[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.

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.

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.

Sonographic detection of acute parenchymal injury in an experimental porcine model of renal hemorrhage: gray-scale imaging using a sonographic contrast agent.

OBJECTIVE: The purpose of this study was to determine the usefulness of contrast-enhanced sonography in the detection of acute parenchymal injury. SUBJECTS AND METHODS: In a model of acute renal injury in pigs, four separate renal parenchymal bleeds were created by puncturing an interlobar artery of the upper and lower poles of the kidneys. B-mode gray-scale scans of the kidneys before and after injury, and after the administration of i.v. and intraarterial (i.a.) contrast agents were recorded on videotape for 5 min for each condition (baseline, after injury, after i.v. contrast administration, and after i.a. contrast administration). For each condition and injury, selected frames were analyzed with regions of interest of the normal renal parenchyma, the area of injury, and the perinephric space. Randomized videotape clips from each of the experimental conditions were rated by three sonologists as to the presence or absence of increased intrarenal parenchymal echogenicity, perinephric echogenicity, and confidence as to whether renal injury was present. RESULTS: Areas of renal injury were isoechoic with normal parenchyma on unenhanced scans. After both i.v. and i.a. contrast material injection, areas of injury were visible as areas of increased echogenicity. Contrast increased from 0.2 on unenhanced images to 4.0 and 4.5, respectively, after i.v. and i.a. administration of the new contrast agent. The three observers' ability to diagnose renal injury increased from 0.61, 0.64, and 0.54 to 0.71, 0.70, and 0.74 after i.v. injection and to 0.93, 0.92, and 0.97 after i.a. injection as indicated by the area under the curve in the receiver operating characteristic analysis. CONCLUSION: Transabdominal contrast-enhanced gray-scale sonography can reveal the area of acute renal hemorrhage. This procedure may be applicable in patients when sonographic contrast agents, imaging procedures, and modes of contrast administration are optimized for clinical use in trauma.

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.

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.

Insomnia in young men and subsequent depression. The Johns Hopkins Precursors Study.

The Johns Hopkins Precursors Study, a long-term prospective study, was used to study the relation between self-reported sleep disturbances and subsequent clinical depression and psychiatric distress. A total of 1,053 men provided information on sleep habits during medical school at The Johns Hopkins University (classes of 1948-1964) and have been followed since graduation. During a median follow-up period of 34 years (range 1-45), 101 men developed clinical depression (cumulative incidence at 40 years, 12.2%), including 13 suicides. In Cox proportional hazards analysis adjusted for age at graduation, class year, parental history of clinical depression, coffee drinking, and measures of temperament, the relative risk of clinical depression was greater in those who reported insomnia in medical school (relative risk (RR) 2.0, 95% confidence interval (CI) 1.2-3.3) compared with those who did not and greater in those with difficulty sleeping under stress in medical school (RR 1.8, 95% CI 1.2-2.7) compared with those who did not report difficulty. There were weaker associations for those who reported poor quality of sleep (RR 1.6, 95% CI 0.9-2.9) and sleep duration of 7 hours or less (RR 1.5, 95% CI 0.9-2.3) with development of clinical depression. Similar associations were observed between reports of sleep disturbances in medical school and psychiatric distress assessed in 1988 by the General Health Questionnaire. These findings suggest that insomnia in young men is indicative of a greater risk for subsequent clinical depression and psychiatric distress that persists for at least 30 years.

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.

Exercise and depression in midlife: a prospective study.

OBJECTIVES: This study examined the relationship of self-reported physical activity with subsequent depression and psychiatric distress. METHODS: Physical activity was assessed in medical school and midlife in 973 physicians as part of a prospective observational study. Outcome measures were the incidence of self-reported clinical depression and psychiatric distress on the General Health Questionnaire. RESULTS: The risk of depression was similar for nonexercisers and exercisers. No relationship was observed between physical activity level and subsequent psychiatric distress. CONCLUSIONS: This study found no evidence that exercise reduces risk for depression or psychiatric distress.

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.

Epstein-Barr virus clonality in lymphomas occurring in patients with rheumatoid arthritis.

OBJECTIVE: A causative role for Epstein-Barr virus (EBV) in the development of lymphoma in patients with rheumatoid arthritis (RA) has been proposed. We investigated the molecular features of EBV-positive diffuse large cell lymphomas in 2 patients with RA. METHODS: Southern blot analysis for immunoglobulin gene rearrangements, terminal repeat analysis for clonality of the EBV genome, and double-labeling of the lymphoma cells by in situ hybridization and immunoperoxidase staining were performed. RESULTS: In both cases, double-labeling studies localized the EBV genome to the malignant B cells. Both neoplasms contained clonal immunoglobulin gene rearrangements and clonal EBV genomes. CONCLUSION: Our data indicate that EBV infection was an early step in the development of these neoplasms. The findings further extend knowledge on the similarity of this subset of lymphomas to posttransplantation lymphomas and emphasize the role of immunosuppression in their genesis.

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.

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.

Percutaneous hepatic venous reanastomosis in a patient with Budd-Chiari syndrome.

A 33-year-old woman with Budd-Chiari syndrome for 9 years presented with worsening right upper quadrant pain and progressive liver dysfunction. Hepatic venography demonstrated hepatic vein occlusions, without significant IVC obstruction. Attempts at stenting a stenotic middle hepatic vein were unsuccessful. Transjugular access, however, allowed puncture from the stump of the right hepatic vein into the engorged right intrahepatic vein that had been demonstrated by retrograde hepatic venography. Two Palmaz stents were used to form the veno-venous reanastomosis. Initial success was documented angiographically and by pressure measurements before and after shunting. Followup at 7 and 16 months confirmed patency of the anastomosis without intimal hyperplasia. The patient noted near-complete resolution of her pain, and her liver function stabilized.

Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells.

ADP-ribosylation factors (ARFs) are a family of highly conserved, 20-kDa guanine nucleotide-binding proteins that participate in protein trafficking and enhance cholera toxin-catalyzed ADP-ribosylation. ARF 2 from bovine retinal cDNA was expressed in Sf9 insect cells using recombinant baculovirus and compared to the major insect cell ARF (Sf9 ARF) and to recombinant ARF 2 expressed in Escherichia coli (E. coli rARF 2). The 150000g supernatant and particulate fractions of freeze-thawed, recombinant ARF 2 baculovirus-infected cells contained immunoreactive proteins of 20 and 21 kDa at significantly higher levels than were found in uninfected cells. Infected Sf9 cells incorporated [3H]myristate only into the 20-kDa protein. Sf9 cell recombinant ARF 2 (Sf9 rARF 2) and Sf9 ARF were separated by isoelectric focusing or ion-exchange chromatography and identified by microsequencing of HPLC-purified tryptic peptides. Sf9 ARF displayed considerable sequence identity to mammalian class I ARFs. Both Sf9 ARF and Sf9 rARF 2 stimulated in a GTP-dependent manner cholera toxin-catalyzed ADP-ribosylation. The Ka for GTP of Sf9 ARF was, however, significantly lower than that of Sf9 rARF 2 or E. coli rARF 2. Myristoylation did not significantly affect the ability of ARF 2 to enhance cholera toxin-catalyzed ADP-ribosylation or the Ka for GTP. Despite the sequence identities and the fact that both were synthesized in insect cells, the endogenous Sf9 ARF was functionally different from Sf9 rARF 2.

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.

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.