta5 integrin is known to promote Ad cell entry. Cryo-EM has shown that the integrin-binding RGD (Arg-Gly-Asp) protrusion of the Ad2 penton base protein is highly mobile (P. L. Stewart, C. Y. Chiu, S. Huang, T. Muir, Y. Zhao, B. Chait, P. Mathias, and G. R. Nemerow, EMBO J. 16:1189-1198, 1997). Sequence analysis indicated that the Ad12 RGD surface loop is shorter than that of Ad2 and probably less flexible, hence more suitable for structural characterization of the Ad-integrin complex. The cryo-EM structures of the two virus-receptor complexes revealed a ring of integrin density above the penton base of each virus serotype. As expected, the integrin density in the Ad2 complex was diffuse while that in the Ad12 complex was better defined. The integrin consists of two discrete subdomains, a globular domain with an RGD-binding cleft approximately 20 A in diameter and a distal domain with extended, flexible tails. Kinetic analysis of Ad2 interactions with alphavbeta5 indicated approximately 4.2 integrin molecules bound per penton base at close to saturation. These results suggest that the precise spatial arrangement of five RGD protrusions on the penton base promotes integrin clustering and the signaling events required for virus internalization.

Adenoviruses, Human↗

PubMed1998

Interactions of soluble recombinant integrin alphav beta5 with human adenoviruses.

alphav integrins have been identified as coreceptors for adenovirus (Ad) internalization; however, direct interactions of these molecules with Ad have not been demonstrated. We report here the expression of soluble integrin alphav beta5, which retains the ability to recognize the Ad penton base as well as vitronectin, an Arg Gly Asp (RGD)-containing extracellular matrix protein. Soluble integrin alphav beta5 reacted with seven different Ad serotypes (subgroups A to E) in solid-phase binding assays. The soluble integrin exhibited different levels of binding to each Ad serotype; however, binding to multiple Ad types required the presence of divalent metal cations and was inhibited by a synthetic RGD peptide, indicating that RGD and cation-binding sequences regulate Ad interactions with alphav beta5. Incubation of Ad particles with soluble alphav beta5 integrin also inhibited subsequent Ad internalization into epithelial cells as well as virus attachment to monocytic cells. These findings suggest that soluble alphav integrins or antagonists of these coreceptors could be used to limit infection by multiple Ad types. The generation of soluble alphav integrins should also permit further detailed kinetic and structural analysis of Ad interactions with its coreceptors.

Adenoviruses, Human↗

PubMed1997

Growth arrest of Epstein-Barr virus immortalized B lymphocytes by adenovirus-delivered ribozymes.

Epstein-Barr virus (EBV) infection is associated with several human diseases that involve unrestricted proliferation of B lymphocytes. EBV nuclear antigen 1 (EBNA-1) is expressed in all EBV-infected cells and plays an essential role in persistence of the EBV genome. EBNA-1 has also been reported to have oncogenic potential. As an approach for treating EBV infections, we examined the capacity of EBNA-1 ribozymes delivered by recombinant adenoviruses to suppress EBNA-1 expression and to block virus-induced B cell proliferation. In contrast to primary B cells, EBV-transformed B lymphoblastoid cell lines expressed alphav integrins, the adenovirus internalization receptors, and were also susceptible to adenovirus-mediated gene delivery. Adenovirus delivery of a specific ribozyme (RZ1) to lymphoblastoid cell lines, suppressed EBNA-1 mRNA and protein expression, significantly reduced the number of EBV genomes, and nearly abolished cell proliferation in low serum. Adenovirus delivery of RZ1 also prevented EBV infection of an established EBV-negative B cell line. These studies demonstrate the potential use of adenovirus-encoded ribozymes to treat EBV-induced lymphoproliferative disorders.

Adenoviridae↗

PubMed1997

Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization.

Interaction of the adenovirus penton base protein with alpha v integrins promotes virus entry into host cells. The location of the integrin binding sequence Arg-Gly-Asp (RGD) on human type 2 adenovirus (Ad2) was visualized by cryo-electron microscopy (cryo-EM) and image reconstruction using a mAb (DAV-1) which recognizes a linear epitope, IRGDTFATR. The sites for DAV-1 binding corresponded to the weak density above each of the five 22 A protrusions on the adenovirus penton base protein. Modeling of a Fab fragment crystal structure into the adenovirus-Fab cryo-EM density indicated a large amplitude of motion for the Fab and the RGD epitope. An unexpected finding was that Fab fragments, but not IgG antibody molecules, inhibited adenovirus infection. Steric hindrance from the adenovirus fiber and a few bound IgG molecules, as well as epitope mobility, most likely prevent binding of IgG antibodies to all five RGD sites on the penton base protein within the intact virus. These studies indicate that the structure of the adenovirus particle facilitates interaction with cell integrins, whilst restricting binding of potentially neutralizing antibodies.

