The effect of autoimmune blistering diseases on work productivity.

BACKGROUND: Autoimmune blistering diseases (AIBD) are known to negatively impact upon quality of life (QoL); however, there is a paucity of research on the effect of AIBD on work productivity. AIBD can be quite disfiguring in terms of a patient's appearance due to their blistering nature. OBJECTIVE: To determine the impact of AIBD on work productivity and to determine whether patients are stigmatized at work due to their appearance. METHODS: Sixty-one patients with AIBD completed the Work Productivity and Activity Impairment Questionnaire-Specific Health Problem (WPAIQ-SHP), the Dermatology Life Quality Index (DLQI), the Autoimmune Bullous Disease Quality of Life (ABQOL) and the Treatment of Autoimmune Bullous Disease Quality of Life questionnaires (TABQOL). RESULTS: Non-responders to treatment had more work and activity impairment compared to responders. Worse WPAIQ-SHP scores were correlated with higher ABQOL, TABQOL and DLQI scores. Approximately 14.8% of subjects experienced stigmatization at work due to their appearance. The most common body areas stigmatized were easily visible sites, particularly the hands, arms and feet, with the majority of occurrences related to co-workers; for some patients, this stigmatization occurred on a daily basis. Loss of productivity at work was statistically much higher in those with higher disease severity, ABQOL & TABQOL scores and in non-responders to treatment. CONCLUSION: Autoimmune blistering diseases negatively impacts upon work productivity and activity. Stigmatization was common in the workplace which leads to increased stress, itself a stimulator of pemphigus.

TSG101 protein steady-state level is regulated posttranslationally by an evolutionarily conserved COOH-terminal sequence.

Antisense inactivation of the tsg101 tumor susceptibility gene in murine NIH3T3 fibroblasts leads to neoplastic transformation and tumorigenesis, which are reversed by restoration of tsg101 activity. tsg101 deficiency is associated with a series of mitosis-related abnormalities, whereas overexpression of TSG101 can also result in neoplastic transformation and the perturbation of cell cycling. Together, these observations imply that TSG101 production outside of a narrow range can lead to abnormal cell growth. We report here that the TSG101 protein is maintained at an almost constant steady-state level in cultured murine and human cells and that this occurs through a posttranslational process involving TSG101 protein degradation. Sustained overproduction of TSG101 from chromosomally inserted adventitious constructs resulted in compensatory down-regulation of endogenous TSG101 and replacement of the native protein by the adventitious one. Using deletion mutants of TSG101, we mapped the region responsible for autoregulation of the TSG101 steady-state level to an evolutionarily conserved sequence, here termed the "steadiness box," located near TSG101's COOH-terminal end. Our results suggest a model in which the biological effects of TSG101 are modulated either by self-promoted proteolysis or participation with other cellular protein(s) in a proteolytic feedback-control loop.

The Hermansky-Pudlak syndrome (HPS) protein is part of a high molecular weight complex involved in biogenesis of early melanosomes.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder in which oculocutaneous albinism, bleeding tendency and a ceroid-lipofuscin lysosomal storage disease result from defects of multiple cytoplasmic organelles: melanosomes, platelet dense granules and lysosomes. The HPS polypeptide, a 700 amino acid protein which is unrelated to any known proteins, is likely to be involved in the biogenesis of these different organelles. Here, we show that HPS is a non-glycosylated, non-membrane protein which is a component of two distinct high molecular weight complexes. In non-melanotic cells the HPS protein is contained almost entirely in an approximately 200 kDa complex that is widely distributed throughout the cytosol. In melanotic cells the HPS protein is partitioned between this cytosolic complex and a >500 kDa complex that appears to consist of the approximately 200 kDa complex in association with membranous components. Subcellular fractionation, immunofluorescence and immunoelectron microscopy studies indicate that the membrane-associated HPS complex of melanotic cells is associated with tubulovesicular structures, small non-coated vesicles, and nascent and early-stage melanosomes. These findings suggest that the HPS complex is involved in the biogenesis of early melanosomes.

Culture conditions control expression of the genes for aflatoxin and sterigmatocystin biosynthesis in Aspergillus parasiticus and A. nidulans.

