Coping strategies and risk of cardiovascular disease incidence and mortality: the Japan Public Health Center-based prospective Study.

AIMS: Coping strategies may be significantly associated with health outcomes. This is the first study to investigate the association between baseline coping strategies and cardiovascular disease (CVD) incidence and mortality in a general population cohort. METHODS AND RESULTS: The Japan Public Health Center-based prospective Study asked questions on coping in its third follow-up survey (2000-04). Analyses on CVD incidence and mortality included 57 017 subjects aged 50-79 without a history of CVD and who provided complete answers on approach- and avoidance-oriented coping behaviours and strategies. Cox regression models, adjusted for confounders, were used to determine hazard ratios (HRs) according to coping style. Mean follow-up time was 7.9 years for incidence and 8.0 years for mortality.The premorbid use of an approach-oriented coping strategy was inversely associated with incidence of stroke (HR = 0.85; 95% CI, 0.73-1.00) and CVD mortality (HR = 0.74; 95% CI, 0.55-0.99). Stroke subtype analyses revealed an inverse association between the approach-oriented coping strategy and incidence of ischaemic stroke (HR = 0.79; 95% CI, 0.64-0.98) and a positive association between the combined coping strategy and incidence of intra-parenchymal haemorrhage (HR = 2.03; 95% CI, 1.01-4.10). Utilizing an avoidance coping strategy was associated with increased mortality from ischaemic heart disease (IHD) only in hypertensive individuals (HR = 3.46; 95% CI, 1.07-11.18). The coping behaviours fantasizing and positive reappraisal were associated with increased risk of CVD incidence (HR = 1.24; 95% CI, 1.03-1.50) and reduced risk of IHD mortality (HR = 0.63; 95% CI, 0.40-0.99), respectively. CONCLUSION: An approach-oriented coping strategy, i.e. proactively dealing with sources of stress, may be associated with significantly reduced stroke incidence and CVD mortality in a Japanese population-based cohort.

Sugar-sweetened beverage and diet soda consumption and the 7-year risk for type 2 diabetes mellitus in middle-aged Japanese men.

PURPOSE: This cohort study investigated the association between sugar-sweetened beverage (SSB) and diet soda consumption and the incidence of type 2 diabetes in Japanese men. METHODS: The participants were 2,037 employees of a factory in Japan. We measured consumption of SSB and diet soda using a self-administered diet history questionnaire. The incidence of diabetes was determined in annual medical examinations over a 7-year period. Hazard ratios (HRs) with 95 % confidence intervals (CIs) for diabetes were estimated after adjusting for age, body mass index, family history, and dietary and other lifestyle factors. RESULTS: During the study, 170 participants developed diabetes. The crude incidence rates (/1,000 person-years) across participants who were rare/never SSB consumers, <1 serving/week, ≥ 1 serving/week and <1 serving/day, and ≥ 1 serving/day were 15.5, 12.7, 14.9, and 17.4, respectively. The multivariate-adjusted HR compared to rare/never SSB consumers was 1.35 (95 % CI 0.80-2.27) for participants who consumed ≥ 1 serving/day SSB. Diet soda consumption was significantly associated with the incident risk of diabetes (P for trend = 0.013), and multivariate-adjusted HRs compared to rare/never diet soda consumers were 1.05 (0.62-1.78) and 1.70 (1.13-2.55), respectively, for participants who consumed <1 serving/week and ≥ 1 serving/week. CONCLUSIONS: Consumption of diet soda was significantly associated with an increased risk for diabetes in Japanese men. Diet soda is not always effective at preventing type 2 diabetes even though it is a zero-calorie drink.

Serum gamma-glutamyltransferase and the risk of hyperuricemia: a 6-year prospective study in Japanese men.

