[Lethal case of chronic necrotizing pulmonary aspergillosis (CNPA)]. / Chronisch nekrotisierende pulmonale Aspergillose (CNPA).

Though not overtly immunosuppressed, an elderly female patient suffered from chronic pleurisy due to Aspergillus fumigatus. In spite of nearly six months of itraconzole-therapy, she eventually succumbed to CNPA. Pleurisy may be a typical manifestation of this rare mycosis which is often ill fated.

Properties of a purified proteinase from the yeast Candida albicans.

The proteinase from culture supernatants of Candida albicans strain CBS-2730 was purified virtually to homogeneity by ion-exchange chromatography and affinity chromatography. The enzyme consists of a single polypeptide chain with tryptophan at the N- and leucine at the C-terminus. Its molecular weight is approx. 45,000 and the isoelectric point is at pH 4.4. With albumin as a substrate an apparent Km was determined to be 7 . 10(-5) M. The enzyme is inhibited by pepstatin at equimolar ratio and thus is a carboxyl proteinase (EC 3.4.23.6). Other group-specific inhibitors, though, did not efficiently block the enzyme. Above pH 8.4 the enzyme undergoes alkaline denaturation which is accompanied by dimerization. The enzyme is a glycoprotein. It is stable in presence of non-ionic detergents and can be freeze-dried. The enzyme clots milk at pH 5.5 and has trypsinogen kinase activity. Among several purified proteins that have been tested as a substrate, only horse ferritin was resistant to proteolysis, while myeloma proteins of the A1- and A2-type were readily cleaved, as were two proteinase inhibitors of human serum. Antibodies against purified enzyme did not react with several commercial Candida antigen preparations; antibodies against the enzyme, though, have been detected repeatedly in sera from patients with manifest candidiasis.

Detection of Candida proteinase by enzyme immunoassay and interaction of the enzyme with alpha-2-macroglobulin.

Double antibody immunoassay of secretory aspartic proteinases from 2 strains of the yeast Candida albicans was performed in microtest plates with rabbit antibody and peroxidase conjugated rabbit antibody. The test detects approximately 0.1 ng of proteinase and is 2 orders of magnitude more sensitive than direct measurement of enzymic activity with hemoglobin as a substrate. The immunoassay was affected both by alkaline denaturation, which is a common feature of aspartic proteinases, and by the presence of the proteinase scavenger alpha-2-macroglobulin. Conditions are described that allow for comparison of proteinases from different strains of yeast and minimize the interference of macroglobulin.

Sequential protein analysis from single identified neurons of Aplysia californica. A microelectrophoretic technique involving polyacrylamide gradient gels and isoelectric focusing.

Electrophoresis in polyacrylamide gradient gels and isoelectric focusing in polyacrylamide gels of capillary size are powerful tools for the analysis of molecular weight and charge properties of small protein samples. This is demonstrated using identified neurons from the abdominal ganglion of the sea hare Aplysia californica. Certain cell-specific peptides, which are considered to be neurosecretory, have been shown to be water soluble when ethylene glycol was employed as a mobilizing agent. Although the mode of action of ethylene glycol is not yet understood, this method may be of value for various extraction procedures. The application of a new staining method that is preferential for separations of sodium dodecyl sulfate-proteins yields information about the charge of water-insoluble proteins which has so far been inaccessible. Preliminary results gained by a small, two-dimensional mapping procedure as well as optical density separation patterns of two different nuclear protein fractionation from a single isolated nucleus outline further possibilties of the microgel techniques.

Characterization of the estrogen-induced uterine peroxidase by microelectrophoresis.

Estrogen-dependent peroxidase from rat uterine fluid has been investigated by microelectrophoretic techniques. The molecular weight of the enzyme has been determined in the range of 100,000 by using polyacrylamide gradient gels in the absence and presence of nonionic and anionic detergent. The isoelectric points are located between pH 4.5 and 5.9. Employing the two-dimensional combination of isoelectric focusing and gel gradient electrophoresis, the enzyme was separated into two subunits, one having a molecular weight of 70,000, the other less than 20,000. The large subunit has slight enzymatic activiy, while the smaller subunit may be responsible for the charge difference in the holoenzyme pattern. The glycoprotein pattern of the uterine fluid peroxidase is further defined by its separation by affinity chromatography using a concanavalin A-Sepharose column and by its susceptibility to neuraminidase treatment.