Adenoviridae↗

PubMed1997

Apoptosis signaling pathway in T cells is composed of ICE/Ced-3 family proteases and MAP kinase kinase 6b.

Fas/APO-1(CD95) ligation activates programmed cell death, a cellular process that plays an important role in the maturation of the host immune response. We show that activation of a specific MAP kinase kinase (MKK), MKK6b, is necessary and sufficient for Fas-induced apoptosis of Jurkat T cells. MKK6b activation occurs downstream of an interleukin-1 converting enzyme-like (ICE-like) protease(s), while execution of the apoptotic pathway by MKK6b requires both ICE- and CPP32-like proteases. Surprisingly, the p38 MAP kinase protein, a known substrate of MKK6b, does not participate in Fas/MKK6b-mediated apoptosis. These findings indicate a divergence of the MKK6b signaling pathways, one of which activates p38 and leads to regulation of gene expression, and one of which activates the ICE/Ced-3 family of proteases and leads to cell death. These studies represent a demonstration of an apoptotic pathway that is comprised of both the ICE/Ced-3 family of proteases and MAP kinase kinase 6.

Amino Acid Sequence↗

PubMed1995

Arachidonic acid disrupts calcium dynamics in neonatal rat cardiac myocytes.

OBJECTIVES: The purpose of this study was to investigate the effects of prolonged arachidonic acid (AA) exposure on electrically induced fluctuations of cytosolic free Ca2+ concentration ([Ca2+]i) in cardiac myocytes and to identify intracellular biochemical events that may play a role in the actions of AA on [Ca2+]i dynamics. METHODS: Electrically induced [Ca2+]i transients were investigated in cultured single neonatal rat ventricular myocytes using spectrofluorometric analysis of fura-2-[Ca2+]i binding. KCl-induced depolarization, caffeine and ryanodine were used to assess the effects of AA on Ca2+ handling by the sarcolemma and the sarcoplasmic reticulum. Prostanoid formation was measured with an ELISA technique. alpha-Tocopherol was used to determine if free radical formation was a factor in the AA effects on [Ca2+]i. RESULTS: Exposure to 10-30 microM AA produced a concentration-dependent and reversible configuration change and eventually a cessation of [Ca2+]i transients. Continued exposure resulted in a Ca2+ overload (tonic [Ca2+]i greater than peak systolic [Ca2+]i). AA did not influence KCl-induced [Ca2+]i increase but did eliminate caffeine-induced [Ca2+]i transients. AA exposure stimulated the formation of 6-oxo-prostaglandin F1 alpha in a concentration-dependent manner, but thromboxane B2 formation was not influenced. alpha-Tocopherol pretreatment significantly delayed times till cessation of [Ca2+]i transients and Ca2+ overload, whereas ryanodine and cyclo-oxygenase inhibitors were without effect. CONCLUSIONS: The present data provide evidence that the initial action of AA on [Ca2+]i transients during excitation-contraction coupling involves an effect of AA on sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ handling. AA-induced cessation of electrically induced [Ca2+]i transients and Ca2+ overload may involve the formation of free radicals.

6-Ketoprostaglandin F1 alpha↗

PubMed1994

Multiple adenovirus serotypes use alpha v integrins for infection.

The nucleotide sequence encoding the penton base integrin-binding domains of several human adenoviruses was obtained by homology PCR. Each of the penton base proteins contains a conserved Arg-Gly-Asp (RGD) sequence that is predicted to lie at the apex of two extended alpha helices. The penton base RGD domain promotes efficient infection of host cells by multiple adenovirus serotypes via interaction with alpha v integrins, thus indicating that alpha v integrins play a central role in the entry of adenoviruses into host cells.

Adenoviruses, Human↗

PubMed1993

Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment.

Adenovirus contains a heterodimeric protein complex consisting of 186 kd fiber protein that mediates high affinity virus attachment to cells and a 400 kd pentavalent subunit (penton base) that contains five Arg-Gly-Asp sequences, implying a role for integrins in adenovirus infection. We demonstrate that the vitro-nectin-binding integrins alpha v beta 3 and alpha v beta 5 promote viral infection in a novel way since antibodies against these receptors or soluble penton base block virus internalization without affecting attachment. Moreover, adenovirus binds to cultured cells lacking alpha v integrins but fail to become internalized, thus restricting infection of these cells. Transfection of alpha v(-) cells with a cDNA encoding alpha v results in the expression of integrins alpha v beta 3 and alpha v beta 5 and allows virus internalization and infection. These data indicate that adenovirus attachment and uptake into cells are separate but cooperative events that result from the interaction of distinct viral coat proteins with a receptor for attachment and alpha v integrin receptors for internalization.