We conducted a longitudinal study to investigate whether increased serum gamma-glutamyltransferase independently predicts subsequent development of hyperuricemia. The study participants included 3,310 Japanese men without hyperuricemia, aged 20-54 years. The participants had annual heath examinations for 6 years to assess incident hyperuricemia (defined as serum uric acid>416.4 µmol/l and/or taking medication for hyperuricemia). The risk of incident hyperuricemia was compared in participants grouped according to their baseline serum gamma-glutamyltransferase level. During follow-up, there were 529 incident cases of hyperuricemia. A positive, dose-response relationship was observed between serum gamma-glutamyltransferase and the risk of incident hyperuricemia. The hazard ratios (95% confidence intervals) for hyperuricemia, compared with a serum gamma-glutamyltransferase level ≤19 U/l, were 1.32 (1.05-1.67) for 20-39 U/l, 1.28 (0.90-1.83) for 40-59 U/l, 1.56 (0.98-2.47) for 60-79 U/l, and 1.57 (1.02-2.41) for ≥80 U/l after adjustment for baseline serum uric acid, creatinine, total cholesterol, and glycated hemoglobin levels, ln(serum alanine aminotransferase), age, systolic blood pressure, medications for hypertension, hypercholesterolemia, and diabetes, body mass index, and smoking and exercise habits. A similar positive relationship was observed regardless of the presence or absence of alcohol drinking, obesity, metabolic disorders (any combination of hypertension, hypercholesterolemia and/or diabetes), or clinically high serum aminotransferases, without evidence of a significant interaction between increased serum gamma-glutamyltransferase and risk factors for incident hyperuricemia. These findings indicate that increased serum gamma-glutamyltransferase is an independent predictor of subsequent development of hyperuricemia.

A novel protein that participates in nonself discrimination of malignant cells by homologous complement.

The human complement (C) system protects an individual against substances of nonself origin, including xenografts and microbial pathogens. Human cells express C-regulatory proteins, CD46 and CD55, thereby circumventing attack by C3, a major effector of C. Nevertheless, certain malignant cells, particularly those undergoing apoptotic stress, can activate homologous C, overcoming the regulatory actions of CD46 and/or CD55. The molecular mechanisms whereby malignant cells are tagged by homologous C3 remain largely unknown. We identified a novel gene product that converts human cells into targets for homologous complement. Only malignant cells and cell lines exposed to Fas or X-irradiation stimuli produced this protein, designated M161Ag, which was an unglycosylated 43-kDa protein. Analysis of cloned cDNAs indicated that this molecule was a secretory protein containing five amino acids encoded by TGA codons. Its functions were unique in that once secreted from the tumor cells, it bound back to the surface of these cells and activated homologous complement (C3) via the alternative pathway, allowing for C3 deposition on the membrane. This molecule may offer new insight into innate immunity; surveillance of tumor cells by complement is a common feature in the human immune system.

Bilirubin excretion in rats with normal and impaired bilirubin conjugation: effect of phenobarbital.

The effect of phenobarbital on bilirubin excretion was studied in rats with different capacities for bilirubin conjugation. Drug treatment induced substantial increases in bilirubin UDP-glucuronyl transferase activity in the liver of both normal and heterozygous Gunn rats, but not homozygous Gunn rats in which enzyme activity is completely absent. However, enhancement of bilirubin excretion in vivo was observed only in heterozygous Gunn rats. In these animals the maximum capacity to excrete bilirubin into bile (T(max)), like the activity of the conjugating enzyme, was half normal; phenobarbital caused an increase in T(max) to levels characteristic of normal animals, with a twofold rise in the excretion of conjugated pigment. This appeared to be largely unrelated to enhancement of bile flow, and there was no stimulation of alternate pathways of bilirubin excretion. Conjugated bilirubin was consistently recovered from the plasma and urine of both untreated normal and heterozygous Gunn rats infused with unconjugated pigment. The quantities thus recovered comprised a similar fraction of the total pigment conjugated in both types of animal. Moreover, there were linear correlations between T(max) and both the rate of bile flow and the activity of the conjugating enzyme over the range of values represented by control rats of both types. These findings suggest that the process by which conjugated bilirubin is secreted into the bile is closely related to conjugation and limits the final excretory rate at different levels of pigment excretion. The phenobarbital effect uniquely observed in heterozygous Gunn rats appears to be mediated primarily by enhancement of the limited capacity for bilirubin conjugation with an associated rise in functional secretory capacity.

Skipping breakfast and 5-year changes in body mass index and waist circumference in Japanese men and women.