High temperature and nitrate supported gene expression for sterigmatocystin biosynthesis in Aspergillus nidulans; ammonium did not. Homologous genes for aflatoxin biosynthesis in A. parasiticus showed the opposite transcript expression pattern, suggesting that the two mycotoxins are regulated differently. The aflR gene is postulated to require additional genetic elements to effect its own activation by the different culture conditions. A patulin polyketide synthase (PKS) gene was found to be regulated differently than the aflatoxin PKS. Thus, the biosyntheses of structurally similar compounds in these two fungi appear to be regulated very differently.

Organization and nucleotide sequence of the human Hermansky-Pudlak syndrome (HPS) gene.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism, bleeding tendency, and lysosomal ceroid storage disease, associated with defects of multiple cytoplasmic organelles-melanosomes, platelet-dense granules, and lysosomes. HPS is frequently fatal and is the most common single-gene disorder in Puerto Rico. We previously characterized the human HPS cDNA and identified pathologic mutations in the gene in patients with HPS. The HPS protein is a novel apparent transmembrane polypeptide that seems to be crucial for normal organellar development. Here we describe the structural organization, nucleotide sequence, and polymorphisms of the human HPS gene. The gene consists of 20 exons spanning about 30.5 kb in chromosome segment 10q23.1-q23.3. One of the intervening sequences is a member of the novel, very rare class of so-called "AT-AC" introns, defined by highly atypical 5' and 3' splice site and branch site consensus sequences that provide novel targets for possible pathologic gene mutations. This information provides the basis for molecular analyses of patients with HPS and will greatly facilitate diagnosis and carrier detection of this severe disorder.

Mouse pale ear (ep) is homologous to human Hermansky-Pudlak syndrome and contains a rare 'AT-AC' intron.

Hermansky-Pudlak syndrome (HPS) is a rare, often fatal, autosomal recessive disorder in which albinism, bleeding and lysosomal storage are associated with defects of diverse cytoplasmic organelles, including melanosomes, platelet dense granules and lysosomes. Similar multi-organellar defects occur in the Chediak-Higashi syndrome (CHS), as well as in a large number of different mouse mutants. The HPS gene is located in 10q23, and two genetically distinct mouse loci, pale ear (ep) and ruby-eye (ru), both with mutant phenotypes similar to human HPS, map close together in the homologous region of murine chromosome 19, suggesting that one of these loci might be homologous to human HPS. We recently identified the human HPS gene, which encodes a novel ubiquitously-expressed transmembrane protein of unknown function. Here, we describe characterization of the mouse Hps cDNA and genomic locus, and identification of pathologic Hps gene mutations in ep but not in ru mice, establishing mouse pale ear as an animal model for human HPS. The phenotype of homozygous ep mutant mice encompasses those of both HPS and CHS, suggesting that these disorders may be closely related. In addition, the mouse and human HPS genes both contain a rare 'AT-AC' intron, and comparison of the sequences of this intron in the mouse and human genes identified conserved sequences that suggest a possible role for pre-mRNA secondary structure in excision of this rare class of introns.

Positional cloning of a gene for Hermansky-Pudlak syndrome, a disorder of cytoplasmic organelles.

Hermansky-Pudlak syndrome (HPS) is an often-fatal autosomal recessive disease in which albinism, bleeding, and lysosomal storage result from defects of diverse cytoplasmic organelles: melanosomes, platelet dense bodies, and lysosomes. HPS is the most common single-gene disorder in Puerto Rico, with an incidence of 1 in 1,800. We have identified the HPS gene by positional cloning, and found homozygous frameshifts in this gene in Puerto Rican, Swiss, Irish and Japanese HPS patients. The HPS polypeptide is a novel transmembrane protein that is likely to be a component of multiple cytoplasmic organelles and that is apparently crucial for their normal development and function. The different clinical phenotypes associated with the different HPS frameshifts we observed suggests that differentially truncated HPS polypeptides may have somewhat different consequences for subcellular function.

alpha-Amylase inhibitors from wheat: amino acid sequences and patterns of inhibition of insect and human alpha-amylases.

