[Clinical and genetic characteristics of children with dopa-responsive dystonia caused by tyrosine hydroxylase gene variations].

Objective: To explore the clinical and genetic characteristics of children with dopa-responsive dystonia (DRD) caused by tyrosine hydroxylase (TH) gene variations. Methods: Clinical data of 9 children with DRD caused by TH gene variations diagnosed in the Department of Children Rehabilitation, the Third Affiliated Hospital of Zhengzhou University from January 2017 to August 2022 were retrospectively collected and analyzed, including the general conditions, clinical manifestations, laboratory tests, gene variations and follow-up data. Results: Of the 9 children with DRD caused by TH gene variations, 3 were males and 6 were females. The age at diagnosis was 12.0 (8.0, 15.0) months. The initial symptoms of the 8 severe patients were motor delay or degression. Clinical symptoms of the severe patients included motor delay (8 cases), truncal hypotonia (8 cases), limb muscle hypotonia (7 cases), hypokinesia (6 cases), decreased facial expression (4 cases), tremor (3 cases), limb dystonia (3 cases), diurnal fluctuation (2 cases), ptosis (2 cases), limb muscle hypertonia (1 case) and drooling (1 case). The initial symptom of the very severe patient was motor delay. Clinical symptoms of the very severe patient included motor delay, truncal hypotonia, oculogyric crises, status dystonicus, hypokinesia, decreased facial expression, and decreased sleep. Eleven TH gene variants were found, including 5 missense variants, 3 splice site variants, 2 nonsense variants, and 1 insertion variant, as well as 2 novel variants (c.941C>A (p.T314K), c.316_317insCGT (p.F106delinsSF)). Nine patients were followed up for 40 (29, 43) months, and no one was lost to follow-up. Seven of the 8 severe patients were treated by levodopa and benserazide hydrochloride tablets and 1 severe patient was treated by levodopa tablets. All the severe patients responded well to levodopa and benserazide hydrochloride tablets or levodopa tablets. Although the weight of the patients increased and the drug dosage was not increased, the curative effect remained stable and there was no obvious adverse reaction. One severe patient developed dyskinesia in the early stage of treatment with levodopa and benserazide hydrochloride tablets and it disappeared after oral administration of benzhexol hydrochloride tablets. Until the last follow-up, motor development of 7 severe patients returned to normal and 1 severe patient still had motor delay due to receiving levodopa and benserazide hydrochloride tablets for only 2 months. The very severe patient was extremely sensitive to levodopa and benserazide hydrochloride tablets and no improvement was observed in this patient. Conclusions: Most of the DRD caused by TH gene variations are severe form. The clinical manifestations are varied and easily misdiagnosed. Patients of the severe patients responded well to levodopa and benserazide hydrochloride tablets or levodopa tablets, and it takes a long time before full effects of treatment become established. Long-term effect is stable without increasing the drug dosage, and no obvious side effect is observed.

Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT.

Depression is a risk factor for coronary artery disease in men: the precursors study.

BACKGROUND: Several studies have found that depression is an independent predictor of poor outcome after the onset of clinical coronary artery disease. There are few data concerning depression as a risk factor for the development of coronary artery disease. OBJECTIVE: To determine if clinical depression is an independent risk factor for incident coronary artery disease. PATIENTS AND METHODS: The Johns Hopkins Precursors Study is a prospective, observational study of 1190 male medical students who were enrolled between 1948 and 1964 and who continued to be followed up. In medical school and through the follow-up period, information was collected on family history, health behaviors, and clinical depression. Cardiovascular disease end points have been assessed with reviews of annual questionnaires, National Death Index searches, medical records, death certificates, and autopsy reports. RESULTS: The cumulative incidence of clinical depression in the medical students at 40 years of follow-up was 12%. Men who developed clinical depression drank more coffee than those who did not but did not differ in terms of baseline blood pressure, serum cholesterol levels, smoking status, physical activity, obesity, or family history of coronary artery disease. In multivariate analysis, the men who reported clinical depression were at significantly greater risk for subsequent coronary heart disease (relative risk [RR], 2.12; 95% confidence interval [CI], 1.24-3.63) and myocardial infarction (RR, 2.12; 95% CI, 1.11-4.06). The increased risk associated with clinical depression was present even for myocardial infarctions occurring 10 years after the onset of the first depressive episode (RR, 2.1; 95% CI, 1.1-4.0). CONCLUSION: Clinical depression appears to be an independent risk factor for incident coronary artery disease for several decades after the onset of the clinical depression.

