Characterization of the gene encoding a histidine and aspartic acid-rich protein from Pneumocystis carinii.

A cDNA clone derived from Pneumocystis carinii contained an unusual sequence (GTGATG)2(ATGGTG)4(ATG)4 and many GAT repeats. It was found to encode a histidine and aspartic acid-rich protein (HARP). The complete cDNA contained an 888-bp open reading frame encoding a putative protein of 32.6 kDa. The deduced HARP protein contained 39 aspartic acid and 22 histidine residues. The genomic copy of the HARP gene (1203 bp in length) was found to contain 3 small introns of 46, 44, and 38 bp, respectively. HARP was predicted by computer programs to be a plasma membrane protein with nickel-binding activity.

Incorporation of fatty acids and amino acids by cultured Pneumocystis carinii.

Cultured P. carinii rapidly took up a variety of fatty acids. The relative rates of uptake for four fatty acids were 18:1 >> 16:0 approximately equal to 18:0 approximately equal to 18:2. Fatty acids were primarily incorporated into phospholipids and the uptake process was specifically inhibited by 2.2 and 22 microM primaquine, a concentration having no effect on host cells. Amino acids were also taken up by cultured P. carinii in a primaquine sensitive process. Radiolabeled leucine was incorporated into the major surface glycoprotein of P. carinii. The formation of radioactive P. carinii-specific proteins indicated that the cultured organisms carried out transcription and translation and that the incorporation of amino acids was dependent upon P. carinii rather than rare HEL human embryonic lung cells. The spinner flask culture system provides convenient access to P. carinii for metabolic studies in defined medium for a period of 5-14 days after inoculation.

Evidence for Pneumocystis carinii binding to a cell-free substrate: role of the adhesive protein fibronectin.

Attachment of Pneumocystis carinii organisms to the alveolar epithelium is probably a prerequisite for the initiation of P. carinii infection. Previous studies have demonstrated a role for the extracellular matrix protein fibronectin in mediating attachment of P. carinii organisms to cultured alveolar cells; however, these studies could not clearly distinguish the role of fibronectin binding to P. carinii organisms and fibronectin binding to the alveolar cells. The current study demonstrates the direct binding of P. carinii organisms to substrate-bound soluble fibronectin P. carinii organisms bound specifically in a concentration-dependent manner to fibronectin-coated plates; maximum binding (19.1% +/- 1.9%) occurred at a fibronectin concentration of 50 micrograms/ml. P. carinii organisms did not bind significantly to bovine serum albumin-coated plates (5.8% +/- 1.2%). Binding of P. carinii organisms to fibronectin-coated plates was inhibited in a concentration-dependent manner by addition of the tetrapeptide arginine-glycine-aspartic acid-serine, which represents the active site of the fibronectin cell-binding domain. Similarly, attachment of P. carinii organisms was significantly inhibited by the addition of monoclonal antibodies directed against the cell-binding domain of fibronectin or by the addition of calcium ion chelators. To evaluate the role of the major P. carinii surface antigen gp120 in attachment of P. carinii organisms, purified gp120 and specific polyclonal anti-gp120 antibodies were used to inhibit attachment of P. carinii organisms to fibronectin-coated plates.(ABSTRACT TRUNCATED AT 250 WORDS)

Total cellular fatty acid composition of cultured Pneumocystis carinii.

Oleic acid makes up > 50% of the total fatty acids of Pneumocystis carinii grown on WI-38 cells. Oleic acid levels increased in parallel with increasing trophozoites over 7 days in culture. The fatty acid composition of P. carinii resembles that of certain fungi but differs from those of lung surfactant lipid, host cells, and fetal bovine serum.

Pneumocystis carinii organisms obtained from rats, ferrets, and mice are antigenically different.

Pneumocystis carinii infections were developed in animals immunosuppressed by dexamethasone treatment either from activation of latent infection (ferret) or by transtracheal inoculation with P. carinii-infected lung tissue from the homologous species (rat or mouse). Convalescent-phase antisera were obtained by stopping dexamethasone treatment after 2 to 4 weeks and allowing animals 5 to 8 weeks for recovery. P. carinii harvests from infected lungs were purified by differential filtration, solubilized in buffer containing urea, sodium dodecyl sulfate (SDS), and 2-mercaptoethanol, subjected to SDS-polyacrylamide gel electrophoresis, and blotted to polyvinylidene difluoride sheets for Western immunoblot analysis. These lung preparations are hereafter referred to as P. carinii antigens. Convalescent-phase antisera from each animal species were reacted on Western blots of P. carinii antigens prepared from organisms isolated from rats, ferrets, or mice. Each combination of P. carinii antigens and antisera from the same species of animal reacted with three or more P. carinii antigen proteins. Convalescent-phase mouse antisera reacted with P. carinii antigens from mice but not rats or ferrets. Convalescent-phase rat antisera reacted with P. carinii antigens from rats and mice but not ferrets, and convalescent-phase ferret antisera showed reactions with ferret and mouse P. carinii antigens but not rat antigens. These findings indicate antigenic differences among P. carinii strains infecting these animals.

