Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins.     

Molecular characterization of the Clostridium difficile toxin A gene

The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs had a mol% G + C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Gal alpha 1-3Gal beta 1-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.     

Adhesion of peripheral blood lymphocytes of children with arthritis to human umbilical vein endothelial cells.

To determine whether adhesion of peripheral blood lymphocytes (PBL) of patients with juvenile rheumatoid arthritis (JRA) may be enhanced, adhesion of PBL of children with JRA, children with seronegative spondyloarthropathies (SSA), age-appropriate and adult controls, to human umbilical vein endothelial cells (HUVEC) was assessed in vitro. B and CD4 T lymphocytes in initial, adherent, and non-adherent cell fraction were identified by flow cytometry. B lymphocytes of all the younger subjects combined had a higher adherence to activated HUVEC compared with B lymphocytes of the adult donors. Except for greater adherence of HLA-DR+ CD4 T cells, lymphocytes of children with JRA showed no enhanced adhesion to either unactivated or activated HUVEC. The percentage of B cells adherent to activated HUVEC in each of the subject groups was 1.5-3.6-fold higher than adherent CD4 T lymphocytes. Surface analyses indicated higher percentages of CD49d (alpha 4)+ and CD29 (beta 1)+ CD4 T lymphocytes in adherent cells, but less of a differential in CD49 (alpha 4)+ and no difference in CD29 (beta 1)+ B lymphocytes. There were fewer Leu-8 (L-selectin)+ B and Leu-8+ CD4 T cells among adherent cells. The data suggest a greater adhesive capacity of B lymphocytes compared with CD4 T lymphocytes which is unrelated to disease, and the possibility that B lymphocytes may utilize adhesion molecules distinct from those of CD4 T lymphocytes. Only a small subset of T cells of patients with JRA may have an enhanced capacity for adhesion to endothelium.     

Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A.

The toxin A gene of Clostridium difficile contains a 2.5-kb region encoding a series of contiguous repeating units located at the COOH terminus of the molecule. We previously showed that the monoclonal antibody (MAb) PCG-4, which neutralizes the enterotoxic activity of toxin A, binds to epitopes located within these repeating units. In the present study, we subcloned a series of fragments from this portion of the gene. The recombinant peptides expressed from the gene fragments were examined for reactivity with MAb PCG-4 to identify the epitopes involved in binding. Our results showed that MAb PCG-4 recognizes epitopes in amino acid residues 2097 through 2141 and amino acid residues 2355 through 2398.     

Cancer of the colon in an egg donor: policy repercussions for donor recruitment

This paper describes the tragic case of a young woman who died of cancer of the colon after successfully donating eggs to her younger sister. Although there is no direct link between her operation and the subsequent development of bowel carcinoma, this case imparts a feeling of unease when seen in conjunction with other cases reported during the last few years. It is a reminder that little is known of the long-term consequences of some aspects of assisted conception. Women undergoing ovarian stimulation for themselves or a matched recipient have the right to be advised, in an agreed format, that there is some concern about unproven potential risks from the stimulatory drugs. The safety of egg donors must assume priority over all other considerations, including lack of donors or any moral position. The recent decision by the Human Fertilisation and Embryology Authority (HFEA) to withdraw any form of payment or recompense to egg donors does not seem to us to be based on a balance of scientific advances, patient needs and the ethics of gamete supply. They state that the intention to withdraw payments was implicit in the 1990 Human Fertilisation and Embryology (HFE) Act. However the Act was based on the Warnock report made 6 years earlier. Even in 1990 ovum donation was uncommon and fertility drugs had not yet caused any unease. The Act provided the HFEA with discretionary powers to issue directions so that the future policies would be consistent with any emerging new medical evidence. It is imperative that the HFEA provide convincing evidence on how the current policy of payment to donors harms society, donors or recipients, and how in the UK the new policy will improve medical practice in assisted conception. Successful pilot studies must precede the implementation of any new policy. Failure to do this could cause irreversible harm to the practice of assisted conception using donor gametes, which will ultimately be against the basic aims of the 1990 HFE Act.      