Primary low-grade endometrial B-cell lymphoma.

We describe three cases of primary low-grade B-cell lymphoma of the endometrium and contrast the histological, immunohistochemical, and molecular features with two examples of benign endometrial lymphoid infiltrates. The first case was an incidental finding in a curettage specimen, confirmed on a subsequent hysterectomy. The other two cases of lymphoma were incidental findings on hysterectomy procedures performed for prolapse and cervical dysplasia, respectively. All three lymphomas occurred in patients in their sixties; none formed gross tumors. Histologic examination revealed lymphoid nodules adjacent to endometrial glands. The lymphoid cells showed mild nuclear enlargement and slight irregularities of the nuclear contour. None of the three patients had evidence of disease outside the endometrium by physical examination, bone marrow biopsy, or sampling of pelvic lymph nodes. Immunohistochemistry demonstrated a B-cell phenotype of the lymphoid cells (CD20 positive, CD79a positive) with aberrant coexpression of the T-cell-associated marker CD43. Polymerase chain reaction (PCR) amplification of the VDJ region of the immunoglobulin heavy-chain was performed on DNA isolated from paraffin sections. These studies demonstrated a clonal proliferation of B-lymphocytes in two cases. In the third case, a faint band was found superimposed on a background smear, suggesting the presence of a B-cell clone. In contrast, the two examples of histologically benign lymphoid aggregates of the endometrium consisted predominantly of T cells with rare B-lymphocytes; there was no evidence of coexpression of CD43 by B-cells. The PCR amplification from the benign lymphoid aggregates did not support a clonal process. Primary lymphoid neoplasms of the endometrium are rare, and all cases described so far have been high-stage, high-grade neoplasms. To our knowledge, this is the first report of primary low-grade B-cell lymphoma of the endometrium, presumably arising from endometrial lymphoid tissue.

Detection of Epstein-Barr virus in cardiac biopsies of heart transplant patients with lymphoproliferative disorders.

We investigated whether in situ hybridization for EBV RNA on routine cardiac biopsies could be used as a predictive test for the development of posttransplant lymphoproliferative disorder (PTLD) in cardiac transplant recipients. We examined the sensitivity of the test by determining the frequency of EBV-positive cells in cardiac biopsy specimens from patients with a known history of PTLD. Biopsy specimens obtained during routine monitoring for rejection before or shortly after the diagnosis of PTLD from 10 pediatric heart transplant patients were examined. Four of 74 specimens (5.4%) demonstrated EBV-positive lymphocytes in the cardiac biopsy rejection infiltrates. The four positive specimens were obtained from 3 different patients, all before the diagnosis of PTLD. Given the low number of cardiac biopsy specimens with EBV-positive lymphocytes, as well as the low incidence of PTLD in cardiac transplant patients, we conclude that a routine screening of all cardiac biopsy specimens using in situ hybridization for EBV with the intention of predicting PTLD is not warranted. However, in situ hybridization for EBV might be used in selected cases, such as those in which the transplant patient does not respond to immunosuppressive therapy for rejection. In these patients, the presence of EBV-positive lymphocytes in biopsy specimens initially interpreted as showing rejection might instead raise the suspicion of incipient PTLD.

Clonal VDJ recombination of the immunoglobulin heavy chain gene by PCR in classical Hodgkin's disease.

Although Hodgkin's disease (HD) has been a subject of much investigation, fundamental questions remain unanswered regarding its lineage and clonality. The authors used a polymerase chain reaction (PCR) technique to investigate whether clonal Variable-Diversity-Joining recombination of the immunoglobulin heavy (IgH) chain gene, a phenomenon that characterizes clonal B-cell proliferations, exists in nodular sclerosing (NSHD) and mixed cellularity (MCHD) Hodgkin's disease (so-called "classical" Hodgkin's disease). The isolation of DNA from paraffin-embedded tissue sections allowed for direct correlation of PCR results with the cell populations that were analyzed. Thirty-two cases were studied. These included 12 cases in which the Reed-Sternberg (RS) cells expressed the B-cell antigen, CD20, and 10 cases that were classified as syncytial variant of NSHD (3 CD20+, 7 B-cell antigen negative). Overall, clonal patterns of VDJ PCR products were found in 14 of 32 (44%) cases. These clonal patterns were identified in 7 of 12 (58%) cases of CD20+ classical HD and in 7 of 20 (35%) cases of B-antigen-negative classical HD. Clonal patterns were found in 3 of 10 cases of syncytial variant of NSHD, including 2 of 3 (67%) CD20+ cases and 1 of 7 (14%) B-cell antigen-negative cases. The results of this study provide support that a subset of HD represents a clonal B-cell neoplasm, and indicate that clonal IgH VDJ sequences are more frequently found in CD20+ HD.