OBJECTIVE: This study investigated the relationship between frequency of skipping breakfast and annual changes in body mass index (BMI) and waist circumference (WC). METHODS: The participants were 4,430 factory employees. BMI and WC were measured repeatedly at annual medical examinations over a 5-year period. The association between frequency of skipping breakfast at the baseline examination and annual changes in anthropometric indices was evaluated using the generalized estimating equation method. RESULTS: The mean (standard deviation) BMI was 23.3 (3.0) kg m-2 for men and 21.9 (3.6) kg m-2 for women; and the mean WC was 82.6 (8.7) cm for men and 77.8 (9.8) cm for women. During the follow-up period, mean BMI increased by 0.2 kg m-2 for men and women, and mean WC increased by 1.1 cm for men and 1.0 cm for women. The annual change in the BMI of men who skipped breakfast four to six times per week was 0.061 kg m-2 higher, and that of those who skipped breakfast seven times per week was 0.046 kg m-2 higher, compared with those who did not skip breakfast. Annual changes in the WC of male participants who skipped breakfast seven times per week was 0.248 cm higher than that of those who did not skip breakfast. Skipping breakfast was not associated with changes in BMI or WC in women. CONCLUSIONS: Skipping breakfast was closely associated with annual changes in BMI and WC among men, and eating breakfast more than four times per week may prevent the excessive body weight gain associated with skipping breakfast.

Purification and characterization of a novel substrate for plasma kallikrein (PK-120) in human plasma.

A 120 kDa plasma protein, which is susceptible to plasma kallikrein, was purified from human plasma by polyethylene glycol fractionation followed by ion exchange chromatography using Q-Sepharose, S-Sepharose, and hydroxyapatite and gel filtration on Sephacryl S-200. The 120 kDa protein, termed PK-120 in this paper, was a single polypeptide chain containing about 20% sugar by weight and its concentration in plasma was estimated to be 80 micrograms/ml by ELISA. At least three fragments, 100, 70, and 35 kDa, were produced from PK-120 by plasma kallikrein. The N-terminal sequence and Western blot demonstrated that PK-120 was first cleaved to yield the 100 and 35 kDa fragments, then the 100 kDa fragment was cleaved into the 70 kDa fragment. N-Terminal sequence analyses of PK-120 and its fragments demonstrated that it is a novel plasma protein, distinct from high molecular weight kininogen, a natural substrate for plasma kallikrein.

Purification and characterization of recombinant hamster tissue complement C1s.

Hamster complement C1s cDNA was inserted into expression plasmid BCMGSNeo, and transfected to SEA7 cells, A31 mouse fibroblasts transformed by polyoma virus. The transfectant secreted a large amount of recombinant C1s that was activated in the serum free culture medium and hydrolyzed acetyl-Gly-L-Lys-naphthyl ester (AGLNE). C1s was purified to a homogeneity from the culture medium of the transfectant by DEAE-Sephadex, Dymatrex orange A and size-exclusion HPLC. Purified hamster C1s consumed human complement in hemolytic assay and hydrolyzed gelatin in enzymography. To investigate the enzymic action of C1s at molecular levels, several antibodies were prepared against hamster C1s. One peptide (amino-acid residues 379-391) and two peptides (amino-acid residues 478-496 and 560-583) corresponding to the heavy and the light chain, respectively, were synthesized. The amino-acid sequences of these regions is not conserved between hamster and human C1s. Antibodies against these peptides were raised in rabbits. The anti-peptide antibodies bound specifically to hamster serum and recombinant C1s but not to human C1s. They inhibited the esterase activity of recombinant C1s to varying degrees depending on each antibody's binding site.

Immune complex independent activation of complement, C1s secreted from hamster embryo malignant fibroblasts, Nil2C2 in serum free culture medium.

Antibody independent activation of complement C1s was examined by immunoblot analysis using an antibody against a synthetic peptide of hamster C1s L chain. Approx. 50% of C1s secreted from hamster embryo malignant fibroblasts Nil2C2 was functionally active in its two-chain form in the serum free culture medium. In contrast, no active C1s was found in a culture medium of hamster embryo fibroblasts (HEF). Active C1s was detectable, however, in the culture medium after HEF became a cell line. The immune complex independent activation of C1s was also observed in rat cell lines but not in secondary rat embryo fibroblasts. C1s in a membrane fraction of Nil2C2 was a proenzyme form and was not activated by incubation of the membrane itself suggesting that C1s was activated after secretion. The activation of C1s was not inhibited by human C1 inhibitor (C1-INH), benzamidine or soy bean trypsin inhibitor (SBTI) but was inhibited by leupeptin, nitrophenyl guanidinobenzoate and DFP. Our results suggest that C1s is activated either by a serine proteinase(s) other than those reported to cleave C1s or by an activator which directly stimulates autoactivation of C1s.