Adenoviruses, Human↗

PubMed1993

Murine C4b-binding protein. Mapping of the ligand binding site and the N-terminus of the pre-protein.

We have constructed cell surface-bound forms of murine C4b-binding protein (mC4BP) that allowed us to monitor the binding of mC4BP to C4b with relatively simple erythrocyte rosette assays. We used two types of surface-bound mC4BP: one in which segments of mC4BP were fused directly to a peptide containing the transmembrane and cytoplasmic domains of human complement receptor CR2 (BPR1-type); and a second in which the same segments were fused to a longer peptide containing the five membrane-proximal short consensus repeats (SCR) of CR2 as well as the transmembrane and cytoplasmic domains (BPR2-type). COS cells transfected with either construct carrying all six mC4BP SCR rosetted with C4b-bearing EAC14 cells but not with C4b-lacking EAC1 cells; and rosetting was inhibited by excess inactivated C4 but not inactivated C3. COS cells transfected with BPR2 constructs carrying only SCR 1-3 or 1-4 gave similar rosetting behavior. However, rosetting was not observed with BPR2 constructs carrying only SCR 1-2 or 2-6, or with BPR1 constructs carrying only SCR 1-2, 1-3, 1-4, or 2-6. Finally, we found that alteration of the AUG sequence 56 triplet codons upstream of the putative N-terminus of mature mC4-BP eliminates rosetting whereas alteration of a second AUG sequence 13 codons upstream has no effect on rosetting. These results indicate that 1) SCR 1-3 of mC4BP are necessary and adequate for binding to C4b, 2) steric effects close to the cell surface may interfere with binding, and 3) mC4BP has an extraordinarily long 56 amino acid residue signal peptide.

Amino Acid Sequence↗

PubMed1993

Blockade of the 32P phosphate flush of pancreatic beta cells from adult rats who received a low-protein diet during early lactation.

In order to study the effect of nutrition on the onset of disturbances in Wistar rat pancreatic beta cells, we compared the effects of a low protein diet (8% protein) and a normal protein diet (25% protein) supplied to the dams (6 in each group) during the first 12 days of lactation. The parameter evaluated was the beta cells phosphate flush in response to stimulatory concentration of glucose (16.7 mM) of isolated islets of Langerhans from 60-day old pups. Using a collagenase digestion technique, islets were isolated from the pups and the 32P fractional outflow rate (FOR) of the beta cells was used as a metabolic index in both experimental groups (N = 36). We observed that although the weights of the pups of the two groups were not significantly different at 60 days of age (control = 186 +/- 18 g; undernourished during lactation = 179 +/- 19 g), the typical phosphate flush response (FOR = 2.4 +/- 0.4%/min) to a stimulatory glucose concentration (16.7 mM) was abolished in the rats from undernourished mothers. Our data are consistent with the hypothesis that undernutrition may be an important cause of diabetes mellitus type II.

Animals↗

PubMed1992

Mutants of complement component C3 cleaved by the C4-specific C1-s protease.

To identify some of the structural features determining specific protease recognition of complement components C3 and C4, we used site-specific mutagenesis to construct mutants of murine C3 that are cleaved by the C4-specific C1-s protease. Insertion of three amino acid residues corresponding to residues at the C1-s cleavage site of human C4 into murine C3 at the analogous C3 convertase cleavage site was adequate to render the mutant protein susceptible to C1-s cleavage. In addition, insertion of C3-specific residues at the same site or introduction of the C4-specific residues as substitutions rather than as an insertion also rendered the site susceptible to cleavage, but with 10- to 50-fold lower efficiencies, and insertion of even a single amino acid residue affected recognition by C1-s. Finally, insertion of amino acid residues into mC3 partially inhibited cleavage by the alternative-pathway C3 convertase, with insertion of C3- or C4-specific residues giving about the same level of inhibition. A simple interpretation of these data is that C1-s cleavage is dependent primarily on steric accessibility and on recognition of specific amino acid residues at the cleavage site, whereas C3 convertase cleavage is dependent primarily on specific interactions distal to the cleavage site, with only relatively weak, non-C3-specific interactions at the cleavage site itself.

Amino Acid Sequence↗

PubMed1992

Serum selenium levels in individuals on PKU diets.

