Increased turnover of T lymphocytes in HIV-1 infection and its reduction by antiretroviral therapy.

The mechanism of CD4(+) T cell depletion in human immunodeficiency virus (HIV)-1 infection remains controversial. Using deuterated glucose to label the DNA of proliferating cells in vivo, we studied T cell dynamics in four normal subjects and seven HIV-1-infected patients naive to antiretroviral drugs. The results were analyzed using a newly developed mathematical model to determine fractional rates of lymphocyte proliferation and death. In CD4(+) T cells, mean proliferation and death rates were elevated by 6.3- and 2.9-fold, respectively, in infected patients compared with normal controls. In CD8(+) T cells, the mean proliferation rate was 7.7-fold higher in HIV-1 infection, but the mean death rate was not significantly increased. Five of the infected patients underwent subsequent deuterated glucose labeling studies after initiating antiretroviral therapy. The lymphocyte proliferation and death rates in both CD4(+) and CD8(+) cell populations were substantially reduced by 5-11 weeks and nearly normal by one year. Taken together, these new findings strongly indicate that CD4(+) lymphocyte depletion seen in AIDS is primarily a consequence of increased cellular destruction, not decreased cellular production.

Localization of caldesmon and its dephosphorylation during cell division.

Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle caldesmon to dissociate from microfilaments during prometaphase. (Yamashiro, S., Y. Yamakita, R. Ishikawa, and F. Matsumura. 1990. Nature (Lond.). 344:675-678; Yamashiro, S., Y. Yamakita, H. Hosoya, and F. Matsumura. 1991. Nature (Lond.) 349:169-172). To explore the functions of caldesmon phosphorylation during cytokinesis, we have examined the relationship between the phosphorylation level, actin-binding, and in vivo localization of caldesmon in cultured cells after their release of metaphase arrest. Immunofluorescence studies have revealed that caldesmon is localized diffusely throughout cytoplasm in metaphase. During early stages of cytokinesis, caldesmon is still diffusely present and not concentrated in contractile rings, in contrast to the accumulation of actin in cleavage furrows during cytokinesis. In later stages of cytokinesis, most caldesmon is observed to be yet diffusely localized although some concentration of caldesmon is observed in cortexes as well as in cleavage furrows. When daughter cells begin to spread, caldesmon shows complete colocalization with F-actin-containing structures. These observations are consistent with changes in the levels of microfilament-associated caldesmon during synchronized cell division. Caldesmon is missing from microfilaments in prometaphase cells arrested by nocodazole treatment, as shown previously (Yamashiro, S., Y. Yamakita, R. Iskikawa, and F. Matsumura. 1990. Nature (Lond.). 344:675-678). The level of microfilament-associated caldesmon stays low (12% of that of interphase cells) when some cells start cytokinesis at 40 min after the release of metaphase arrest. When 60% of cells finish cytokinesis at 60 min, the level of microfilament-associated caldesmon is recovered to 50% of that of interphase cells. The level of microfilament-associated caldesmon is then gradually increased to 80% when cells show spreading at 120 min. Dephosphorylation appears to occur during cytokinesis. It starts when cells begin to show cytokinesis at 40 min and completes when most cells finish cytokinesis at 60 min. These results suggest that caldesmon is not associated with microfilaments of cleavage furrows at least in initial stages of cytokinesis and that dephosphorylation of caldesmon appears to couple with its reassociation with microfilaments. Because caldesmon is known to inhibit actomyosin ATPase and/or regulate actin assembly, its continued dissociation from microfilaments may be required for the assembly and/or activation of contractile rings.

A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage of cell-substrate interaction.

Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of beta1 integrin, seems to be a key component of FAs, but its exact role in cell-substrate interaction remains to be clarified. Here, we identified a novel ILK-binding protein, affixin, that consists of two tandem calponin homology domains. In CHOcells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin-ILK signaling required for the establishment of cell-substrate adhesion.

Fertilization of sea urchins needs magnesium ions in seawater.

When sea urchin eggs were inseminated in seawater free of magnesium, the fertilization rate was very low. Spermatozoa that had been treated with egg jelly to induce the acrosome reaction also failed to fertilize eggs in seawater free of magnesium. These results indicate that magnesium is indispensable for some process or processes at fertilization, such as membrane, fusion or sperm penetration.