Effects of coupling methods on galvanic corrosion behavior of commercially pure titanium with dental precious alloys.

BACKGROUND: When a dissimilar couple is exposed to corrosive environment, it will normally exhibit a galvanic corrosion. The galvanic corrosion might be influenced by various factors, including type and concentration of electrolyte, surface area ratio between anode and cathode, type of coupling material, and coupling manner. PURPOSE: The purpose of this study was to investigate and compare the galvanic corrosion behavior of commercially pure titanium when coupled with type IV Au alloy, Au-Ag-Pt alloy, and Ag-Au-Pd alloy by different coupling methods. MATERIALS AND METHODS: Couples were prepared by a laser welding or a mechanical adhering method. Electrochemical corrosion studies were conducted in a Ringer's solution at a scanning rate of 0.1 mV/sec in a range from -250 mV to +250 mV with respect to E(OCP). Corrosion parameters (E(OCP), I(CORR), E(CORR)) were obtained. RESULTS: It was found that (i) there was a significant difference between LWC and AJC for three couples (p<0.05), (ii) the crevice line caused all three couples more corrosive than weld joint line, (iii) for both joint, it was found that type (IV) Au alloy exhibited discoloration to some extent. CONCLUSIONS: It is concluded that among the three couples with two different coupling methods, Ti/Ag-Au-Pd couple exhibited best corrosion resistance in a room temperature Ringer's solution.

Regulatory system of guinea-pig complement C3b: tests for compatibility of guinea-pig factors H and I with human factors.

Two proteins that are involved in cleavage of methylamine-treated C3 of guinea-pig origin (C3(MA)gp) have been isolated from guinea-pig serum. One of them functioned as a cofactor of human factor I (Ihu) for cleavage of C3(MA)gp and its molecular size was 150 kDa. The other was functionally pure and able to cleave C3(MA)gp together with human factor H (Hhu). They appear to be analogous to human factors H and I in the guinea-pig and will be referred to as Hgp and Igp. Methylamine-treated human C3 [C3(MA)hu] was not a compatible substrate for Hgp or Igp: little cleavage of C3(MA)hu was observed if human factor H (Hhu) or I was substituted with the guinea-pig counterpart. C3(MA)gp, on the other hand, served as a substrate, though less efficiently, for Hhu and Ihu. Human C4b-binding protein (C4bp) and membrane cofactor protein (MCP) as well as Hhu could participate in cleavage of C3(MA)gp by Igp or Ihu. In these assays, C3(MA)gp was degraded again less efficiently than C3(MA)hu. Interestingly, human C3b/C4b receptor (CR1) mediated factor I-dependent cleavage of C3(MA)hu and C3(MA)gp to a similar extent regardless the sources of factor I. These results suggest that factor I-dependent C3b regulatory system is species-specific except in the case of CR1, which may function as a cofactor irrespective of species.

Monoclonal anti-human C4b antibodies: stabilization and inhibition of the classical-pathway C3 convertase.

Two IgG mouse monoclonal antibodies (MAbs), Abs 242 and 463, were prepared by fusion of spleen cells from mice immunized with human C4b with a myeloma cell line, P3/ X 63-Ag 8.653. They were assessed for their effect on the activation and stability of the cell-bound classical-pathway C3 convertase, EAC14b2a and on the binding of C2 and C4bp to EC4b. Ab 242 recognized a conformational neoantigen which appeared upon activation of C4 with C-1s and disappeared after chain separation of C4b, while Ab 463 recognized a linear epitope in the beta-chain of C4b. Ab 242 was found to be a C4bp-like MAb: it accelerates the decay-dissociation of C3 convertase and interferes with the binding of C2 to C4b. It also interfered with the binding of C4bp to C4b. These results suggest that Ab 242 recognizes an epitope which is closely related to the C2- and C4bp-binding sites in C4b. Ab 463, on the other hand, was found to be a nephritic factor like MAb: it prolongs the half-life of C3 convertase from 8 to 30 min at 37 degrees C.

Human factor H and C4b-binding protein serve as factor I-cofactors both encompassing inactivation of C3b and C4b.