Four alpha-amylase inhibitors, WRP24, WRP25, WRP26, and WRP27, were purified from wheat flour by preparative, reversed-phase high performance liquid chromatography. All have polypeptide molecular masses of about 14 kDa and are members of the cereal superfamily of protease and alpha-amylase inhibitors. Sedimentation velocity analysis indicated that WRP25 and WRP27 are monomeric proteins, whereas WRP24 is a dimer. WRP24 is identical in N-terminal amino acid sequence to the well characterized 0.19 dimeric inhibitor from wheat kernels. WRP25 and WRP26 differ in sequence from each other at only three positions and represent previously unseparated forms of the 0.28 wheat inhibitor. WRP27 is a previously uncharacterized inhibitor and is more similar in sequence to the 0.28 inhibitor than to the 0.19 inhibitor. WRP25 and WRP26 inhibited alpha-amylases from the rice weevil, red flour beetle, and the yellow meal worm, but did not inhibit human salivary alpha-amylase. WRP24 inhibited the human as well as the insect alpha-amylases, but inhibited one of the two rice weevil alpha-amylases much more strongly than the other. WRP27 was notable in that, of the enzymes tested, it strongly inhibited only the rice weevil alpha-amylases. We observed that the growth rate of red flour beetle larvae was slowed when purified WRP24 was included in the diet at a level of 10%. Addition of WRP24 to corn starch resulted in greater weight loss of red flour beetle adults than occurred on control diets. Our results support the hypothesis that these alpha-amylase inhibitors provide wheat seeds with a selective evolutionary advantage since the inhibitors can slow the growth of insect pests that attack cereal grains.

Characterization of the polyketide synthase gene (pksL1) required for aflatoxin biosynthesis in Aspergillus parasiticus.

Aflatoxins are potent toxic and carcinogenic compounds, produced by Aspergillus parasiticus and A. flavus as secondary metabolites. In this research, a polyketide synthase gene (pksL1), the key gene for aflatoxin biosynthesis initiation in A. parasiticus, has been functionally identified and molecularly characterized. PCR-derived DNA probes were used to find the pksL1 gene from subtracted, aflatoxin-related clones. Gene knockout experiments generated four pksL1 disruptants which lost both the ability to produce aflatoxins B1, B2, and G1 and the ability to accumulate norsolorinic acid and all other intermediates of the aflatoxin biosynthetic pathway. A pksL1 DNA probe detected a 6.6-kb poly(A)+ RNA transcript in Northern (RNA) hybridizations. This transcript, associated with aflatoxin production, exhibited a regulated expression that was influenced by growth phase, medium composition, and culture temperature. DNA sequencing of pksL1 revealed an open reading frame for a polypeptide (PKSL1) of 2,109 amino acids. Sequence analysis further recognized four functional domains in PKSL1, acyl carrier protein, beta-ketoacyl-acyl carrier protein synthase, acyltransferase, and thioesterase, all of which are usually present in polyketide synthases and fatty acid synthases. On the basis of these results, we propose that pksL1 encodes the polyketide synthase which synthesizes the backbone polyketide and initiates aflatoxin biosynthesis. In addition, the transcript of pksL1 exhibited heterogeneity at the polyadenylation site similar to that of plant genes.

GreA-induced transcript cleavage in transcription complexes containing Escherichia coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure.

The Escherichia coli GreA and GreB proteins induce cleavage of 3' fragments from nascent transcripts in halted transcription complexes. We have overproduced and purified the GreA protein and tested how it affects initiation, pausing, and termination by E. coli RNA polymerase. Recombinant GreA induced cleavage of two to three nucleotide fragments in two promoter-proximal complexes, whereas an apparently endogenous cleavage removed a single larger fragment. Both types of cleavage stopped once the transcript was shortened to approximately 10 nucleotides. However, during initiation, GreA induced cleavage of transcripts as short as four nucleotides, inhibiting their release as abortive products and stimulating both productive initiation and "primer-shifting" at a weak promoter. GreA induced repetitive cleavage over a long distance in complexes containing a long G-less nascent transcript. However, reverse translocation was inhibited in transcription complexes that contained a G-rich, C-less nascent transcript. Substituting IMP for GMP in the transcript relieved inhibition. Finally, GreA had little effect on transcription through the his and trp leader pause sites or on termination at nine different p-independent terminators. We propose that transcript cleavage and reverse translocation are controlled in part by backsliding of the nascent transcript through an RNA-binding site.

Molecular cloning of genes related to aflatoxin biosynthesis by differential screening.