Outbreak of methicillin-resistant Staphylococcus aureus in a German tertiary-care hospital.

A biphasic outbreak of methicillin-resistant Staphylococcus aureus in intensive-care units of a German tertiary-care hospital afflicted 89 patients within 4 years. The spread of the outbreak most likely was facilitated by the contamination of mobile radiograph equipment. The outbreak was controlled by measures of hospital hygiene.

Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase from Aspergillus fumigatus.

Oligonucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergillus Fumigatus. The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre-proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene.

Purification and characterisation of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue.

A serine proteinase (Alp) from the culture supernate of a clinical isolate of Aspergillus fumigatus was purified to virtual homogeneity at a yield of 41%. The procedure involved affinity chromatography on agarose-epsilon-amino-caproyl-D-tryptophan methyl ester. Alp had an estimated mol. wt of 32 Kda and the pI was determined at pH 7.9. The enzyme was fully inhibited by phenylmethyl sulphonyl fluoride, chymostatin and alpha-1-proteinase inhibitor, and it was largely inhibited by alpha-1-anti-chymotrypsin. Partial inhibition was observed with tosyl-phenylalanine chloromethyl ketone, but tosyl-lysine chloromethyl ketone was ineffective. Thus, Alp may be identical with the major chymotryptic activity of A. fumigatus, which has already been described. The N-terminal sequence of 25 amino acids revealed an 88% homology of Alp with the subtilisin-related proteinase of A. oryzae. Alp acted on casein over a broad range from pH 5.5 to 11.5 and also acts to a lesser extent on haemoglobin and serum albumin. The enzyme degraded elastin and a synthetic elastase substrate; hence, it may be identical with the previously described elastinolytic activity of the fungus. At pH 7.3 and a concentration of 1 microgram/ml, Alp was not toxic for Vero cells, but it efficiently detached such cells from a plastic surface. Specific antibodies against Alp were detected by enzyme immunoassay in the sera of patients and Alp-antigen was demonstrated by immunofluorescence in mycotic human lung. In addition, a second proteinase (Exalp) with extremely alkaline activity, and an aspartic proteinase of A. fumigatus are described.

Cervical mucus peroxidase is a reliable indicator for ovulation in humans.

This study was designed to establish whether fluctuation in cervical mucus peroxidase concentration correlates with the cyclic pattern of the menstrual cycle hormones in the human female. Forty healthy, normal-cycling women between the ages of 19 and 29 years were chosen for the study. Blood samples and cervical mucus were collected on days 6, 9 through 15, and 17 through 19 of the menstrual cycle for three consecutive cycles in each volunteer. Blood estrogen, progesterone, follicle-stimulating hormone, and luteinizing hormone levels were quantitated by radioimmunoassay, and cervical mucus peroxidase concentrations were measured spectrophotometrically. The data showed that in the typical menstrual cycle the mucus peroxidase peak was reached immediately prior to the luteinizing hormone/follicle-stimulating hormone surge and coincided with the estrogen peak. Consistent data in three consecutive menstrual cycles in each volunteer led us to postulate that the mucus peroxidase peak during the menstrual cycle precedes the ovulatory period in the normal healthy woman.

PIP: This study was designed to establish whether fluctuations in cervical mucus peroxidase concentration correlates with the cyclic pattern of the menstrual cycle hormones in the human female. 40 healthy, normal cycling women between ages 19-29 were chosen for the study. Blood samples and cervical mucus were collected on days 6, 9-15, and 17-19 of the menstrual cycle for 3 consecutive cycles in each volunteer. Blood estrogen, progesterone, follicle stimulating hormone (FSH), and luteinizing hormone (LH) levels were quantitated by radioimmunoassay, and cervical mucus peroxidase concentrations were measured spectrophotometrically. Data showed that in the typical menstrual cycle the mucus peroxidase peak was reached immediately prior to the LH/FSH surge and coincided with the estrogen peak. Consistent data in 3 consecutive menstrual cycles in each volunteer led the authors to postulate that the mucus peroxidase peak during the menstrual cycle precedes the ovulatory period in the normal healthy woman.

