Lichenoid vulvar disease: A review.

Vulvar dermatoses are common, potentially debilitating conditions that can be seen by a variety of medical specialists. Lichenoid vulvar diseases, namely lichen sclerosus (LS), lichen planus (LP), and lichen simplex chronicus (LSC), can all negatively impact patients' quality of life and LS and LP also have an association with squamous cell carcinoma. It is essential that dermatologists are familiar with the unique features of each of these conditions to ensure the appropriate management and follow up. Herein, we provide an update on the epidemiology, clinical presentation, histopathology, and treatment of patients with vulvar LS, LP, and LSC.

An Escherichia coli expression assay and screen for human immunodeficiency virus protease variants with decreased susceptibility to indinavir.

We have developed a recombinant Escherichia coli screening system for the rapid detection and identification of amino acid substitutions in the human immunodeficiency virus (HIV) protease associated with decreased susceptibility to the protease inhibitor indinavir (MK-639; Merck & Co.). The assay depends upon the correct processing of a segment of the HIV-1 HXB2 gag-pol polyprotein followed by detection of HIV reverse transcriptase activity by a highly sensitive, colorimetric enzyme-linked immunosorbent assay. The highly sensitive system detects the contributions of single substitutions such as I84V, L90M, and L63P. The combination of single substitutions further decreases the sensitivity to indinavir. We constructed a library of HIV protease variant genes containing dispersed mutations and, using the E. coli recombinant system, screened for mutants with decreased indinavir sensitivity. The discovered HIV protease variants contain amino acid substitutions commonly associated with indinavir resistance in clinical isolates, including the substitutions L90M, L63P, I64V, V82A, L24I, and I54T. One substitution, W6R, is also frequently found by the screen and has not been reported elsewhere. Of a total of 12,000 isolates that were screened, 12 protease variants with decreased sensitivity to indinavir were found. The L63P substitution, which is also associated with indinavir resistance, increases the stability of the isolated protease relative to that of the native HXB2 protease. The rapidity, sensitivity, and accuracy of this screen also make it useful for screening for novel inhibitors. We have found the approach described here to be useful for the detection of amino acid substitutions in HIV protease that have been associated with drug resistance as well as for the screening of novel compounds for inhibitory activity.

Fluorescent light-induced chromatid breaks distinguish Alzheimer disease cells from normal cells in tissue culture.

The neurodegeneration and amyloid deposition of sporadic Alzheimer disease (AD) also occur in familial AD and in all trisomy-21 Down syndrome (DS) patients, suggesting a common pathogenetic mechanism. We investigated whether defective processing of damaged DNA might be that mechanism, as postulated for the neurodegeneration in xeroderma pigmentosum, a disease with defective repair not only of UV radiation-induced, but also of some oxygen free radical-induced, DNA lesions. We irradiated AD and DS skin fibroblasts or blood lymphocytes with fluorescent light, which is known to cause free radical-induced DNA damage. The cells were then treated with either beta-cytosine arabinoside (araC) or caffeine, and chromatid breaks were quantified. At least 28 of 31 normal donors and 10 of 11 donors with nonamyloid neurodegenerations gave normal test results. All 12 DS, 11 sporadic AD, and 16 familial AD patients tested had abnormal araC and caffeine tests, as did XP-A cells. In one of our four AD families, an abnormal caffeine test was found in all 10 afflicted individuals (including 3 asymptomatic when their skin biopsies were obtained) and in 8 of 11 offspring at a 50% risk for AD. Our tests could prove useful in predicting inheritance of familial AD and in supporting, or rendering unlikely, the diagnosis of sporadic AD in patients suspected of having the disease.

The gene clusters ARC and COR on chromosomes 5 and 10, respectively, of Saccharomyces cerevisiae share a common ancestry.