Human factor H in the complement (C) system has been characterized as a decay-accelerator for the alternative C pathway C3 convertase and a cofactor for factor I-mediated inactivation of C3b. The current concept is that it does not serve as a C4b-inactivating cofactor. In the present study, we demonstrated that in fluid-phase, factor H and Factor I can cleave methylamine-treated C4(C4ma), a C4b analogue, to C4d, regardless of its isotype. The buffer pH and ionic strength were critical factors for the C4ma cleavage, which proceeded at around pH 6.0 and low conductivity around 3.0 mS. Similar results were obtained with fluid-phase C4b. Cell-bound C4b, however, did not undergo factor I-mediated inactivation by factor H. Hence, all of the human cofactors reported to date can mediate factor I-mediated cleavage of both C3b and C4b at least in the fluid-phase.

A monomeric human C4b-binding protein (C4bp) more efficiently inactivates C3b than natural C4bp: participation of C-terminal domains in factor I-cofactor activity.

We designed a cDNA construct encoding an artificial membrane molecule consisting of all 8 short consensus repeats (SCRs) of human monomeric C4b-binding protein (C4bp) followed by DAF's GPI anchor, named mC4bp, and expressed the protein on swine endothelial cells (SEC). At the same level of expression, mC4bp protected host cells as effectively as DAF, the most potent complement (C) regulator on the membrane. This result was unexpected from the reported functional properties of natural multimeric C4bp. Here, we investigated the mechanism whereby mC4bp has potent cell-protective activity. Our results were as follows: (1) mC4bp serves more efficiently as a methylamine-treated C3 (C3ma)-inactivating factor I-cofactor than natural C4bp and as efficiently as MCP as a methylamine-treated (C4ma)-inactivating cofactor by fluid-phase cofactor assay: (2) the potency of C3ma inactivation by mC4bp and factor I is quite high compared to those of other cofactors: (3)blocking studies using mAbs against C4bp suggested that both the 48 kDa N-terminal fragment and the C-terminal domain near the portion responsible for bundle formation participate in the high C3ma-inactivating capacity of mC4bp. Thus, acquiring high C3ma-inactivating capacity secondary to monomeric alteration leads to high C regulatory activity of mC4bp. These results infer that mC4bp differs from C4bp in its potent factor I-cofactor activity and is a good candidate as a safeguard against hyperacute rejection of xenografts.

Improvement of learning and increase in dopamine level in the frontal cortex by methylphenidate in mice lacking dopamine transporter.

The symptoms of attention-deficit/hyperactivity disorder (ADHD) are characterized by inattention and hyperactivity-impulsivity. It is a common childhood neurodevelopmental disorder that often persists into adulthood. Improvements in ADHD symptoms using psychostimulants have been recognized as a paradoxical calming effect. The psychostimulant methylphenidate (MPH) is currently used as the first-line medication for the management of ADHD. Recent studies have drawn attention to altered dopamine-mediated neurotransmission in ADHD, particularly reuptake by the dopamine transporter (DAT). This hypothesis is supported by the observation that DAT knockout mice exhibit marked hyperactivity that is responsive to acute MPH treatment. However, other behaviors relevant to ADHD have not been fully clarified. In the present study, we observed learning impairment in shuttle-box avoidance behavior together with hyperactivity in a novel environment in DAT knockout mice. Methylphenidate normalized these behaviors and enhanced escape activity in the tail suspension test. Interestingly, the effective dose of MPH increased extracellular dopamine in the prefrontal cortex but not striatum, suggesting an important role for changes in prefrontal dopamine in ADHD. Research that uses rodent models such as DAT knockout mice may be useful for elucidating the pathophysiology of ADHD.

Effect of high-impact and low-repetition training on bones in ovariectomized rats.

This study was designed to investigate the effect of high-impact and low-repetition jump training on bones in ovariectomized (OVX) rats. Forty female Wistar rats were sham-operated (sham) or OVX at the age of 11 weeks. The rats were divided randomly into the following four groups: sham-sedentary (SS; n = 10), sham-exercised (SE; n = 10), OVX-sedentary (OS; n = 10), and OVX-exercised (OE; n = 10). The rats started the jump training at the age of 12 weeks. The jump-training protocol was 10 times/day, 5 days/week and the jumping-height was 40 cm. After 8 weeks of training, the mass and breaking force in the tibia and ulna, cross-sectional areas of diaphysis in the tibia, and serum bone turnover markers were measured. The jump training significantly increased the fat-free dry weight, ash weight, and ultimate breaking force in the tibia. The rate of increase in these parameters was similar in both the sham and the OVX groups. On the other hand, in the ulna, there were no significant changes in the ultimate breaking force. The jump training significantly increased the periosteal perimeter and cortical area, although the increase in these parameters in OE compared with OS was lower than that in SE compared with SS. The jump training significantly increased serum osteocalcin in the OVX groups, as well as in the sham groups. These results suggest that high-impact and low-repetition training had beneficial effects on bone formation and bone biomechanical properties in OVX rats, as well as in sham rats.

