Distribution of angiotensin type 1a receptor-containing cells in the brains of bacterial artificial chromosome transgenic mice.

In the central nervous system, angiotensin II (AngII) binds to angiotensin type 1 receptors (AT(1)Rs) to affect autonomic and endocrine functions as well as learning and memory. However, understanding the function of cells containing AT(1)Rs has been restricted by limited availability of specific antisera, difficulties discriminating AT(1)R-immunoreactive cells in many brain regions and, the identification of AT(1)R-containing neurons for physiological and molecular studies. Here, we demonstrate that an Agtr1a bacterial artificial chromosome (BAC) transgenic mouse line that expresses type A AT(1)Rs (AT1aRs) identified by enhanced green fluorescent protein (EGFP) overcomes these shortcomings. Throughout the brain, AT1aR-EGFP was detected in the nuclei and cytoplasm of cells, most of which were neurons. EGFP often extended into dendritic processes and could be identified either natively or with immunolabeling of GFP. The distribution of AT1aR-EGFP cells in brain closely corresponded to that reported for AngII binding and AT1aR protein and mRNA. In particular, AT1aR-EGFP cells were in autonomic regions (e.g., hypothalamic paraventricular nucleus, central nucleus of the amygdala, parabrachial nucleus, nuclei of the solitary tract and rostral ventrolateral medulla) and in regions involved in electrolyte and fluid balance (i.e., subfornical organ) and learning and memory (i.e., cerebral cortex and hippocampus). Additionally, dual label electron microscopic studies in select brain areas demonstrate that cells containing AT1aR-EGFP colocalize with AT(1)R-immunoreactivity. Assessment of AngII-induced free radical production in isolated EGFP cells demonstrated feasibility of studies investigating AT1aR signaling ex vivo. These findings support the utility of Agtr1a BAC transgenic reporter mice for future studies understanding the role of AT(1)R-containing cells in brain function.

Characterization of a soluble ternary complex formed between human interferon-beta-1a and its receptor chains.

The extracellular portions of the chains that comprise the human type I interferon receptor, IFNAR1 and IFNAR2, have been expressed and purified as recombinant soluble His-tagged proteins, and their interactions with each other and with human interferon-beta-1a (IFN-beta-1a) were studied by gel filtration and by cross-linking. By gel filtration, no stable binary complexes between IFN-beta-1a and IFNAR1, or between IFNAR1 and IFNAR2 were detected. However, a stable binary complex formed between IFN-beta-1a and IFNAR2. Analysis of binary complex formation using various molar excesses of IFN-beta-1a and IFNAR2 indicated that the complex had a 1:1 stoichiometry, and reducing SDS-PAGE of the binary complex treated with the cross-linking reagent dissucinimidyl glutarate (DSG) indicated that the major cross-linked species had an apparent Mr consistent with the sum of its two individual components. Gel filtration of a mixture of IFNAR1 and the IFN-beta-1a/IFNAR2 complex indicated that the three proteins formed a stable ternary complex. Analysis of ternary complex formation using various molar excesses of IFNAR1 and the IFN-beta-1a/IFNAR2 complex indicated that the ternary complex had a 1:1:1 stoichiometry, and reducing SDS-PAGE of the ternary complex treated with DSG indicated that the major cross-linked species had an apparent Mr consistent with the sum of its three individual components. We conclude that the ternary complex forms by the sequential association of IFN-beta-1a with IFNAR2, followed by the association of IFNAR1 with the preformed binary complex. The ability to produce the IFN-beta-1a/IFNAR2 and IFN-beta-1a/IFNAR1/IFNAR2 complexes make them attractive candidates for X-ray crystallography studies aimed at determining the molecular interactions between IFN-beta-1a and its receptor.

Pleiotrophin is an abundant protein in dissociative extracts of bovine fetal epiphyseal cartilage and nasal cartilage from newborns.

An abundant protein that is identical to the growth-associated protein pleiotrophin (PTN) has been isolated from dissociative extracts of bovine nasal and fetal epiphyseal cartilage. The yield from these tissues was at least 15 micrograms/g wet weight of cartilage. PTN was absent or was present only in trace amounts in mature articular cartilage. An analysis of tryptic fragments of PTN, held together with disulfide bonds, did not indicate any set pattern of cystine cross-links, which suggests a propensity for rapid refolding of the protein. PTN could not be isolated from thin (10 microns) slices of nasal cartilage in physiological extraction buffers, which indicates that it was tightly associated with the cell surface, was tightly associated with nonextractable matrix, or was an intracellular protein. Its appearance in various extraction media parallels that of histone H2b, a nucleosomal protein; this suggests a possible intracellular location for the protein. Immunohistochemical analysis of its distribution in fetal epiphysis indicated that it is associated with chondrocytes.