120-kD surface glycoprotein of Pneumocystis carinii is a ligand for surfactant protein A.

Pneumocystis carinii is the most common cause of life-threatening pneumonia in immunocompromised patients. In the current study, surfactant protein A (SP-A), the major nonserum protein constituent of pulmonary surfactant, is demonstrated to bind P. carinii in a specific and saturable manner. SP-A is surface bound and does not appear to be internalized or degraded by the P. carinii organism. Furthermore, SP-A binding to P. carinii is time- and calcium-dependent and is competitively inhibited by mannosyl albumin. In the absence of calcium or the presence of excess mannosyl albumin, SP-A binding to P. carinii is reduced by 95 and 71%, respectively. SP-A avidly binds P. carinii with a Kd of 8 x 10(-9) M and an estimated 8.4 x 10(6) SP-A binding sites per P. carinii organism, as determined from Scatchard plots. SP-A is shown to bind P. carinii in vivo, and a putative binding site for SP-A on P. carinii is demonstrated to be the mannoserich surface membrane glycoprotein gp120. These findings suggest that P. carinii can interact with the phospholipid-rich material in the alveolar spaces by specifically binding a major protein constituent of pulmonary surfactant.

Isolation and characterization of rat lung Pneumocystis carinii gp120.

The principal glycoprotein (gp120) of Pneumocystis carinii obtained from infected rat lung was isolated by differential extraction and size-exclusion chromatography. The purified glycoprotein was cleaved with CNBr to two peptides of approximately 27 and 33 kDa. Amino acid sequences were obtained from both peptides. Proteolytic digestion with V8 protease yielded several peptides and sequences were obtained from peptides of 10 and 19 kDa. The cyanogen bromide cleavage results led to the conclusion that gp120 exists as a homodimer.

Immunologic comparisons of Pneumocystis carinii strains obtained from rats, ferrets, and mice using convalescent sera from the same sources.

Pneumocystis carinii (Pc) infections were developed in animals immunosuppressed by dexamethasone treatment either from activation of latent infection (ferret) or trans-tracheal inoculation of Pc obtained from infected lungs of the homologous species (rat, mouse). Convalescent antisera were obtained by stopping dexamethasone treatment after 2-4 wk and allowing 5-8 wk for recovery. Parasites from infected lungs were purified by differential filtration, solubilized in loading buffer, subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis, and blotted to polyvinylidene fluoride sheets for Western analysis. Antisera from each animal species were reacted on Western blots of antigens from rat, ferret, and mouse. Each combination of antigen and antibody from the same species of animal showed reaction with 5 or more bands of Pc antigen. Convalescent mouse antibody did not react with rat or ferret antigens. Convalescent rat antibody reacted with a mouse antigen at about 66 kDa but not with ferret antigen, and convalescent ferret antibody showed minimal, probably non-specific reactions with both rat and mouse antigens. Variations in reactions indicate antigenic differences in Pc strains infecting these animals.

Tricalcium phosphate ceramic--a resorbable bone implant: review and current status.

Fourteen animal studies involving the implantation of beta-tricalcium phosphate ceramic (TPC) in rats, dogs, and primates have shown the material to be effective in repairing many types of bony defects. Histological examinations confirm that the implant is resorbed and concomitantly replaced by normal bone when firmly fixed to freshly cut and bleeding bone. Tissue compatibility has been shown to be superior to other synthetic materials. TPC has been used in human clinical studies to repair marginal and periapical periodontal defects, as well as apexification and miscellaneous alveolar bony defects. When used to repair marginal periodontal defects, degrees of repair equaled or exceeded those obtained using autogenous bone. Complete bone fill, as evidenced by radiography, was observed in the repair of two-and three-walled periapical defects. TPC afforded a better barrier than calcium hydroxide in the obturation of open apexes and provided equivalent repair. No adverse reactions attributable to TPC have been reported.

Analyses of renal medullary lipid droplets from normal, hydronephrotic, and indomethacin treated rabbits.

Lipid droplets isolated from rabbit renal medullary tissue were analyzed and found to be composed of triglyceride and free fatty acids in a ratio of 2.9:1. These triglycerides were unique when compared to triglycerides of other rabbit tissues examined, in that they contained high percentages of octadecanoic acid (stearic acid, 9.8%), 5,8,11,14-eicosatetraenoic acid (arachidonic acid, 6.8%), and 7,10,13,16-docosatetraenoic acid (adrenic acid, 10%). Lipid droplet triglycerides were found to increase during experimental hydronephrosis and after administration of indomethacin, a prostaglandin synthetase and phosphodiesterase inhibitor. From gas liquid chromatography of fatty acid methyl esters of these triglycerides, it was determined that they were enriched further in their percent composition of 9,12-octadecadienoic acid (linoleic acid) and arachidonic acid, a prostaglandin precursor. The inverse relationship between lipid droplets and prostaglandin content in the inner medulla suggested a significant role of lipid droplet triglycerides as storage pools for prostaglandin precursors.