Rapid turnover of T lymphocytes in SIV-infected rhesus macaques.

Studies of lymphocyte turnover in animal models have implications for understanding the mechanism of cell killing and the extent of lymphocyte regeneration in human immunodeficiency virus infection. Quantitative analyses of the sequential changes in bromodeoxyuridine labeling of CD4 and CD8 T lymphocytes not only revealed the normal proliferation and death rates of these cell populations in uninfected macaques, but also showed a substantial increase in these rates associated with simian immunodeficiency virus (SIV) infection. Faster labeling and delabeling in memory and naïve T lymphocyte subpopulations as well as in NK (natural killer) and B cells were also observed in infected macaques, suggesting a state of generalized activation induced by SIV.

Is outer arm dynein intermediate chain 1 multifunctional?

The outer arm dynein of sea urchin sperm axoneme contains three intermediate chains (IC1, IC2, and IC3; M(r) 128,000, 98,000, and 74,000, respectively). IC2 and IC3 are members of the WD family; the WD motif is responsible for a protein-protein interaction. We describe here the molecular cloning of IC1. IC1 has a unique primary structure, the N-terminal part is homologous to the sequence of thioredoxin, the middle part consists of three repetitive sequences homologous to the sequence of nucleoside diphosphate kinase, and the C-terminal part contains a high proportion of negatively charged glutamic acid residues. Thus, IC1 is a novel dynein intermediate chain distinct from IC2 and IC3 and may be a multifunctional protein. The thioredoxin-related part of IC1 is more closely related to those of two redox-active Chlamydomonas light chains than thioredoxin. Antibodies were prepared against the N-terminal and middle domains of IC1 expressed as His-tagged proteins in bacteria. These antibodies cross-reacted with some dynein polypeptides (potential homologues of IC1) from distantly related species. We propose here that the three intermediate chains are the basic core units of sperm outer arm dynein because of their ubiquitous existence. The recombinant thioredoxin-related part of IC1 and outer arm dyneins from sea urchin and distantly related species were specifically bound to and eluted from a phenylarsine oxide affinity column with 2-mercaptoethanol, indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction.

A dynein light chain of sea urchin sperm flagella is a homolog of mouse Tctex 1, which is encoded by a gene of the t complex sterility locus.

The outer-arm dynein of sea urchin sperm flagella contains six light chains with molecular masses of 23.2, 20.8, 12.3, 11.5, 10.4 and 9. 3kDa. We have cloned a cDNA for the 12.3kDa polypeptide (light chain 3) and found that this protein is highly homologous to mouse Tctex1, a protein encoded by a member of the multigene family in the t complex region that is involved in male sterility and the development of the germ cells. Tctex1 has recently been shown to be homologous to a light chain of cytoplasmic dynein. Therefore, the cytoplasmic dynein light chain has been implicated in the mechanism for the transmission ratio distortion (meiotic drive) that is characteristic of t haplotypes in mice. Our present finding, however, indicates that axonemal light chain 3 must be considered equally important.

A novel 15 kDa Ca2+-binding protein present in the eggs of the sea urchin, Hemicentrotus pulcherrimus.

A novel Ca2+-binding protein, different from calmodulin, has been purified to homogeneity from the soluble cytoplasmic protein fraction of the egg of the sea urchin, Hemicentrotus pulcherrimus. This protein, designated as 15 kDa protein, shows a Ca2+-dependent mobility shift upon SDS-gel electrophoresis and has Ca2+-binding ability. This protein did not resemble the sea urchin egg calmodulin in either molecular mass or amino acid composition. The 15 kDa protein could not activate cyclic adenosine 3',5'-monophosphate-dependent phosphodiesterase from bovine brain and did not bind to fluphenazine-Sepharose 6B. Antibodies against the 15 kDa protein did not react with sea urchin egg calmodulin. These results suggest that the 15 kDa protein is a novel Ca2+-binding protein in the sea urchin egg.

Surgical correction in infancy to reduce mortality in transposition of the great arteries.