Hyaluronan-binding region of aggrecan from pig laryngeal cartilage. Amino acid sequence, analysis of N-linked oligosaccharides and location of the keratan sulphate.

The hyaluronan-binding region (HABR) was prepared from pig laryngeal cartilage aggrecan and the amino acid sequence was determined. The HABR had two N-termini: one N-terminal sequence was Val-Glu-Val-Ser-Glu-Pro (367 amino acids in total), and a second N-terminal sequence (Ala-Ile-Ser-Val-Glu-Val; 370 amino acids in total) was found to arise due to alternate cleavage by the signal peptidase. The N-linked oligosaccharides were analysed by examining their reactivity with a series of lectins. It was found that the N-linked oligosaccharide on loop A was of the mannose type, while that on loop B was of the complex type. No reactivity was detected between the N-linked oligosaccharide on loop B' and any of the lectins. The location of keratan sulphate (KS) in the HABR was determined by Edman degradation of the immobilized KS-containing peptide. The released amino acid derivatives were collected and tested for the presence of epitope to antibody 5-D-4. On the basis of 5-D-4 reactivity and sequencing yields, the KS chains are attached to threonine residues 352 and 357. There is no KS at threonine-355. This site is not in fact in G1, but about 16 amino acid residues into the interglobular domain. Comparison of the structure of the KS chain from the HABR and from the KS domain of pig laryngeal cartilage aggrecan was made by separation on polyacrylamide gels of the oligosaccharides arising from digestion with keratanase. Comparison of the oligosaccharide maps suggests that the KS chains from both parts of the aggrecan molecule have the same structure.

Primary structure of a protein isolated from reef shark (Carcharhinus springeri) cartilage that is similar to the mammalian C-type lectin homolog, tetranectin.

During the course of characterization of low molecular weight proteins in cartilage, we have isolated a protein from reef shark (Carcharhinus springeri) cartilage that bears a striking resemblance to the tetranectin monomer originally described by Clemmensen et al. (1986, Eur. J. Biochem. 156, 327-333). The protein was isolated by extraction of neural arch cartilage with 4 M guanidine hydrochloride, dialysis of the extract to bring the guanidine to 0.4 M (reassociating proteoglycan aggregates), followed by cesium chloride density gradient removal of the proteoglycans. The amino acid sequence had 166 amino acids and a calculated molecular weight of 18,430. The shark protein was 45% identical to human tetranectin, indicating that it was in the family of mammalian C-type lectins and that it was likely to be a shark analog of human tetranectin. The function of tetranectin is unknown; it was originally isolated by virtue of its affinity for the kringle-4 domain of plasminogen. Sequence comparison of human tetranectin and the shark-derived protein gives clues to potentially important regions of the molecule.

Isolation and primary structure of PARP, a 24-kDa proline- and arginine-rich protein from bovine cartilage closely related to the NH2-terminal domain in collagen alpha 1 (XI).

A protein rich in proline and arginine (proline/arginine-rich protein (PARP] has been isolated from dissociative extracts of bovine nasal and articular cartilage, and its primary structure has been determined. The protein has 218 amino acids, giving a calculated protein Mr of 24,075. In nasal cartilage, this protein is in molar concentrations equivalent to 1/20-1/10 that of the link protein of cartilage proteoglycan aggregates. PARP has also been isolated from bovine articular cartilage, bovine fetal epiphysis, and nonossified human tarsal bones. PARP is similar to various collagen NH2-terminal domains. It is 49% identical to the NH2-terminal end of collagen alpha 1 (XI), 17% identical to the NC4 domain of collagen alpha 1 (IX), and 14% identical to the NC3 domain of collagen alpha 1 (XII). Four cysteines are conserved between type XI collagen and PARP, and these form two disulfide bonds. Two of the cysteines are also conserved between PARP and collagens IX and XII. The homology between the collagens and PARP makes it possible to speculate on the likely disulfide bond pattern in the collagen NH2-terminal domains. It is probable that PARP is a collagen fragment removed during processing in a manner analogous to chondrocalcin (the C-terminal propeptide of type II collagen).

An 18-kDa glycoprotein from bovine nasal cartilage. Isolation and primary structure of small, cartilage-derived glycoprotein.