Gain of 1q and loss of 22 are the most common changes detected by comparative genomic hybridisation in paediatric ependymoma

Ependymomas are the third most common brain tumour in the paediatric population. Although cytogenetic and molecular analyses have pinpointed deletions of chromosomes 6q, 17, and 22 in a subset of tumours, definitive patterns of genetic aberrations have not been determined. In the present study, we analysed 40 ependymomas from paediatric patients for genomic loss or gain using comparative genomic hybridisation (CGH). Eighteen of the tumours (45%) had no detectable regions of imbalance. In the remaining cases, the most common copy number aberrations were loss of 22 (25% of tumours) and gain of 1q (20%). Three regions of high copy number amplification were noted at 1q24-31 (three cases), 8q21-23 (two cases), and 9p (one case). Although there was no association with the loss or gain of any chromosome arm or with benign versus anaplastic histologic characteristics, the incidence of gain of 7q and 9p and loss of 17 and 22 was significantly higher in recurrent versus primary tumours. This study has identified a number of chromosomal regions that may contain candidate genes involved in the development of different subgroups of ependymoma.     

The Devereux Adolescent Behavior Rating Scale: a tentative model of second-order factor structure across independent clinical samples.

The second-order factor structure of the Devereux Adolescent Behavior Rating Scale was examined in (a) a sample of 254 adolescents who were receiving residential treatment for severe emotional/behavior disorders and (b) an independent sample of 404 adolescents hospitalized for substance abuse. A plausible range of factors was estimated for each group through parallel and average partial analyses and suggested wither a two- or three-factor solution. Subsequent congruence analyses provided tentative support for a three-factor model: (a) undercontrolled/disruptive behaviors; (b) withdrawn/psychotic behaviors; and (c) needs approval/dependent behaviors. The third factor was poorly defined in the residential treatment sample, but extraction of the third factor increased the across-group replicability of the first two factors.     

Morphological and immunohistological study of testicular lymphomas

We have studied normal testis, seminomas and malignant lymphomas of the testis using routine stains and a panel of antibodies directed against lymphoid and basement membrane antigens. The results show that normal testis contains, at most, a minor population of T-lymphocytes: seminomas contain mixed T- and B-cell populations with a predominance of B-lymphocytes; and most primary lymphomas are B-cell tumours of large centroblastic type. Solid testicular lymphomas presenting secondarily to acute lymphoblastic leukaemias showed intact seminiferous tubular basement membranes with predominantly interstitial lymphomatous infiltrates, whereas the tubules in primary cases were over-run by lymphoma cells and basement membranes were disrupted.     

Clostridium difficile toxin-induced reactive arthritis in a patient with chronic Reiter's syndrome

The first case ofClostridium difficile toxin-induced reactive arthritis in a patient with chronic Reiter's syndrome is described and compared with previous cases of reactive arthritis associated with this organism. This case demonstrates how distinct clinical manifestations may develop at different times in Reiter's syndrome, according to the infecting organism. Diagnostic terminology is discussed in this context.Clostridium difficile should now be considered a firmly established cause of reactive arthritis.     

Nitric oxide is not permissive for cutaneous active vasodilatation in humans

The precise role of nitric oxide (NO) in cutaneous active vasodilatation in humans is unknown. We tested the hypothesis that NO is necessary to permit the action of an unknown vasodilator. Specifically, we investigated whether a low-dose infusion of exogenous NO, in the form of sodium nitroprusside (SNP), would fully restore vasodilatation in an area of skin in which endogenous NO was inhibited during hyperthermia. This finding would suggest a 'permissive' role for NO in active vasodilatation. Eight subjects were instrumented with three microdialysis fibres in forearm skin. Sites were randomly assigned to (1) Site A: control site; (2) Site B: NO synthase (NOS) inhibition during established hyperthermia; or (3) Site C: NOS inhibition throughout the protocol. Red blood cell flux was measured using laser-Doppler flowmetry (LDF) and cutaneous vascular conductance (CVC; LDF/mean arterial pressure) was normalized to maximal vasodilatation at each site. In Site B, N ^G-nitro- l -arginine methyl ester ( l -NAME) infusion during hyperthermia reduced CVC by ∼32 % (65 ± 4 % CVC_max vs. 45 ± 4 % CVC_max; P < 0.05). Vasodilatation was not restored to pre-NOS inhibition values in this site following low-dose SNP infusion (55 ± 4 % CVC_max vs. 65 ± 4 % CVC_max; P < 0.05). CVC remained significantly lower than the control site with low-dose SNP infusion in Site C ( P < 0.05). The rise in CVC with low-dose SNP (ΔCVC) was significantly greater in Site B and Site C during hyperthermia compared to normothermia ( P < 0.05). No difference in ΔCVC was observed between hyperthermia and normothermia in the control site (Site A). Thus, NO does not act permissively in cutaneous active vasodilatation in humans but may directly mediate vasodilatation and enhance the effect of an unknown active vasodilator.