Topical fluconazole for experimental candida keratitis in rabbits.

Using a reproducible model of Candida albicans keratitis in rabbits we studied the effect of topical fluconazole, a new triazole. Candida albicans DSM 70010 (2.5 X 10(5) cells) was injected into the corneal stroma of both eyes of 21 rabbits. All eyes developed a corneal ulcer. Forty-eight hours after inoculation the animals were divided into three groups: (1) 14 eyes, received fluconazole (2 mg/ml) and the epithelium subsequently removed; (2) 14 eyes, received only fluconazole drops; (3) 14 eyes, received 0.9% NaCl: half of this group was also debrided. We applied one drop of either substance 10 times a day for 24 days. A further six rabbits were used to judge if the drug penetrated into the cornea and aqueous humour. There was a highly significant difference between the fluconazole groups (1,2) and the control group (3) as to hypopyon and complications (descemetocele, corneal perforation) as well as recultivation of C. albicans from corneal tissue. The difference between the fluconazole groups with and without debridement was not significant. The drug penetrated into the cornea and aqueous humour of both uninflamed and inflamed eyes.

[Optical brighteners in fungal diagnostics]. / Optische Aufheller in der Pilzdiagnostik.

Fluorescent staining using optical brighteners (diaminostilbenes) affords the semispecific and rapid detection of fungal elements in clinical specimens. After yielding a first hint of mycosis, the identification of the involved fungal genus is often desirable in cases when culture proves unsuccessful. In such cases, immunohistochemistry or in situ hybridisation may further the diagnosis with respect to establish an appropriate therapy.

Identification of certain false-positive results in the Cand-Tec test for candidal antigen.

By repeated testing of sera after heating (56 degrees C, 15 min) certain false-positives in the Cand-Tec test can be identified from the thermo-resistance of the reaction. True titers are drastically reduced by such pretreatment of sera. The thermostable antigen giving rise to falsely positive agglutination is not identical with mannan.

A variety of Candida proteinases and their possible targets of proteolytic attack in the host.

Acid proteinases are secreted by the majority of strains of Candida albicans, C. tropicalis and C. parapsilosis, reflecting the sequence of virulence of Candida spp. for man. As revealed by exposure to various substrates and inhibitors, Candida proteinases are strain-specific, although close relationships exist between enzymes from strains of the same species. Possible targets of hydrolytic attack by Candida proteinases in the host are immunoglobulins, the proteinase scavenger alpha-2-macroglobulin, zymogens of regulatory serine proteinases such as coagulation factor X, and angiotensinogen. The involvement of Candida proteinase in the pathogenesis of acronecrosis is discussed.

Two-dimensional micro-separation technique for proteins and peptides, combining isoelectric focusing and gel gradient electrophoresis.

The two-dimensional combination of gel electrofocusing and gel gradient electrophoresis yields separate information about differences in net charge and molecular weight of macro-ions. A micro-version of this technique is described. The first dimension electrofocusing run is performed in cylindrical polyacrylamide gel of capillary size, using xylene cyanole FF as a reliable marker dye to indicate the final separation stage when proteins reach their isoelectric points. In the second-dimension run the focused proteins are separated in a continuous slab gel gradient of less than stamp size with total acrylamide concentration ranging from ca. 1% to more than 40%. Spreading of peaks in the gradient slab gel is shown to be related to the approach of protein to its pore limit in the gel. This spreading is a useful indicator of the final position of a protein in the gel if molecular weights are estimated according to the separation pattern in the gradient gel. The method described has been developed for the separation of proteins extracted from large single cells (i.e., neurons of the mollusc Aplysia californica.

On the role of proteinases from Candida albicans in the pathogenesis of acronecrosis.

Evidence is presented for the involvement of proteinases from Candida albicans in the pathogenesis of acronecrosis that occurred in a young woman and which coincided with Candida sepsis. Secretory acid Candida proteinase by immunofluorescence was traced in the obstructed blood vessels of necrotic skin that was infested with yeast. The specificity of immunofluorescence was proven by exclusion of cross reactivity with pepsin, cathepsin-D, acid erythrocyte proteinase and porcine renin. The possible molecular mechanisms of interference of fungal proteinases are discussed with respect to the renin-angiotensin system and blood coagulation.