Thresholds for inertial cavitation in albunex suspensions under pulsed ultrasound conditions.

Stabilized microbubbles used as echo-contrast agents can be destroyed by ultrasonic irradiation. We have identified two pressure thresholds at which these microbubbles undergo inertial cavitation (here, defined as the collapse of gas bubbles followed by emission of an acoustic broadband noise). The first threshold (P1) corresponds to the pressure at which all the microbubbles in a cavitation field lose their property as an effective scatterer because of fragmentation or deflation. The second threshold (P2) is associated with the acoustic reactivation of the remnants of the contrast agents and is related to the onset of more violent inertial cavitation. P1 and P2 were measured as a function of the concentration of Albunex (Molecular Biosystems Inc., San Diego, CA) contrast agent, the number of transmitting acoustic cycles, and the pulse repetition frequency (PRF). The ultrasound frequency used was 1.1 MHz, and the peak negative acoustic pressures ranged from 0 to 8 MPa. Our results, measured in Isoton II (Coulter Diagnostics, Miami, FL) and whole blood solutions, showed that P1 increased with increasing Albunex concentration and decreased with increasing PRF, whereas P2 decreased with increasing Albunex concentration and was independent of the PRF. Both P1 and P2 decreased with increasing number of acoustic cycles N for N < 10 and were independent of the number of cycles for N > 10. Ultrasound images of Albunex acquired by a commercial scanner showed echo enhancement not only at pressure levels below P1 but also at levels above P2. The threshold P2 was achieved at ultrasound energies above the diagnostic level. Inertial cavitation produced at P2 was associated with a higher level of hemolysis compared with P1. The results of this investigation have potential significance for both diagnostic and therapeutic ultrasound applications.

Controlled failure mechanisms toughen the dentino-enamel junction zone.

STATEMENT OF PROBLEM: The dentino-enamel junction (DEJ) durably unites dissimilar hard brittle enamel and tough flexible dentin. In contrast to artificial bonds between restorations and dentin, the DEJ rarely fails except when it is affected by inherited disorders. Knowledge of DEJ toughening mechanisms is important in understanding inherited disorders, in biomimetic engineering of junctions between artificial restorations and teeth, and in tissue-engineering a DEJ. PURPOSE: The purpose of this study was to identify specific DEJ-zone failure mechanisms and to survey the fracture toughness of the human DEJ zone. MATERIAL AND METHODS: Fracture toughness indentations were made at 3 sites across the DEJ zone of 10 human incisor teeth. Failure modes identified using optical microscopy and fracture toughness (MPa.m(1/2)) were calculated following Vickers microindentation. Site mean values were then calculated and compared using 1-way analysis of variance (alpha=.05). RESULTS: The DEJ did not undergo catastrophic interfacial delamination; instead, damage was distributed over a broad zone. The primary damage mode involved cracking and damage dispersion in the specialized first-formed enamel close to the DEJ. Multiple, somewhat convoluted and sometimes branching, cracks spread and diffused damage over a wide area of adjacent enamel rather than producing catastrophic interfacial failure. Other secondary mechanisms included short microcracks in the DEJ adjacent dentin with possible cracked bridging, as well as plastic deformation of the DEJ without delamination. A DEJ-zone fracture toughness of approximately 0.8 to 0.9 MPa.m(1/2) was calculated. CONCLUSION: DEJ-zone damage occurred primarily within the adjacent layer of specialized first-formed enamel, and the optical DEJ interface resisted delamination.

Cardiogenic shock complicates successful treatment of refractory thrombotic thrombocytopenia purpura with rituximab.