A glycosylated protein (small, cartilage-derived glycoprotein, SCGP) of approximately 18 kDa with unknown function has been isolated from dissociative extracts of bovine nasal cartilage and its primary structure determined. The protein has 121 amino acids, giving a calculated protein molecular weight of 13,878, four disulfide bonds, two N-linked oligosaccharides and one O-linked oligosaccharide. In nasal cartilage, this glycoprotein is in molar concentrations equivalent to 1/5-1/2 that of the link protein of cartilage proteoglycan aggregates, and it has also been isolated from bovine articular cartilage and from bovine fetal epiphysis. The N-terminal, glycosylated region of the molecule is relatively rich in arginine, proline, glycine, and threonine. The C-terminal 82 amino acids (which contains all four of the disulfide bonds and none of the carbohydrate) can be found as a discrete entity in cartilage extracts, indicating that the N-terminal domain is readily removed by extracellular proteolytic attack.

Blastomyces dermatitidis infections in the RSA.

Twenty cases of blastomycosis have been confirmed in the RSA, 9 of which are presented for the first time. Patients came from all four provinces and the mean age was 40 years. Six cases were diagnosed between 1985 and 1987. Differences between strains of Blastomyces dermatitidis isolated in the RSA and in North America include morphological and cultural characteristics, mycelial-yeast conversion, antigenic structure, and compatibility in cross-mating experiments. The diagnosis of this disease can be made by direct examination of unstained specimens, by histological examination or by culture of the organism. Culture should be attempted in all cases for confirmation of microscopic findings.

Blastomycosis in Africa. A review of known cases diagnosed between 1951 and 1987.

The known African cases of blastomycosis to 1987 are presented, including thirteen previously undescribed cases. This brings to 81 the total number of cases known to have occurred in Africa. The question of whether the disease in Africa is the same in all respects as that in North America is addressed; the age and sex distributions of patients are similar. Minor differences in the clinical features relate particularly to the type of skin lesion, the more frequent bone involvement and the less frequent central nervous system involvement in the African patient. Little is known about the epidemiology of blastomycosis in Africa; one noteworthy feature is the apparent absence of the disease in dogs. Isolates of Blastomyces dermatitidis from the two continents, although closely related, differ in some respects.

Opportunistic fungal infection by Fusarium oxysporum in a renal transplant patient.

A white female with chronic glomerular nephritis received a renal transplant in 1971. After 1 year, Livido Reticularis (LR) developed and in 1976 erythematous, painful nodules formed on the LR and ulcerated. The patient also suffered diffuse calcification of the major blood vessels and small arterioles of the extremities with progressive necrosis and gangrene of the fingers. Hyperparathyroidism was evident. The necrotic ulcers yielded Candida albicans and Fusarium oxysporum; both organisms were seen in histological preparations. The ulcers were excised and grafted; no specific antifungal therapy was given.

Trichophyton ajelloi isolated from a child.

A case of widespread tinea corporis due to Trichophyton ajelloi in a young child is presented. The fungal isolate was unusual in that abundant microconidia were produced together with typical macroconidia. It was pathogenic for the guinea pig.

Early diagnosis of opportunistic systemic fungal and nocardial infections.

An approach to the expediting of the diagnosis of opportunistic systemic mycoses is presented. Communication between clinician and microbiologist is basic to this approach. The importance of the clinical assessment of the individual patient, coupled with a high index of suspicion, is stressed. Our experience with 11 of 42 cases of systemic mycosis over a 28-month period is analysed. For the diagnosis of fungaemia a method for the microscopical examination of peripheral blood is briefly evaluated, and a membrane filter blood culture technique is shown to be valuable, yielding results in 16-24 hours. In the absence of fungaemia the considered microscopical examination of suitable specimens, when feasible, is the most rapid method available. Serological methods may be helpful in early diagnosis, but this is often hampered by the absence of baseline sera and by the lengthy nature of some tests. Newer indirect methods such as gas chromatography are being developed but have not yet been used routinely.

Blastomycosis of the tongue: a case report.

A clinically malignant tongue ulcer in a 63-year-old White man was proved by histological examination and culture to be due to Blastomyces dermatitidis. In addition, pulmonary lesions caused by B. dermatitidis were found at autopsy.

Torulopsis glabrata fungaemia. A report of two cases.

Two cases of Torulopsis glabrata fungaemia are presented. The literature on detection of micro-organisms in peripheral blood and on systemic T. glabrata infection is briefly reviewed. Microscopical examination of a buffy coat preparation, a simple and rapid procedure for diagnosing this condition, is described. A scheme of criteria which may be helpful in the diagnosis of clinically significant fungaemia is offered.