The yeast Saccharomyces cerevisiae contains two clusters of eight genes each on chromosome 10 and 5, denoted, respectively, the COR and ARC regions. The genes in the COR region include TRS1 (a tRNA(Ser) gene), ANB1, CYC1, UTR1, UTR3, OSM1, tRNA(Gly) and RAD7 whereas the genes in the ARC region include TRS2 (a tRNA(Ser) gene), TIF51A, UTR5, ANP1, RAD23, UTR4, CYC7 and UTR2. We have performed a physical analysis of the ARC region, including determining DNA sequence of the 7529 nucleotides; the open reading frames; the size and orientation of the transcripts; and the phenotypes resulting from deletions or gene disruptions. The ARC region was systematically compared to the COR region which was previously described. The gene pairs CYC1-CYC7 and ANB1-TIF51A were previously shown to be, respectively, approximately 80% and 90% identical. tRNA(Ser) genes, TRS1 and TRS2, are located in both clusters 953 nt and 344 nt downstream of ANB1 and TIF51A, respectively. Some of the other gene pairs of these clusters are related in function and share only short segments of similarity distributed within the regions. The best alignment based on amino acid and nucleotide sequences indicates that the ARC and COR regions are ancestrally related by a duplication, a transposition, and a single rearrangement, followed by extensive divergence. These comparisons allowed an evaluation of distantly related sequences not obviously revealed by standard computer analysis. Surprisingly, the alignment suggested that a translated region of the ARC ANP1 gene and the COR tRNA(Gly) gene are ancestrally related. Also translated regions of the COR gene RAD7 share similarities with both of the two adjacent ARC genes, ANP1 and RAD23. Five examples of simple repeated amino acid and DNA sequences occurred in the ARC region but none in the COR region. We suggest that these repeated sequences played a role in the divergence of ARC genes.

Optimization of pro-urokinase secretion from recombinant Saccharomyces cerevisiae.

Secretion of a nonglycosylated form of human pro-urokinase, also known as single-chain urinary plasminogen activator (scu-PA), from Saccharomyces cerevisiae is described. A "supersecreting" yeast strain harboring multiple copies of integrated plasmids was grown batchwise and at constant respiratory quotient (RQ) in 20-L fermenters. Because the promoters used to drive expression of the pro-urokinase genes are not tightly regulated, secretion into the culture supernatant was growth associated. Although the final cell density achieved in the perturbed-batch fermentation (45 g dry wt/L) was less than that observed in the RQ-controlled culture (77 g dry wt/L), the scu-PA titer in the perturbed-batch fermentation (1863 IU/mL) was nearly twice that attained at constant RQ (1108 IU/mL). The effects on cell growth and scu-PA titer of other process variables (pH, temperature, phosphate concentration, and medium composition) are also discussed.

Nucleotide sequence of the COR region: a cluster of six genes in the yeast Saccharomyces cerevisiae.

We have determined the nucleotide (nt) sequence of the 7.5-kb COR segment that encompasses a cluster of six genes (CYC1, UTR1, UTR3, OSM1, tRNA(Gly) and RAD7) located on chromosome X of the yeast Saccharomyces cerevisiae. This sequence revealed five open reading frames and a tRNA gene which correspond in position, size and orientation to the transcripts previously identified by Barry et al. [Mol. Cell. Biol. 7 (1987) 632-638]. The extensively studied CYC1 gene encodes iso-1-cytochrome c; the UTR1 and UTR3 genes encode dispensible proteins whose functions are unknown; the OSM1 gene encodes a protein required for growth on hypertonic media; the tRNA(Gly) gene encodes a glycine tRNA; and the RAD7 gene encodes a protein required for repair of UV-induced damage. The OSM1 protein contains a signal sequence for secretion and a region similar to GTP-binding domains. The RAD7 protein displays 5'-untranslated elements similar to those of the stress-inducible gene UB14. The nt sequence upstream from the tRNA(Gly) gene contains a diverged copy of the sigma repeated element. This cluster of COR genes appears to have an ancestral relationship with the cluster of ARC genes on chromosome V.

Characterization of a nonglycosylated single chain urinary plasminogen activator secreted from yeast.

Using site-directed mutagenesis, we have changed the asparagine in human single-chain urinary plasminogen activator (u-PA) at position 302 to an alanine. This alteration removes the only known amino acid residue glycosylated in the protein. The single-chain u-PA containing an alanine residue at position 302 instead of asparagine (scu-PA(N302A] cDNA gene was expressed in the yeast Saccharomyces cerevisiae. Secretion of the protein product into the culture broth was achieved by replacing the human secretion signal codons with those from yeast invertase, adding a yeast promoter from the constitutively expressed glycolytic genes triosephosphate isomerase or phosphoglycerate kinase, and integrating multiple copies of these transcriptional units into the genome of yeast strains carrying the "supersecreting" mutation ssc1. When fermented in a fed-batch mode, these recombinant baker's yeast strains secreted scu-PA(N302A) in a strongly growth-associated manner. Greater than 90% of the u-PA found in the culture broth was in the single-chain form. Scu-PA(N302A) was purified to homogeneity using two chromatography steps. The purified protein had a molecular weight of 47,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and lacked any detectable N-linked glycosylation. The in vitro fibrinolytic properties of scu-PA(N302A) were found to be essentially equivalent to those of natural single-chain u-PA derived from the human kidney cell line TCL-598. Since scu-PA(N302A) lacks the immunogenic N-linked carbohydrate pattern of yeast, it may be a useful therapeutic agent which can be produced economically by yeast fermentation.