Regulation of pancreastatin release from a human pancreatic carcinoid cell line in vitro.

The objective of these experiments was to investigate the influence of activation of three second messenger systems (protein kinase-C, adenylate cyclase-cAMP, and calcium mobilization) on the secretion of pancreastatin (PST) and chromogranin-A (CGA) by a human pancreatic carcinoid cell line (BON) in tissue culture. Stimulation of protein kinase-C by a phorbol ester (0.025-7.5 microM) caused a significant dose-related release of PST (186 +/- 22-4271 +/- 228% over controls). Treatment of BON cells with graded doses of 8-bromo-cAMP (0.14-3.0 mM) and isobutylmethylxanthine (IBMX; 0.01-1.0 mM) also stimulated a dose-related release of PST (107 +/- 22-284 +/- 28 and 16 +/- 12-1076 +/- 100% over controls, respectively). Incubation of BON cells with ionomycin (0.134-13.4 microM) increased the release of PST (102 +/- 15-554 +/- 21% over controls) in a dose-related manner. A combination of IBMX and ionomycin resulted in an additive effect, whereas treatment with a phorbol ester plus IBMX resulted in a synergistic effect on PST release. Pretreatment of BON cells with monensin, an agent that prevents processing of precursors to smaller peptides, significantly decreased PST, but not CGA, secretion in response to phorbol ester or ionomycin. These findings indicate that protein kinase-C, cAMP, and Ca2+ mobilization participate in CGA and PST secretion. Although the observation that secretions of PST and CGA in response to theophylline are quantitatively associated, the absence of a quantitative relationship in the release patterns of PST and CGA in response to phorbol ester and ionomycin do not support a simple precursor-product relationship between CGA and PST. The monensin experiments are consistent with the notion that PST is derived from CGA in BON cells.

Hemodynamic simulation study of cerebral arteriovenous malformations. Part 2. Effects of impaired autoregulation and induced hypotension.

The hemodynamic changes occurring during obliteration procedures for arteriovenous malformations (AVM) have not been fully elucidated. Therefore, we undertook a simulation study using a compartmental flow model to investigate the role of altered autoregulatory conditions in the development of hyperperfusion during obliteration of large high-flow AVM. Induced hypotension was also simulated to evaluate its usefulness in reducing the incidence and severity of the event. As the AVM flow was decreased during the obliteration procedures, feeder pressure increased and drainer pressure decreased, with a concomitant increase in the perfusion pressure in the brain tissue surrounding the AVM. Cerebral blood flow (CBF) remained constant at 50 ml 100 g-1 min-1 in the presence of autoregulation and increased to 67 ml 100 g-1 min-1 in its absence. When the lower limit of the autoregulatory pressure range (LAR) was shifted from 60 to 50 or 40 mm Hg, the flow volume increased markedly from 67 to 77 ml 100 g-1 min-1 or to 92 ml 100 g-1 min-1 after complete obliteration. Decrease in LAR would be a cause of the hyperperfusion. Induced systemic hypotension was found to be effective in reducing the magnitude of these hemodynamic changes, when induction was appropriately performed in a stepwise fashion. A simulation study is useful in clarifying the various hemodynamic changes that develop during the treatment of AVM.

Evidence that a nicked C4b, C4b', is a functionally active C4b derivative.

Factor I-catalyzed C4b cleavage is a regulatory reaction for the classical pathway of the complement system. Although the reaction was shown to be a two-step reaction, production of a nicked form of C4b, C4b', as an intermediate cleavage product and subsequent splitting of C4b' into C4c and C4d, it is not known which of the two steps represents the inactivation of the C4b function in the assembly of C3 convertase, C4b,2a. We have purified C4b' and assessed the ability of C4b' to assemble C3 convertase with C2 by utilizing size exclusion high performance liquid chromatography. Evidence was obtained demonstrating that C4b' still retains the function of C4b to assemble C3 convertase. Thus, the substantial step for the inactivation of the C4b function appears to be the second cleavage reaction, that is, the cleavage of C4b' into C4c and C4d.