The mortality for infants with transposition of the great arteries (TGA) prior to the introduction of medical and surgical septostomy was over 80% by 6 months of age. The mortality has fallen recently, due to balloon atrial septostomy (BAS), but still exceeds 25% by 6 months of age in major centers. The cumulative mortality rises to 40% by 1 year of age with or without additional surgical atrial septectomy. In our center, 12 patients with an average age of 3 months were operated on for interatrial baffle correction of their TGA under surface-induced deep hypothermia. BAS had been done preliminarily in those patients without an adequate atrial communication. Ten of these 12 patients (83%) survived. These patients have been followed up from one month to eight years with an average follow-up of over four years. Surgical correction during infancy offers a better survival rate for infants with TGA than does medical management, with or without palliative surgery.

Effects of hybrid peptide analogs to receptor recognition domains on alpha- and gamma-chains of human fibrinogen on fibrinogen binding to platelets.

We synthesized a series of hybrid peptides that correspond to the gamma-chain dodecapeptide (400-411), variable numbers of glycine residues, and the RGDS peptide [Y-HHLGGAKQAGDV(G)nRGDS] to investigate the relationship of these receptor recognition domains of fibrinogen to platelet membrane glycoprotein IIb/IIIa. The tetrapeptide RGDS, the GRGDSPA peptide and the dodecapeptide inhibited binding of fibrinogen to GPIIb/IIIa by 50% (IC50) at concentrations of 17 +/- 1.6 microM, 15 +/- 2.1 microM, and 87 +/- 6.8 microM, respectively. The inhibitory effect of hybrid peptides increased as the number of glycine residues increased, plateauing with 9-11 glycine residues in hybrid peptide analogs, which had an IC50 of 0.68 +/- 0.14 microM. These hybrid peptides completely inhibited the binding of fibrinogen to activated platelets when used in sufficient concentrations. The peptide Y-HHLGGAKQAGDV(G)9RGDS blocked ADP-induced aggregation in citrated platelet-rich plasma with IC50 of 3.5 +/- 0.6 microM. When the peptide Y-HHLGGAKQAGDV(G)9RGDS was labeled with 125I to quantify its binding to platelets, maximal binding was observed within 30 min. The binding sites of the hybrid peptide were 43,600 molecules/platelet (Kd = 3.1 +/- 0.5 x 10(-7) M) to stimulated platelets and 12,500 molecules/platelet (Kd = 1.4 +/- 0.2 x 10(-7) M) to nonstimulated platelets. The hybrid peptides had the same binding affinity to platelets as fibrinogen and inhibited platelet function. Moreover, anti-GPIIb/IIIa antibody inhibited the binding of the labeled hybrid peptide to stimulated platelets. These results indicate that in the native fibrinogen molecule the presence of both RGD sequence or gamma-chain domain at optimal distances increased the binding affinity to GPIIb/IIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibody selectively inhibits binding of von Willebrand factor to glycoprotein ib. Recognition site is located in the A1 loop of von Willebrand factor.

A 20-year-old man with severe von Willebrand disease recently presented a progressive bleeding tendency, characterized recurrent subcutaneous hemorrhages and cerebral hemorrhage. Mixing and infusion studies suggested the presence of an inhibitor directed against vWF:RCo activity of von Willebrand factor (vWF) without significant inhibition of the FVIII:C. The inhibitor was identified as an antibody of IgG class. The inhibitor inhibited the interaction of vWF in the presence of ristocetin and that of asialo-vWF with GPIb while it partially blocked botrocetin-mediated interaction of vWF to GPIb. The inhibitor reacted with native vWF, the 39/34 kDa fragment (amino acids [aa] 480/ 481-718) and the recombinant vWF fragment (MalE-rvWF508-704), but not with Fragment III-T2 (heavy chains, aa 273-511; light chains, aa 674-728). A synthetic peptide (aa 514-542) did not inhibit vWF-inhibitor complex formation. We conclude that this is the first auto-antibody of class IgG from human origin that recognizes the sequence in the A1 loop of vWF, resulting in a virtual absence of functional vWF and a concomitant severe bleeding tendency although recognition site is different from the residues 514-542 which is crucial for vWF-GPIb interaction.

Acquired von Willebrand syndrome: data from an international registry.

