Cloning of the PYR4 gene encoding orotidine-5′-phosphate decarboxylase in Cephalosporium acremonium

Summary We have cloned the Cephalosporium acremonium pyr4 gene by cross-hybridization with the equivalent gene from Neurospora crassa, the closest relative from which this gene is available. The C. acremonium pyr4 gene complements an E. coli pyrF mutant lacking orotidine-5-phosphate decarboxylase (OMPdecase), and most probably does not contain introns. Maxicell analysis in E. coli shows that it encodes a 46 kDa polypeptide. The C. acremonium OMPdecase contains a highly conserved pentadecapeptide characteristic for this category of enzyme. Extensive sequence comparison suggests an important role of this region in enzymatic activity.     

Wiedemann‐Steiner syndrome in two patients from Portugal

Wiedemann‐Steiner syndrome (WSS) is a rare genetic disorder characterized by growth retardation, facial dysmorphism, hypertrichosis cubiti and neurodevelopment delay. It is caused by pathogenic variants in the KMT2A gene. This report describes two unrelated Portuguese patients, age 11 and 17 years, with a phenotype concordant with WSS and clinical and molecular diagnosis of WSS by the identification of two novel frameshift variants in the KMT2A gene. This work also highlights the presence of certain clinical features in patients with growth retardation and development delay and should draw attention to the diagnosis of WSS, when hirsutism, particularly hypertrichosis cubiti is present.     

Clinical evaluation of a commercial ligase-based gene amplification method for detection ofMycobacterium tuberculosis

The purpose of this study was to evaluate the clinical usefulness of a commercial ligase-based gene amplification method (LCxMycobacterium tuberculosis test; Abbott Laboratories, USA) for detection ofMycobacterium tuberculosis. The tuberculosis infection rate among clinical samples was 10.6%. The sensitivity, specificity, and positive and negative predictive values were 23.5%, 100%, 100%, and 91.7%, respectively, with the fluorochrome auramine stain; 32.4%, 100%, 100%, and 92.6%, respectively, with culture; and 76.5%, 95.8%, 68.4% and 97.2%, respectively, with the gene amplification method. When only samples from patients without current or previous treatment were studied, the sensitivity was 36.4% with the auramine stain, 63.6% with culture, and 100% with the gene amplification assay. The mean treatment time for culture-negative and assay-negative samples was greater than that of culture-negative and assay-positive samples. The LCxMycobacterium tuberculosis test is a sensitive method for detection and identification ofMycobacterium tuberculosis. It produces few false-positive results. However, as it can remain positive after the culture becomes negative, it is not recommended for evaluation of treatment efficiency.     

Polyamine N-acetyltransferase in Leishmania amazonensis

 N-Acetyltransferase, which is suggested to be responsible for the production of N ¹-acetylspermidine in Leishmania amazonensis and to be involved in the process of inactivation and degradation of excessive polyamines, was partially purified and characterized. Among the substrates tested, sym-norspermidine, sym-norspermine, and 1,3-diaminopropane had the highest reaction rates, but the naturally occurring polyamines spermine and spermidine were also acetylated at considerable rates, whereas putrescine was a poor substrate. The Michaelis constants (K _m values) for spermine and spermidine were 0.66 and 3.3 mM, respectively. The K_m value for acetylcoenzyme A (acetyl-CoA) was determined to be 34 μM. CoA inhibited the reaction in a competitive manner; the inhibition constant was 5 μM. The enzyme showed an apparent relative molecular mass of 35,000. Received: 16 November 1995 / Accepted: 10 January 1996     

Detecting pre-ovulatory luteinizing hormone surges in urine

The study objectives were to determine (i) if pre-ovulatory luteinizing hormone (LH) surges, undetected in urine by two immunoradiometric assays (IRMA), were detectable by an ultrasensitive immunofluorometric assay (IFMA) and (ii) the influence of creatinine adjustment on the detection and timing of the urinary LH surges. Daily urine specimens were contributed by healthy 25-36 year old volunteers during 14 ovulatory menstrual cycles for an epidemiological study conducted in 1983-1985. Specimens were selected as having been previously assayed by two IRMA without consistently detecting LH surges. These urine specimens were remeasured using an IFMA and adjusted for creatinine concentration. IFMA measurements revealed unambiguous LH surges in all cycles. Adjusting IRMA urinary LH values for creatinine concentrations revealed previously undetected LH surges in four of eight cycles. Creatinine adjustment also altered the timing of IRMA and IFMA LH surges by 1-5 days. These results demonstrate an IFMA that detects pre- ovulatory LH surges in unpreserved, frozen urine from cycles where such surges were previously undetectable. Further, creatinine adjustment can markedly affect detection and timing of the onset and peak of the urinary LH surge. While our analysis suggests that this adjustment improves the validity of the LH measure, this requires further investigation.      

Use of interleukin 15 to enhance interferon-gamma production by antigen-specific stimulated lymphocytes from rhesus macaques

The enzyme-linked immune spot (ELISPOT) assay is receiving increased attention as a means for quantifying antigen-specific CD8 T-cell responses in rhesus macaques. Further improving the sensitivity of this assay could aid in the evaluation of vaccine candidates and/or immune therapeutic candidates. Interleukin (IL)-15 has been demonstrated to stimulate expansion of human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) and to regulate homeostatic proliferation of CD8+ memory cells. We evaluated the in vitro effect of IL-15 to increase the detection of interferon-gamma (IFN-gamma) production by antigen-specific stimulated lymphocytes from a group of rhesus macaques exposed to simian-human immunodeficiency virus (SHIV) and a second group infected with SIVmac251, before and after antiretroviral treatment (ART). Results from these studies demonstrate that the presence of IL-15 during stimulation in a peptide-based ELISPOT assay greatly enhanced IFN-gamma production in both SHIV and simian immunodeficiency virus (SIV)-infected macaques. IFN-gamma production was mainly mediated by CD8 lymphocytes. The optimal concentrations of IL-15 that give enhancement of IFN-gamma production to specific antigen, without a significant increase in the spontaneous IFN-gamma release, ranged from 0.5 to 2.5 ng/ml. The mean number of IFN-gamma spots was increased 3.1- to 3.6-fold in response to SIV gag or HIV env peptide pools, respectively, in peripheral blood mononuclear cells (PBMC) from SHIV-infected macaques. Similarly, in SIV-infected macaques, IL-15 increased the mean number of IFN-gamma spots 2.7-fold in response to both SIV gag and env peptide pools. In samples obtained after ART in the same macaques, the increase factor was 2.5 for SIV gag and 1.8 for the env peptide pools. Thus, the sensitivity of the ELISPOT assay can be enhanced by addition of IL-15. This modified assay will be useful for detection of low frequencies of IFN-gamma producing cells in rhesus macaques.