The biosynthesis of 1a, 1b-dihomo-PGF2 and 1a,1b-dihomo-PGF2 alpha from 7, 10, 13, 16-docosatetraenoic acid by an acetone-pentane powder of sheep vesicular gland microsomes.

Thin-layer chromatographic (t.l.c.) analysis of the products formed from the incubation of an acetone-pentane powder of sheep vesicular gland microsomes with 7,10,13,16-docosatetraenoic acid (adrenic acid) revealed the presence of two products having Rf values identical to PGE2 and PGF2alpha. These products were purified by t.l.c., derivatized by treatment with methoxyamine, diazomethane, and N,O-bis-(trimethylsily1)-trifluoroacetamide, and these derivatives used for gas chromatography and gas chromatography-mass spectrometry. The results were consistent with 1a, 1b-dihomo-PGF2 and 1a, 1b-dihomo-PGF2alpha proposed structures. Formation of 1a, 1b-dihomo-PGF2 alpha could be increased, at the expense of 1a, 1b-dihomo-PGE2 by the addition of copper and reduced glutathione to the incubation mixture. Reduction of 1a, 1b-dihomo-PGE2 with NaBH4 in methanol resulted in total conversion to two products having chemical and physical properties consistent with 1a, 1b-dihomo-PGF2alpha and 1a, 1b-dihomo-PGF2beta proposed structures. The initial rate of adrenic acid-dependent oxygen uptake was determined to be 25% of that of arachidonic acid. The prostaglandin synthetase inhibitors, naproxen and 5,8,11,14-eicosatetraynoic acid (Ro 3-1428) inhibited adrenic acid-dependent oxygen uptake; Ro 3-1428 was shown to be a time-dependent inhibitor.

Stereoisomeric relationships among anti-inflammatory activity, inhibition of platelet aggregation, and inhibition of prostaglandin synthetase.

This study compares the affects of a new non-steroidal anti-inflammatory drug, d,l-6-chloro-alpha-methyl-carbazole-2-acetic acid, its enantiomers, and indomethacin on platelet aggregation, prostaglandin synthetase, adjuvant arthritis, gastric ulceration and arachidonic acid induced diarrhea. In the adjuvant arthritic rat, doses producing anti-inflammatory activity were similar for all compounds with the exception of the l-isomer which was much less active. On the other hand, indomethacin was 10 to 25 times more potent with regard to inhibition of platelet aggregation, inhibition on prostaglandin synthetase, inhibition of arachidonic acid induced diarrhea, and induction of gastric ulceration than the racemate and its isomers. Such divergence of potencies suggests that the racemate, unlike indomethacin, would have no affect on platelet aggregation and, hence, produce no prolongation of bleeding time at doses possessing anti-inflammatory activity. The data also suggest that the racemate and d-isomer have greater specificity toward anti-arthritic acitvity and are less ulcerogenic than indomethacin. The d-isomer apparently is the more active component of the racemate in all the systems tested since: (a) the d-isomer has 2 to 3 times the inhibitory potency of the racemate and (b) the l-isomer, at high dosages or high concentrations had considerable less affect. Comparison of potencies relative to inhibition of platelet aggregation and of prostaglandin synthetase, are quite close; therefore, mechanistically, the anti-aggregatory affects of these drugs, or lack thereof, may be related to inhibition of prostaglandin synthetase.

Chromatography of prostaglandins utilizing silicic acid impregnated-glass fiber sheets.

A chromatographic method has been developed which offers rapid and convenient monitoring of prostaglandin biosynthesis from arachidonic acid.

Circum-annual changes in triglyceride fatty acids of bat brown adipose tissue.

A circum-annual study of the fatty acids of brown adipose tissue triglycerides ofEptesicus fuscus has demonstrated a rhythmic pattern of change. This is seen as a reciprocal shift of the levels of oleic and linoleic acids. Oleic acid levels are lower during the summer months and higher in the winter months. Levels of palmitic and linoleic acids reach maximal values in midsummer and fall significantly during the winter.Homogenates of brown adipose tissue produce more(14)CO(2) from 1-(14)C-palmitic acid than from 1-(14)C-oleic acid when incubated at temperatures below 20C. The formation of(14)CO(2) from either substrate was maximal in the neighborhood of 30C, and the temperature effect was enhanced by stimulation with DL-carnitine.It is proposed that the rhythmic change in brown adipose tissue triglyceride composition is a reflection of the different rates of fatty acid oxidation and the absence of normal food intake for extended periods of time.