The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity

Sterol 14alpha-demethylase (P45051) is the target for azole antifungal compounds, and resistance to these drugs and agrochemicals is of significant practical importance. We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyces cerevisiae to probe a model structure for the enzyme. The change T315A reduced enzyme activity 2-fold as predicted for the removal of the residue that formed a hydrogen bond with the 3-OH of the sterol substrate and helped to locate it in the active site. This alteration perturbed the heme environment, causing an altered reduced carbon monoxide difference spectrum with a maximum at 445 nm. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 4-5-fold azole resistance in transformants of S. cerevisiae. This is the first example of a single base change in the target enzyme conferring resistance to azoles through reduced azole affinity.     

A mutation in a purported regulatory gene affects control of sterol uptake in Saccharomyces cerevisiae

Aerobically growing wild-type strains of Saccharomyces cerevisiae are unable to take exogenously supplied sterols from media. This aerobic sterol exclusion is vitiated under anaerobic conditions, in heme-deficient strains, and under some conditions of impaired sterol synthesis. Mutants which can take up sterols aerobically in heme-competent cells have been selected. One of these mutations, designated upc2-1, gives a pleiotropic phenotype in characteristics as diverse as aerobic accumulation of sterols, total lipid storage, sensitivity to metabolic inhibitors, response to altered sterol structures, and cation requirements. During experiments designed to ascertain the effects of various cations on yeast with sterol alterations, it was observed that upc2-1 was hypersensitive to Ca2+. Using resistance to Ca2+ as a screening vehicle, we cloned UPC2 and showed that it is YDR213W, an open reading frame on chromosome IV. This belongs to a fungal regulatory family containing the Zn(II)2Cys6 binuclear cluster DNA binding domain. The single guanine-to-adenine transition in upc2-1 gives a predicted amino acid change from glycine to aspartic acid. The regulatory defect explains the semidominance and pleiotropic effects of upc2-1.     

Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Delta strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4beta, 14alpha-dimethyl-4alpha-carboxy-cholesta-8,24-dien-3be ta-ol and 4beta-methyl-4alpha-carboxy-cholesta-8,24-dien-3beta-o l, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.     

A single point mutation in the embB gene is responsible for resistance to ethambutol in Mycobacterium smegmatis

Ethambutol [EMB; dextro-2,2'-(ethylenediimino)-di-1-butanol] is an effective drug when used in combination with isoniazid for the treatment of tuberculosis. It inhibits the polymerization of arabinan in the arabinogalactan and lipoarabinomannan of the mycobacterial cell wall. Recent studies have shown that arabinosyltransferases could be targets of EMB. These enzymes are encoded by the emb locus that was identified in Mycobacterium smegmatis, Mycobacterium leprae, Mycobacterium avium, and Mycobacterium tuberculosis. We demonstrate that a missense mutation in the M. smegmatis embB gene, one of the genes of the emb locus, confers resistance to EMB. The level of resistance is not dependent on the number of copies of the mutated embB gene, indicating that this is a true mechanism of resistance. The mutation is located in a region of the EmbB protein that is highly conserved among the different mycobacterial species. We also identified in this region two other independent mutations that confer EMB resistance. Furthermore, mutations have recently been described in the same region of the EmbB protein from clinical EMB-resistant M. tuberculosis isolates. Together, these data strongly suggest that one of the mechanisms of resistance to EMB consists of missense mutations in a particular region of the EmbB protein that could be directly involved in the interaction with the EMB molecule.     

Alternative pre-mRNA splicing of the sterol 27-hydroxylase gene (CYP 27) caused by a G to A mutation at the last nucleotide of exon 6 in a patient with cerebrotendinous xanthomatosis (CTX)

Proline effectively inhibits protein aggregation during the refolding of bovine carbonic anhydrase. Other osmolytes used such as glycine and ethylene glycol fail to exhibit the 'aggregation-blockade' role shown by proline. Results of viscosity and ANS fluorescence (1-anilino-8-naphthalene sulphonic acid) experiments suggest that proline at high concentrations forms an ordered supramolecular assembly. Based on these results, it is proposed that proline behaves as a protein folding chaperone due to the formation of an ordered, amphipathic supramolecular assembly. To our knowledge, this is the first report wherein proline is proposed as a protein folding aid.     