The acquired von Willebrand syndrome (AvWS) is a rare bleeding disorder with laboratory findings similar to those of congenital von Willebrand disease (vWD). Despite the numerous cases reported in the literature until 1999 (n = 266), large studies on AvWS are not available. Moreover, diagnosis of AvWS has been difficult and treatment empirical. These considerations prompted us to organize an international registry. A questionnaire, devised to collect specific information on AvWS, was sent to all the members of the International Society on Thrombosis and Haemostasis (ISTH), who were invited to respond if they had diagnosed cases with the AvWS cases. 156 members answered the questionnaire and 54 of them sent information on 211 AvWS cases from 50 centers. Data were compared with those already published in the literature and 25 cases already described or not correctly diagnosed were excluded. The 186 AvWS cases that qualified for the registry were associated with lymphoproliferative (48%) and myeloproliferative disorders (15%), neoplasia (5%), immunological (2%), cardiovascular (21%) and miscellaneous disorders (9%). Ristocetin cofactor activity (vWF:RCo) or collagen binding activity (vWF:CBA) were usually low in AvWS (median values 20 U/dL, range 3-150), while factor VIII coagulant activity was sometimes normal (median 25 U/dL, range 3-191). FVIII/vWF inhibiting activities were present in only a minority of cases (16%). Bleeding episodes in AvWS were mostly of mucocutaneous type (68%) and were managed by DDAVP (32%), FVIII/vWF concentrates (37%), intravenous immunoglobulins (33%), plasmapheresis (19%), corticosteroids (19%) and immunosuppressive or chemotherapic agents (35%). Based upon the data of this international registry, it appears that AvWS is especially frequent in lympho- or myeloproliferative and cardiovascular diseases. Therefore, AvWS should be suspected and searched with the appropriate laboratory tests especially when excessive bleeding occurs in patients with these disorders. On the basis of the information provided by this registry guidelines for diagnosis and management of the AvWS are given.

Biological cloning of functionally diverse quasispecies of HIV-1.

Twenty putative clones of HIV-1, derived by biological cloning from two different individuals, were characterized in vitro for functional activity and genotypic diversity. Many of the biological clones isolated by this method were genetically distinct, and could be further distinguished by differences in replication kinetics, host cell range, and/or cytopathicity. Our findings indicate that biological cloning by limiting dilution cultures is an effective method for isolating replication-competent, functionally diverse quasispecies of HIV-1.

Experimental study of cerebral autoregulation during cardiopulmonary bypass with or without pulsatile perfusion.

Twenty-four adult mongrel dogs were divided into four equal groups according to the following method of cardiopulmonary bypass: normothermic continuous (so-called nonpulsatile) perfusion, normothermic pulsatile perfusion, hypothermic continuous perfusion, and hypothermic pulsatile perfusion. Cerebral blood flow was determined by measuring the volume of sagittal sinus venous blood outflow with a transit-time ultrasonic flowmeter. Cardiopulmonary bypass was initiated at a flow rate of 80 ml/kg per minute. Cerebral temperature was maintained at 37 degrees C in the normothermic groups and at 25 degrees C in the hypothermic groups. Arterial pH and carbon dioxide were maintained within the physiologic range by alpha-stat acid-base regulation. Mean cerebral perfusion pressure and blood flow were not affected during 90 minutes of the bypass. The respective values were 67.1 mm Hg and 37.1 ml/100 gm brain per minute with normothermic continuous perfusion, 72.8 mm Hg and 39.0 ml/100 gm per minute with nonpulsatile perfusion, 98.0 mm Hg and 23.0 ml/gm per minute with hypothermic continuous perfusion, and 86.8 mm Hg and 22.3 ml/100 gm per minute with hypothermic pulsatile perfusion. Pump flow rates were altered from 10 to 120 ml/kg per minute in a stepwise fashion to obtain graded changes of perfusion pressure. Cerebral blood flow, however, was not changed significantly by cerebral perfusion pressure so long as perfusion pressure was greater than 50 mm Hg. Conversely, cerebral blood flow changed proportionally with cerebral perfusion pressure at a pressure less than 50 mm Hg. The correlation between cerebral blood flow and perfusion pressure was described as two separate lines determined by linear regression. The slope of the regression line relating cerebral blood flow to perfusion pressure was 0.16 +/- 0.08 for a cerebral perfusion pressure above 50 mm Hg and 0.68 +/- 0.11 below 50 mm Hg in the normothermic continuous perfusion group; 0.14 +/- 0.09 and 0.32 +/- 0.09 with normothermic pulsatile perfusion; 0.10 +/- 0.04 and 0.62 +/- 0.18 with hypothermic continuous perfusion; 0.09 +/- 0.08 and 0.39 +/- 0.04 in the hypothermic pulsatile perfusion group. The slope above 50 mm Hg was significantly smaller and closer to zero in all groups than it was at a perfusion pressure below 50 mm Hg (p < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Indications for surgery based on lung biopsy in cases of ventricular septal defect and/or patent ductus arteriosus with severe pulmonary hypertension.