Serum selenium levels in 73 patients with phenylketonuria were significantly lower than in controls. The phenylketonuric and hyperphenylalaninaemic individuals taking the non-supplemented amino acid mixture generally had lower levels: 36% were below the normal range as defined by our laboratory, compared with 19% in the supplemented group. The low levels were present even in those on diet, who had a greater phenylalanine tolerance--that is, a tolerance for more than 9 x 50 mg phenylalanine exchanges per day, in other words a higher intake of natural protein. Individuals on long-term synthetic diets may be at risk for selenium deficiency even on selenium supplements. In areas where the soil may be low in selenium, the deficiency may be aggravated. Long-term low levels may impair health but the required amount of selenium supplementation remains uncertain.

Adolescent↗

PubMed1992

The role of free radicals as mediators of endothelial cell injury in hyperhomocysteinemia.

Hyperhomocysteinemia has been suggested as a potent new risk factor for premature cardiovascular disease. Homocsyteine can induce endothelial cell injury but the mechanism is not understood. The purpose of this study was to evaluate the role of free radicals as potential causes of endothelial cell injury in a case-control study of obligate heterozygotes for cystathionine beta-synthase deficiency. Firstly, free radical production as measured by neutrophil chemiluminescence in obligate heterozygotes for cystathionine beta-synthase deficiency was compared with age- and sex-matched normal subjects. Secondly, the response of the cellular antioxidant system was examined by measuring the enzymes superoxide dismutase and glutathione peroxidase, their cofactors (selenium, copper), vitamin E and vitamin A in heterozygotes and normal subjects. Analyses of neutrophil chemiluminescence, vitamin A and E, glutathione peroxidase, selenium and copper showed no difference between heterozygotes and controls. While superoxide dismutase activity was higher in heterozygotes than normal subjects, the difference did not reach statistical significance and the hypothesis of excess free radical production as a mechanism of injury was not confirmed. However, further examination of superoxide dismutase activity in a larger number of subjects would be of interest.

Adult↗

PubMed1990

Conversion of the C4d.2 serologic allotype of murine complement component C4 to the C4d.1 allotype by site-specific mutagenesis.

C4d.1 and C4d.2 are serologically defined allotypes of murine complement component C4. Previous studies in Shreffler's laboratory have shown that the structural difference between the two allotypes lies within a single tryptic peptide of the C4 alpha-chain and that the sequences of this fragment from the two allotypes (determined from nucleic acid sequences of genomic clones) differ only by the substitution of arginine in C4d.2 for glutamine in C4d.1. Hence this single amino acid change apparently is responsible for the rather striking serological difference between the two allotypes. To test this conclusion, we have used site-specific mutagenesis to alter the sequence of a full-length C4 cDNA that was derived from a mouse strain expressing the C4d.2 allotype. We substituted a glutamine codon for the arginine codon at the specified site and expressed both mutant and parent recombinant C4 proteins by transient transfection of COS cells. We found that an alloantiserum specific for C4d.1 reacts with the mutant protein but not the parent whereas an alloantiserum specific for C4d.2 reacts with the parent protein, as expected, but not the mutant. These results confirm that a single amino acid difference specifies the C4d.1 and C4d.2 allotypes.

Alleles↗

PubMed1989

Murine complement component C4 and sex-limited protein: identification of amino acid residues essential for C4 function.

Murine sex-limited protein (Slp) is an isotype of murine complement component C4 that shares 95% sequence identity with C4 as well as the intramolecular thioester necessary for C4 function but has no complement activity. Slp is nonfunctional at least in part because it is not cleaved by the activated form of complement protease C1s (C1s), which proteolytically activates C4 in the classical complement pathway. Slp is also distinct from C4 in that its expression in some mouse strains is under testosterone control. In the present studies, we used site-directed mutagenesis of C4 and expression of the mutant proteins in cultured cells to identify the amino acid substitutions in Slp that are responsible for resistance to C1s cleavage. We focused on sequence changes immediately downstream of the cleavage site in C4 because the arginine at that site is conserved in Slp, but the downstream sequences diverge substantially, with six differences in the first 7 residues followed by a 3-residue deletion in Slp. We found that a C4 mutant carrying only the 3-residue deletion is not cleaved by C1s and has essentially no hemolytic activity, whereas a mutant carrying only the six replacement changes is cleaved by C1s and has normal hemolytic activity. Both mutants have intact thioesters. A third mutant in which two acidic residues in the segment deleted in Slp were replaced by aliphatic residues is also cleaved by C1s, has an intact thioester group, and has normal hemolytic activity. These results indicate that the downstream mutations are responsible for the resistance of Slp to C1s cleavage and suggest that the length rather than the specific sequence of this segment is critical in determining susceptibility to the protease.

Amino Acid Sequence↗