Physical analysis of the COR region: a cluster of six genes in Saccharomyces cerevisiae.

Six genes, CYC1, UTR1, UTR3, OSM1, tRNAGly, and RAD7, have been localized within an 8-kilobase region on chromosome X of the yeast Saccharomyces cerevisiae. The physical structures and the transcripts of these genes were identified by analyzing a normal strain and six deletion mutants by genomic blotting, transcriptional analysis, and gene disruption procedures. The well-studied CYC1 gene encodes iso-1-cytochrome c; the tRNAGly gene encodes a tRNA; deletion of OSM1 and RAD7 causes sensitivity to hypertonic medium and UV irradiation, respectively. There were no observable phenotypes in strains having deletions of the UTR1, UTR3, and tRNAGly gene. The high density of transcripts, with little or almost no intragenic regions, indicates that the chromosomal organization of S. cerevisiae resembles the chromosomal organization of procaryotes rather than higher eucaryotes.

Extracellular suppression allows mating by pheromone-deficient sterile mutants of Saccharomyces cerevisiae.

MAT alpha haploids with mutations in the STE13 or KEX2 gene, and MATa haploids with mutations in the STE6 or STE14 gene, do not mate with wild-type cells of the opposite mating type. We found that such mutants were able to mate with partners that carry mutations (sst1 and sst2) that cause cells to be supersensitive to yeast mating pheromone action. Mating ability of MAT alpha ste13 and MAT alpha kex2 mutants could also be restored by adding normal MAT alpha cells to mating mixtures or by adding just the appropriate purified pheromone (alpha-factor). Therefore, the mating deficiencies caused by the ste13 and kex2 lesions, and by inference, the ste6 and ste14 mutations, appear to result only from secretion of an insufficient amount of pheromone or a nonfunctional pheromone.

High-pressure liquid chromatographic analysis of melphalan in plasma.

A new sensitive and rapid high-pressure liquid chromatographic determination of melphalan in plasma was developed. Recovery of 1 microgram added to 1 ml of plasma at 23 degrees was 94% but was greatly reduced at higher temperature. The method has been applied to plasma determinations of melphalan in rats and humans and is currently being utilized for human pharmacokinetic studies.

Hydrolysis and protein binding of melphalan.

Melphalan (30 microgram/ml) is completely hydrolyzed in water at 37 degrees after 8 hr. At lower temperatures, hydrolysis proceeds at slower rates. The presence of bovine serum albumin retards hydrolysis of melphalan (30 microgram/ml) in water. The melphalan hydrolysis rate is directely releated to the bovine serum albumin concentration. At 37 degrees, 8 g of bovine serum albumin/100 ml of water gives a recovery rate of melphalan similar to that of human plasma. In vitro alkylation of melphalan at 37 degrees with human plasma containing 30 microgram/ml, calculated by equilibrium dialysis, methanol extraction, and high-pressure liquid chromatographic analysis, is 30% after 8 hr.

The mating reaction in yeast. I. A new mutation involved in the determination of mating-type.

The isolation and preliminary characterisation of a mutation, not linked to the mating type locus, but which apparently alters the mating type directed sequence of events during sexual conjugation is described. Haploids of the alpha mating type carrying this gene will now mate with other alpha haploids, creating diploids homozygous for the alpha mating type locus. This gene can be carried, but not expressed, in a haploids, however in a/alpha diploids homozygous for this gene mating is now possible with both a and alpha haploids giving either a/a/alpha or a/alpha/alpha triploids. Using these strains mating-deficient mutants have been isolated and preliminary results on their characterisation presented.

DTA-EGA determination of carbides chemically extracted from steel.

Differential thermal analysis-effluent gas analysis (DTA-EGA) techniques have been applied to the determination of specific metal carbides in residues which are isolated from steels by chemical methods. In this method, a weighed portion of sample residue is transferred to the DTA sample-holder and subjected to programmed heating in a dynamic oxygen atmosphere. When combustion of the carbide occurs, a differential temperature response is recorded over a specific temperature range. The temperature range is used as an aid in identifying the specific carbide present. The thermal conductivity of the effluent gas is recorded and the signal resulting from the presence of carbon dioxide in the effluent gas is then used for the quantitative determination of the carbide. The DTA-EGA method has been applied to several experimental steels for determination of the carbides of zirconium, vanadium or titanium individually and for the determination of vanadium and zirconium carbides in mixtures of the two. Results for these metal carbides obtained by DTA-EGA agreed within 15% with those obtained by the lengthy chemical methods. The lower limits of detection, based on the original steel sample weight, were 0.02% for the vanadium and titanium carbides and 0.01% for zirconium carbide.