BACKGROUND: Treatment of thrombotic thrombo-cytopenia purpura (TTP) with daily therapeutic plasma exchange (TPE) may be accompanied by a variety of adjunctive interventions including most recently rituximab. Rituximab, a murine and human monoclonal antibody directed against CD20 antigen on B lymphocytes, is primarily used for treatment of non-Hodgkin's lymphomas. Because of severe and fatal infusion reactions including heart failure, rituximab carries a boxed warning. CASE REPORT: A 20-year-old female presented with TTP. She underwent 17 daily (1 day skipped) TPE. Her platelet (PLT) count reached 150 x 10(9) per L and then gradually declined to 36 x 10(9) per L despite continuing TPE. Because of the refractory behavior of her disease, rituximab was administered. After the rituximab infusion, she developed a nonproductive cough which progressed to a productive cough, acute respiratory failure, chest pain, and hypotension and was moved to intensive care for management of biventricular cardiogenic shock (ejection fraction was 5%-10%). Once stable in the intensive care unit, TPE was resumed. Her PLT count reached 241 x 10(9) per L, and her lactate dehydrogenase decreased to normal after four TPEs. Her heart failure completely resolved and she was discharged. Rituximab was added to her medical record as a drug allergy. CONCLUSION: Refractory TTP has been reported to respond favorably to rituximab when used as an adjunct. Interventions, however, can also carry significant risk as illustrated by the cardiogenic shock in our patient. Use of rituximab for refractory TTP should follow a careful assessment of benefits.

Mechanism of inhibition of transducin guanosine triphosphatase activity by vanadate.

The visual excitation system of the retinal rod outer segments and the hormone-sensitive adenylate cyclase complex are regulated through guanine nucleotide-binding proteins, transducin in the former and inhibitory and stimulatory regulatory components, Gi and Gs, in the latter. These proteins are functionally and structurally similar; all are heterotrimers composed of alpha, beta, and gamma subunits and exhibit guanosine triphosphatase activity stimulated by light-activated rhodopsin or the agonist-receptor complex. Adenylate cyclase can be stimulated by vanadate, which, like NaF, probably acts through Gs. Effects of vanadate on the function of a guanine nucleotide-binding protein were investigated in a reconstituted model system consisting of purified transducin subunits (T alpha, T beta gamma) and rhodopsin in phosphatidylcholine vesicles. Vanadate (decameric) inhibited [3H]GTP binding to T alpha and noncompetitively inhibited GTP hydrolysis in a concentration-dependent manner with maximal inhibition of approximately 90% at 3-5 mM. Vanadate also inhibited release of bound GDP but did not affect the rate of hydrolysis of bound GTP (single turnover rate), indicating that vanadate did not interfere with the intrinsic GTPase activity of T alpha. Binding of T alpha to rhodopsin and the ADP-ribosylation of T alpha by pertussis toxin, both of which are enhanced in the presence of T beta gamma, were inhibited by vanadate. These findings are consistent with the conclusion that vanadate can cause the dissociation of T alpha from T beta gamma, resulting in the inhibition of GDP-GTP exchange and thereby GTP hydrolysis. Adenylate cyclase activation could result from a similar effect of vanadate on Gs.

No role for human herpes virus 8 in the etiology of infantile capillary hemangioma.

Infantile capillary hemangiomas are vascular neoplasms that can appear quite infiltrative histologically and that are characterized by cords of cells with areas of marked cellularity. These lesions have been shown to contain a population of rapidly proliferative endothelial cells. Given the recent association between the presence of human herpes virus 8 (HHV8) and another proliferative vascular lesion, Kaposi's sarcoma, we used polymerase chain reaction technology to examine a series of 16 biopsy specimens from 15 infantile capillary hemangiomas for the presence of HHV8 DNA. We were unable to detect HHV8 DNA in any of the lesions studied. All of the cases were examined in parallel with a case of Kaposi's sarcoma that was known to be positive for HHV8 DNA and a series of negative controls. In all of our cases, amplification of beta-globin gene DNA demonstrated adequate preservation of DNA in the tissue studied. These findings suggest that not all endothelial cell proliferations can be attributed to HHV8 and that the etiology of some of these conditions remains unclear.

Kinetic analysis of agonist-receptor interactions. Model for the "irreversible" binding of choleragen to human fibroblasts.