Mutation of the p53 gene in human astrocytic tumours correlates with increased resistance to DNA-damaging agents but not to anti-microtubule anti-cancer agents

Human brain cancers (gliomas) overexpress large numbers of a receptor for interleukin 13 (IL13), making this receptor an attractive target for anti-glioma therapies. We have recently proposed that the glioma-associated IL13 receptor is different from the one expressed on some hemopoietic and somatic cells. In an attempt to identify an even more glioma-specific target, we have used an antagonist of a related cytokine, IL4, which neutralizes the physiological effects of both IL13 and IL4 on normal cells. Here we demonstrate that the IL4 antagonist also counteracts the action of cytotoxins targeted to the IL13 receptor on normal human cells. Importantly, the IL4 antagonist does not inhibit IL13-based cytotoxins on glioma cells at all. Thus, the IL13 receptor on glioma cells can be categorized as tumor-specific in the presence of an IL4 antagonist. We conclude that IL13 receptor-directed cytotoxins can be delivered to glioma cells without being cytotoxic to normal cells.     

Early-onset Alzheimer''s disease due to mutations of the presenilin-1 gene on chromosome 14: a 7-year follow-up of a patient with a mutation at codon 139

In recent years, several factors have altered the spectrum of respiratory infections and their likelihood of response to empiric treatment. Altered microbial resistance has led to the possible need for specific etiologic diagnosis in some hospital-acquired infections in the normal host. In the immune-compromised host, the spectrum of atypical presentations and unusual organisms limits the clinician's ability to choose effective empiric therapies. In the normal host, bronchoscopic diagnosis seems to be most useful in the groups with severe community-acquired pneumonia or poor response to therapy for community-acquired pneumonia. The group of patients with ventilator-associated pneumonia has been well-researched and the bronchoscopic techniques tend to show increased sensitivity over other diagnostic means, but this has not been proven to alter morbidity, mortality, or cost effectiveness. The immune-compromised host is commonly infected by organisms not easily diagnosed by other means and is thus unable to be treated empirically. Bronchoscopic diagnostic techniques play a larger and more clearly delineated role in these populations, including the patient populations with solid organ transplants, bone marrow transplants, and AIDS.     

Progressive ataxia due to a missense mutation in a calcium-channel gene

We describe a family with severe progressive cerebellar ataxia involving the trunk, the extremities, and speech. The proband, who has prominent atrophy of the cerebellum, shown by magnetic resonance imaging, was confined to a wheelchair at the age of 44 years. Two sons have episodes of vertigo and ataxia that are not responsive to acetazolamide. Quantitative eye-movement testing showed a consistent pattern of abnormalities localizing to the cerebellum. Genotyping suggested linkage to chromosome 19p, and SSCP showed an aberrant migrating fragment in exon 6 of the calcium-channel gene CACNA1A, which cosegregated with the disease. Sequencing of exon 6 identified a G-->A transposition in one allele, at nucleotide 1152, resulting in a predicted glycine-to-arginine substitution at codon 293. The CAG-repeat expansion associated with spinocerebellar ataxia 6 was not present in any family members. This family is unique in having a non-CAG-repeat mutation that leads to severe progressive ataxia. Since a great deal is known about the function of calcium channels, we speculate on how this missense mutation leads to the combination of clinical symptoms and signs.     