Pathologic obstruction of the proximal lumen and secondary atrophy of the media of the peripheral small pulmonary arteries were absolute operative contraindications in cases of VSD and/or PDA with severe pulmonary hypertension. Such patients who were operated on died with no decrease in pulmonary arterial pressure. The index of pulmonary vascular disease (IPVD), a composite and quantitative evaluation of the severity of pulmonary vascular disease, was introduced to determine the operability of other patients. An IPVD rating of 2.2 in Down's syndrome and 2.1 without the syndrome were regarded as the upper permissible limits for surgical intervention based on results of 23 autopsies and 26 lung biopsies of patients operated on before 1981. Open lung biopsy was performed in 51 patients to determine applicability of our operative indications. Twenty-nine cases were considered operable by our criteria, and 28 underwent surgical correction without operative or late death. Twenty-two cases thought inoperable remain under observation. Comparative analysis of the pathology and preoperative hemodynamic data suggested that lung biopsy should be carried out to determine operability in cases with pulmonary vascular resistance greater than 8 units.m2.

Sound spectral analysis of prosthetic valvular clicks for diagnosis of thrombosed Björk-Shiley tilting standard disc valve prostheses.

To evaluate clinical usefulness of sound spectral analysis in the early detection of prosthetic thrombosis, we analyzed specific patterns of thrombus formation in Björk-Shiley tilting standard disc prostheses in relation to the sound spectral analysis of their click sounds. Among a total of 365 Björk-Shiley tilting standard disc prostheses, nine became thrombotic. These consisted of seven prostheses in the mitral position and two in the aortic position, and the affected valves were replaced in all cases. The sound spectral analysis system includes a wide-frequency microphone and a sound spectral analyzer. To quantify the amplitude of the metallic clicks that are specific to the prosthetic valve sounds, we used a normalized maximal frequency as a diagnostic parameter for valve thrombosis. Thrombus formation was localized at the minor strut in two cases, and these demonstrated abnormally low normalized maximal frequency values only on opening clicks. In four cases thrombus formations were noted at both minor and major struts, and these showed abnormally low normalized maximal frequency values on both opening and closing clicks. In two cases with pannus formation, no abnormalities were found by sound spectral analysis. In one case, which had demonstrated abnormally decreased normalized maximal frequency values of both opening and closing clicks, the normalized maximal frequency values were normalized after thrombolytic therapy, suggesting resolved thrombosis. Reoperation, which was performed for concomitant complication of perivalvular leak, revealed no noticeable thrombus formation. The thrombi were seen on the minor strut during surgical procedures in all cases in which valve thrombosis was indicated by the sound spectral analysis. Therefore, the minor strut is considered to be the prevalent area of thrombus formation, and diagnosis could be made after observation of abnormally low normalized maximal frequency on opening clicks. We found the sound spectral analysis to be an extremely useful diagnostic tool for early detection of thrombosis in the Björk-Shiley tilting standard disc prostheses, especially because the opening clicks of the tilting disc valve were too low in amplitude for auscultation to detect the existence of mild abnormality.

A clinical trial of allopurinol (Zyloric) for myocardial protection.