Current studies on receptor-ligand interactions usually employ a "binding" step followed by extensive washing to remove free ligand. This procedure, by definition, removes reversibly bound ligand from the receptor; the equations used by most workers to analyze the data, however, require an equilibrium between free and bound ligand and are not applicable given the design of most binding assays. The assay, in fact, measures binding that is slowly reversible or irreversible. Assuming that ligand receptor interaction involves two stages with a reversible step followed by an "irreversible" event, the Ka for the reversible reaction may be obtained from the rate of the irreversible step. 125I-Choleragen binding to human fibroblasts was only slowly reversible at both 0 and 37 degrees C. The two-step model was, therefore, applied experimentally to determine the Ka for the reversible step in 125I-choleragen binding to human fibroblasts at 0 degrees C (Ka = 1.9 x 10(8) M-1) and at 37 degrees C (Ka = 3.6 x 10(8) M-1). As predicted by the two-step model for ligand binding, the addition of 50 micrograms/ml of unlabeled toxin enhanced the rate of release of radioactivity at 37 degrees C; the rate of radiolabel release remained low at 0 degrees C, even with unlabeled toxin present in the medium. The rate of release of previously incorporated 125I-toxin was accelerated by 50 micrograms/ml of toxin greater than 5 micrograms/ml of toxin. The two-step model for ligand binding appears to be applicable to the study of 125I-choleragen binding to fibroblasts and should be useful, in general, for the analysis of receptor-ligand interaction.

Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin.

Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation.

Type II heat-labile enterotoxin of Escherichia coli activates adenylate cyclase in human fibroblasts by ADP ribosylation.

Type II heat-labile enterotoxin (LT-II) from Escherichia coli causes characteristic morphological changes and accumulation of cyclic AMP in Y-1 adrenal cells, but it is not neutralized by antisera against choleragen (CT) or the classical type I heat-labile enterotoxin (LT-1) from E. coli. The action of purified LT-II on CT- and LT-I-responsive human fibroblasts was investigated and compared with that of CT. Fibroblasts incubated with LT-II or CT had an increased cyclic AMP content as well as a fourfold elevation of membrane adenylate cyclase activity. In membranes, activation of cyclase by toxin was enhanced by NAD, GTP, and dithiothreitol. The effect of LT-II on intact fibroblasts or membranes was increased by trypsin treatment of toxin. Since activation of adenylate cyclase by LT-II was stimulated by NAD, the ability of LT-II to catalyze the [32P]ADP-ribosylation of membrane proteins in the presence of [32P]NAD from control and LT-II- and CT-treated fibroblasts was investigated. Similar proteins were [32P]ADP-ribosylated in membranes exposed to LT-II or CT; LT-II- and CT-specific labeling was significantly decreased in membranes prepared from cells preincubated with either LT-II or CT. These studies are consistent with the hypothesis that LT-II, similar to CT and LT-I, increases cyclic AMP by activating adenylate cyclase through the GTP-dependent ADP-ribosylation of specific membrane proteins.

Identification of thiol:protein disulfide oxidoreductase activity in cultured human fibroblasts: dependence of enzyme activity on growth conditions.

Thiol:protein disulfide oxidoreductase activity was assayed in extracts of cultured normal human skin fibroblasts. Enzyme activity in confluent fibroblasts was dependent on growth conditions. In serum-deprived fibroblasts grown in minimal medium enzyme activity was approximately 40% of that observed in fibroblasts maintained in medium supplemented with 10% fetal calf serum. In fibroblasts cultured in medium supplemented only with insulin, activity was 35% greater than that in fibroblasts cultured in unsupplemented defined medium. Antibodies raised against purified bovine liver thiol:protein disulfide oxidoreductase immunoprecipitated all of the activity present in fibroblast extracts. The thiol:protein disulfide oxidoreductase from human fibroblasts thus appears to share antigenic determinants with the bovine liver enzyme. The human fibroblast may serve as an in vitro model to study the regulation of the oxidoreductase.

Degradation of choleragen bound to cultured human fibroblasts and mouse neuroblastoma cells.

125I-choleragen bound to human fibroblasts was degraded slowly with a t1/2 of 2-3 days; the radiolabel in bound 125I-choleragen was present in both the A and B subunits. During degradation, radiolabel was lost more rapidly from the 125I-A1 (t1/2 approximately 2 days) than from the 125I-B peptides (t1/2 greater than 5 days). 125I-Choleragen bound to neuroblastoma cells showed a considerably shorter t1/2 for both the 125I-A1 and 125I-B peptides; as with the fibroblasts, radiolabel was lost more rapidly from the 125I-A1 than from the 125I-B peptides. The continued presence of choleragen in the fibroblasts and neuroblastoma cells was associated with a prolonged activation of adenylate cyclase. In addition, fibroblasts, previously exposed to toxin and then washed free of unbound choleragen, only slowly recovered their ability to bind 125I-choleragen with a t1/2 of 7 days. Fibroblasts exposed to choleragen also showed evidence of persistent toxin on the surface based on the ability of the cells to bind antitoxin, antisubunit A or antisubunit B antibodies followed by 3H-protein A. It appears that choleragen remains persistently bound to fibroblasts, is degraded at a slow rate, and may prevent the binding of new toxin molecules to the fibroblast. The relatively slow degradation of toxin by fibroblasts may explain the prolonged activation of adenylate cyclase by toxin. The loss of 125I-toxin binding capacity following incubation with toxin may result from continued presence of toxin subunits on the cell surface.