A differential hybridization strategy was used to clone genes associated with aflatoxin biosynthesis. A genomic library, formed between nuclear DNA and the pUC19 plasmid, was screened with three different cDNA probes by the colony hybridization procedure. Nineteen clones were selected; all were positively correlated with and presumably enriched with genes associated with aflatoxin production. Some of these clones were further characterized by using them as probes in Northern (RNA blot) hybridizations. Five clones hybridized strongly with some polyadenylated RNAs formed during the transition to or during idiophase when aflatoxin was produced. However, little or no corresponding hybridization occurred with polyadenylated RNAs formed in early and mid-log growth phase. Two of the clones were further used as probes to hybridize with polyadenylated RNAs formed under aflatoxin-permissive and nonpermissive temperatures. Hybridization occurred with RNA species formed under the permissive temperature only.

Inhibition of digestive proteinases of stored grain Coleoptera by oryzacystatin, a cysteine proteinase inhibitor from rice seed.

Electrophoresis of midgut extracts from the rice weevil, Sitophilus oryzae, and the red flour beetle, Tribolium castaneum, in polyacrylamide gels containing sodium dodecyl sulfate and gelatin revealed there was one major proteinase (apparent molecular mass = 40,000) in the rice weevil and two major proteinases (apparent molecular masses = 20,000 and 17,000) in the red flour beetle. The pH optima using [3H]casein as substrate were about pH 6.8 for the rice weevil and pH 5.2 for the red flour beetle. Use of specific inhibitors, including L-trans-epoxysuccinyl-leucylamino-(4- guanidino)-butane (E-64), p-chloromercuriphenylsulfonic acid (PCMS), and oryzacystatin, indicated that nearly all of the proteinase activity against casein was contributed by cysteine proteinases. The estimated IC50 values for oryzacystatin were 2 x 10(-6) M and 4 x 10(-7) M when tested against midgut extracts from T. castaneum and S. oryzae, respectively.

[Studies on mtDNA of Ustilago maydis. II. Restriction mapping].

A restriction map was constructed for mtDNA of Ustilago maydis. The fragment order for each restriction enzyme was determined by DNA hybridization and fragment overlapping. The restriction sites were located by analysing the secondary digestions of the cloned mtDNA fragments. It was also found that the mtDNA of U. maydis was a circle molecule (60.7 kb), without recognizable repeat sequence.

[Studies of mtDNA of Ustilago maydis. I. Cloning and gene mapping].

This paper covers the following studies of mtDNA of Ustilago maydis. (1) By inserting the Bam HI and Pst I fragments of the mtDNA into the corresponding sites of pBR322, we cloned a unique sequence of 49.6 kb, accounting for 89.3% of the mitochondrial genome (60.7 kb). (2) With heterogenous genes from plants or fungi as probes, we identified seven genes, and mapped them onto the restriction map of the mt DNA. The genes were arranged in such an order: -UmCOB-UmOXII-S-rR NA-UmOXIII-L-rRNA-UmATPase6-UmOXI-. (3) We tried to express the three cloned genes, UmOXII, UmOXIII, and Um-ATPase 6, in E. coli maxcel expression system, but no specific protein was observed.

[Effects of Cymbopogon goeringii (Steud.) A. Camus volatile oil on physiologic properties of the isolated guinea pig myocardium].

In isolated guinea pig papillary muscles and atrium, Cymbopogon goeringii volatile oil (CGCO) markedly inhibited the contraction, prolonged the functional refractory period (FRP) and depressed epinephrine-induced automatically. The results suggested that CGCO may possess antiarrhythmic action.

Small-dose Harringtonine induces complete remission in patients with acute promyelocytic leukemia.

Small-dose Harringtonine (1-3 mg infused during 4-5 hr) was used as a single agent to treat 10 patients with acute promyelocytic leukemia. Every patient received one to three courses, each lasting 13-81 days (mean 33 days). The interval between courses (i.e., interruptions) was 5-11 days. During treatment, marrow aplasia occurred in one patient and hypoplasia in three. Pancytopenia occurred in all 10 patients. Complete remission was achieved in seven patients (70%) and cytoreduction in two. In vitro studies showed that, although Harringtonine produced a decrease in leukemic cells in all five series of marrow cultures from five patients, there was only one wherein the decrease was accompanied by a simultaneous absolute increase in differentiated myeloid cells. Considerable discrepancy existed between the culture results and clinical responses. These results seem to suggest that the therapeutic effect of Harringtonine on acute promyelocytic leukemia originates chiefly from cytotoxicity.