This study explored myocardial protective effects of allopurinol at various doses. Ninety patients undergoing coronary artery bypass or repair or replacement of cardiac valves were divided into three groups of 30 patients each in accordance with the amount of allopurinol administered to patients in each group. Patients in group I received no allopurinol, those in group II received low-dose allopurinol (total dose 1200 mg), and those in group III received high-dose allopurinol (total dose 2400 mg). Aspartate aminotransferase, cardiac isoenzyme of creatine kinase, and lactic dehydrogenase levels were measured up to 5 days after operation. Concentrations of allopurinol and oxypurinol were also measured before initiation of cardiopulmonary bypass and at the start and at the end of aortic crossclamping. Postoperative aspartate aminotransferase, creatine kinase, and lactate dehydrogenase 1 plus lactate dehydrogenase 2 levels in group III were significantly lower than those in groups I and II. Aspartate aminotransferase, creatine kinase, and lactate dehydrogenase 1 plus lactate dehydrogenase 2 levels in group II were lower than those in group I, without statistically significant differences. Plasma oxypurinol concentrations were significantly higher in group III than in group II. It was concluded that allopurinol had resultant high myocardial protective effects in dose-related fashion, but its effect might be attributed to oxypurinol levels formed by its degradation.

Use of athrombogenic tubing for perfusion rewarming following surface-induced deep hypothermia.

A method of heparinless, oxygenatorless, left heart bypass perfusion rewarming following surface hypothermia, with the use of a closed circuit with 130 ml. prime volume including heat exchanger, has been devised. The use of polyurethane-polyvinyl-graphite (PPG)-coated tubing has previously been reported. In this text, the use of an athrombogenic coating with cetyl-pyridinium chloride (CPC) as a regional heparin carrier was studied in dogs, comparing groups with PPG tubing and total systemic heparinization or plain polyvinyl tubing without systemic heparinization. Heparin compounded in the CPC coating eluted into the blood and caused mild transient whole-body heparinization during rewarming from 20 degrees to 25 degrees C., as evidenced by prolongation of the thrombin time. Alterations of hematologic parameters in all three groups were similar to those during surface rewarming except for those affected by heparinization. The left heart bypass method was found useful for hypothermic open-heart surgery when utilized with an athrombogenic surface coating or total body heparinization. It was concluded that the CPC coating is superior to the PPG coating since no cracking surface develops, it is translucent, and it provides a more effective athrombogenic surface.

Indication for total correction of complete transposition of the great arteries with pulmonary hypertension.

Pulmonary vascular disease (PVD) was histologically evaluated and its severity was expressed as an index of PVD in 14 autopsied and eight biopsied cases of complete transposition of the great arteries (TGA) in patients more than 6 months of age. The index varied from 1.0 to 2.3 in six patients who had survived complete surgical repair and ranged from 2.3 to 3.3 in five patients who had died of PVD postoperatively. Consequently, an index of 2.2 could be regarded as an upper limit of PVD for complete surgical repair. The index was significantly correlated to some hemodynamic factors. From the regression equation, the value 2.2 of histologic index could be translated into the clinical factors concerning hemodynamics. We consider that patients with pulmonary vascular resistance of less than 10.6 units . m2, and pulmonary arterial mean pressure less than 51 mm Hg, are suitable candidates for complete surgical repair in TGA, if more than 6 months of age. Thus histologic assessment of PVD in a lung biopsy as an indication for total correction of TGA with pulmonary hypertension appears to be useful in patients who are in borderline hemodynamic condition or in whom the hemodynamics could not be evaluated although pulmonary hypertension was suspected.

Real-time sound spectroanalysis for diagnosis of malfunctioning prosthetic valves.

A newly developed real-time sound spectroanalyzer was found to be capable of diagnosing malfunction of prosthetic heart valves. Sound spectroanalysis is believed to be superior to other methods such as phonocardiography and echocardiography. This system, developed in our institute, allows display on a cathode ray oscilloscope of five different modes of the valvular click, including the sound spectrograph and section pattern. Analysis of the section pattern allows measurement of the high-frequency components of the valvular click, which is normalized so as to be applicable to a click of any intensity. This parameter is called normalized maximal frequency (NMF). Sound spectroanalyses were carried out 228 times on 127 patients having a prosthetic heart valve. NMF values of normally functioning valves differed characteristically according to the type of valve, but did not change during the postoperative time course. Three of seven patients who had cerebral embolism showed significantly lower NMF values than normal, as did all four patients whose valve had become thrombosed.