Absence of human herpesvirus 8 and Epstein-Barr virus genome sequences in cutaneous epithelial neoplasms arising in immunosuppressed organ-transplant patients.

Recent studies have implicated herpesvirus 8 and Epstein-Barr virus in the development of cutaneous malignancies in immunosuppressed patients. In order to examine the strength of this association, we examined 37 malignant, pre-malignant and benign cutaneous epithelial neoplasms removed from immunosuppressed organ recipients for the presence of human herpesvirus 8 and Epstein-Barr viral genome sequences using polymerase chain reaction (PCR) and in situ hybridization. We examined 2 actinic keratoses, 1 benign keratosis, 11 invasive squamous cell carcinomas, 17 squamous cell carcinomas in situ and 6 basal cell carcinomas. We also examined 4 basal cell carcinomas, 1 invasive squamous cell carcinoma and 3 squamous cell carcinomas in immunocompetent hosts. In contrast to findings reported by other investigators, we were unable to detect viral genome sequences in any of the biopsies examined. Our findings suggest that human herpesvirus 8 and Epstein-Barr virus likely do not play an etiologic role in cutaneous epithelial oncogenesis in immunocompromised patients.

Persistent, noncytocidal viral infection: nonsynchrony of viral and cellular multiplication.

Walker, Duard L. (University of Wisconsin Medical School, Madison), Ping-Ping Chang, Robert L. Northrop, and Harry C. Hinze. Persistent, noncytocidal viral infection: nonsynchrony of viral and cellular multiplication. J. Bacteriol. 92:983-989. 1966.-In cultures of human conjunctive cells persistently infected with mumps virus (C-M cultures), the degree to which viral multiplication is linked to cell multiplication was examined. First, cell multiplication was inhibited. This resulted in a 10-fold increase in virus-excreting cells in the culture and an 80-fold increase in virus in the medium as compared with vigorously growing control cultures. Second, by use of elevated incubation temperature, virus multiplication was inhibited without slowing multiplication of the cells, and uninfected cells that appeared in the culture were protected by virus antiserum. This resulted in accumulation of antigen-free cells and, in one experiment, in elimination of the virus. Evidence indicated that uninfected cells accumulated as a result of dilution of virus by repeated cell divisions, but not as a result of selection of uninfected cells. These data indicate that viral multiplication in the C-M system is not closely linked to cell multiplication and that each can proceed at a rate different from the other.

Mechanism of action of cholera toxin on intact cells. Generation of A1 peptide and activation of adenylate cyclase.

When intact mouse neuroblastoma NB cells were incubated with choleragen at 4 degrees C, washed, and incubated at 37 degrees C, activation of adenylate cyclase occurred rapidly after a delay of 15 min. The cells were incubated under the same conditions with 125I-labeled toxin, lysed, and solubilized with sodium dodecyl sulfate under mild conditions. Soluble proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of dithiol reductants to separate labeled toxin products. Initially, only 0.1 to 0.2% of the cell-associated radioactivity migrated on the gels as the A1 peptide of choleragen. After a 15-min delay, the amount of A1 peptide increased rapidly with time and paralleled the activation of adenylate cyclase. Similar results were observed with human skin fibroblasts, Friend erythroleukemic cells, and II3-alpha-N-acetylneuraminosyl-gangliotetraosylceramide-treated rat glioma C6 cells. When toxin-treated NB cells were incubated at increasing temperatures, generation of A1 peptide and activation of adenylate cyclase increased in parallel. Both processes were prevented by incubation of cells at 4 or at 37 degrees C in the presence of anticholeragen antibodies. These results indicate that there is delay both in the formation of A1 peptide and in the activation of adenylase cyclase in intact cells. As A1 is believed to be the catalytically active component of choleragen, it is suggested that the lag period may be related in part to the time required to generate A1 peptide from choleragen.