Ongoing mutation in MALT lymphoma immunoglobulin gene suggests that antigen stimulation plays a role in the clonal expansion

Indirect antigenic stimulation by H. pylori-specific T cells is implicated in the development of low-grade gastric lymphoma of mucosa-associated lymphoid tissue (MALT), however, the role of direct antigen stimulation is unknown. To study the role of direct antigen stimulation in MALT lymphomagenesis and its relationship with the pathogenesis of distinct pathological lesions, which represent different stages of the tumour progression, we cloned and sequenced the rearranged immunoglobulin (Ig) heavy chain gene in three low-grade (two from the lung, one from the stomach) and one high-grade (from the stomach) cases. In the low-grade gastric case, we studied the Ig sequence in primary as well as its disseminated and recurrent tumours. In the high-grade gastric case, we analysed the Ig sequence in tumour cell populations microdissected from the residual diffuse low-grade lesions, diffuse high-grade areas from follicles colonized by high-grade blasts. Compared with the published germline sequences, the heavy chain variable (VH) genes of three MALT lymphomas, in which the putative germline was identified, contained frequent somatic mutations, showing a much higher ratio of replacement/silent mutations in the complementarity determining regions (CDRs) than the framework regions (FRs). Ongoing mutation as indicated by intraclonal variation of the Ig sequence clearly existed in low-grade tumour including its dissemination and recurrence, but was not evident in high-grade tumour cell populations including those microdissected from independent colonized follicles. In addition, the germlines of VH genes used by the three MALT lymphomas are frequently found in autoreactive antibodies. Our results suggest that MALT lymphoma derives from postgerminal centre memory B cells, possibly autoreactive B cell clones, and that direct antigen stimulation may play an important role in the clonal expansion of low-grade MALT lymphoma.     

A Japanese family with paramyotonia congenita which has a mutation in the muscle sodium channel gene

We report here a Japanese family with paramyotonia congenita. The proband was a 42-year-old woman (case 1), who noticed muscle stiffness and weakness in the cold since the age of 7 years. These symptoms were alleviated by warming. Her eldest son (case 2) also experienced similar symptoms, while her younger son and daughter were healthy. Neurological examination in case 1 revealed mild weakness in facial and neck muscles. Cold-induced muscle stiffness and weakness were present. Electromyography showed myotonic discharges, intensified by cooling or repetitive exercise. The amplitude of the compound muscle action potentials was also reduced by the repetitive exercise and cooling. Serum chemistry including potassium and CK was normal. Molecular analysis of SCN4A (exon22-24) by SSCP and nucleotide sequencing revealed a C-to-T transition at nucleotide 3,938, causing a substitution of 1313methionine of threonine in case 1. This mutation was confirmed by PCR-RFLP with a mismatched primer; the proband (case 1) and her eldest son (case 2) had a heterozygous mutation, while the other family members did not. This is the first report that a mutation in SCN4A was identified in a Japanese family with paramyotonia congenita.     

A prion disease with a novel 96-base pair insertional mutation in the prion protein gene

There are coding mutations in the prion protein gene in familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease, and other phenotypes that make up the inherited prion diseases. Insertional mutations consisting of two, five, six, seven, eight, and nine additional octapeptide repeat elements are seen in the inherited prion diseases and usually present as atypical dementias with considerable intrafamilial phenotypic variability. A four-octarepeat insertion was reported previously in an individual without neurodegenerative disease who died of hepatic cirrhosis. Here we report a novel four-octarepeat insertional mutation in a case with classical clinical, electroencephalographic and histopathologic features of CJD with the unusual finding of pronounced prion protein immunoreactivity of the molecular layer of the cerebellum.     

Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtl in transgenic plants showing an increase of beta-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon

The possibility that extracellular matrix-cell surface interaction might lead to signalling was examined in human mononuclear cells. In mononuclear cells loaded with Indo-1 acetoxymethylester, collagen binding elicited a 2-3 fold increase in cytosolic free calcium concentration within seconds. Plasma fibronectin, serum albumin and heparin did not cause any significant change in intracellular calcium levels. Collagen I, collagen IV and glucosylated collagen I evoked an increase in intracellular Ca++ both in the presence and absence of extracellular calcium. However, in the presence of 2mM EDTA, the increase in cytosolic free calcium concentration caused by collagen I and collagen IV was partly abolished, suggesting the requirement of a cation-dependent interaction of collagen with mononuclear cells. Glucosylated collagen induced intracellular calcium mobilization even in the presence of EDTA suggesting a cation-independent interaction. These results indicate that collagen binding induces rapid mobilization of calcium in human mononuclear cells, apparently from intracellular sources.