Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming.

AIM: To determine whether transcriptional reprogramming is capable of reversing the developmental aging of normal human somatic cells to an embryonic state. MATERIALS & METHODS: An isogenic system was utilized to facilitate an accurate assessment of the reprogramming of telomere restriction fragment (TRF) length of aged differentiated cells to that of the human embryonic stem (hES) cell line from which they were originally derived. An hES-derived mortal clonal cell strain EN13 was reprogrammed by SOX2, OCT4 and KLF4. The six resulting induced pluripotent stem (iPS) cell lines were surveyed for telomere length, telomerase activity and telomere-related gene expression. In addition, we measured all these parameters in widely-used hES and iPS cell lines and compared the results to those obtained in the six new isogenic iPS cell lines. RESULTS: We observed variable but relatively long TRF lengths in three widely studied hES cell lines (16.09-21.1 kb) but markedly shorter TRF lengths (6.4-12.6 kb) in five similarly widely studied iPS cell lines. Transcriptome analysis comparing these hES and iPS cell lines showed modest variation in a small subset of genes implicated in telomere length regulation. However, iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines. In order to verify these results in an isogenic background, we generated six iPS cell clones from the hES-derived cell line EN13. These iPS cell clones showed initial telomere lengths comparable to the parental EN13 cells, had telomerase activity, expressed embryonic stem cell markers and had a telomere-related transcriptome similar to hES cells. Subsequent culture of five out of six lines generally showed telomere shortening to lengths similar to that observed in the widely distributed iPS lines. However, the clone EH3, with relatively high levels of telomerase activity, progressively increased TRF length over 60 days of serial culture back to that of the parental hES cell line. CONCLUSION: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.

Preenrichment versus direct selective agar plating for the detection of Salmonella Enteritidis in shell eggs.

The relative effectiveness of two methods for the recovery of Salmonella Enteritidis (SE) from jumbo and medium shell eggs was compared. The first method used in the comparison consisted of a preenrichment of the sample, and the second method was developed by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS). Three bulk lots of blended, pooled eggs, each containing 220 liquid whole eggs that were thoroughly mixed manually were artificially inoculated with different levels of SE cells between approximately 10(0) and 10(3) CFU/ml. Twenty samples containing the contents of approximately 10 eggs each (by weight) were withdrawn from each of the inoculated bulk lots and incubated for 4 days at room temperature (ca. 23 degrees C). For the APHIS method, each sample was cultured by direct plating onto brilliant green (BG), brilliant green with novobiocin (BGN), xylose lysine desoxycholate (XLD), and xylose lysine agar Tergitol 4 (XLT4) agars. For the preenrichment method, 25-g portions from each pool were enriched in modified tryptic soy broth with 30 mg/liter of FeSO4. After 24 h of incubation, the preenrichments were subcultured to tetrathionate and Rappaport-Vassiliadis broths, and streaked to BG, BGN, bismuth sulfite, XLD, and XLT4 agar plates. SE isolates were confirmed biochemically and serologically. In all of the experiments, the preenrichment method recovered significantly more SE isolates (P < 0.05) of all the phage types and inoculum levels than did the APHIS method. From a total of 539 jumbo egg test portions analyzed, 381 (71%) were SE-positive by the preenrichment method and 232 (43%) were positive by the APHIS method. From a total of 360 medium egg test portions analyzed, 223 (62%) were SE-positive by the preenrichment method and 174 (48%) were positive by the APHIS method. The preenrichment method provided greater sensitivity for the isolation of SE in contaminated egg slurries than did the APHIS method.

An improved method for the recovery of Salmonella serovars from orange juice using universal preenrichment broth.

The relative effectiveness of three methods for the recovery of Salmonella serovars from orange juice was determined. One method, a modified Bacteriological Analytical Manual (BAM) procedure consisted of preenrichment in lactose broth at 35 degrees C for 24 h, selective enrichment, and selective plating. Another method, a National Centers for Disease Control and Prevention (CDC 1) procedure, consisted of direct enrichment in tetrathionate broth at 35 degrees C for 24 and 48 h, followed by selective plating. The third method (also from CDC and designated CDC 2) consisted of preenrichment in Universal Preenrichment (UP) broth at 35 degrees C for 24 h, selective enrichment, and selective plating. In 10 experiments encompassing five different Salmonella serovars and 200 test portions per broth, the CDC 1 method recovered 141 Salmonella-positive test portions, the BAM method recovered 151, and the CDC 2 method recovered 171. In 2 of the 10 experiments, with two different Salmonella serovars, the BAM recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did the CDC 2 method. On the basis of the above results, the second phase of this study focused on a comparison of the effectiveness of the BAM-recommended lactose broth and the CDC 2-recommended UP broth as preenrichment media for the recovery of Salmonella serovars from pasteurized and unpasteurized orange juice. Subsequent culture treatment of the two preenrichments was identical so that the effect of other variables (e.g., different selective enrichment media, various incubation temperatures, and different selective plating agars) on the relative performance of these two preenrichment media was excluded. In one of nine experiments, with pasteurized orange juice, lactose broth recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did UP broth. For the combined results of the nine pasteurized orange juice experiments (180 test portions per broth), lactose broth recovered 99 Salmonella-positive test portions, and UP broth recovered 116. In three of seven experiments, with unpasteurized orange juice, lactose broth recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did UP broth. For the combined results of the seven unpasteurized orange juice experiments (140 test portions per broth), lactose broth recovered 73 Salmonella-positive test portions, and UP broth recovered 117. For both pasteurized and unpasteurized orange juice, the total number of Salmonella-positive test portions recovered with UP broth was significantly greater than the number recovered with lactose broth. These results indicate that UP broth is a more effective enrichment broth for the recovery of Salmonella from orange juice than is lactose broth.

Rappaport-Vassiliadis medium for recovery of Salmonella spp. from low microbial load foods: collaborative study.

Twenty-three laboratories participated in a collaborative study to compare the relative effectiveness of Rappaport-Vassiliadis (RV) medium incubated at 42 degrees C, selenite cystine (SC) broth (35 degrees C), and tetrathionate (TT) broth (35 and 43 degrees C) for recovery of Salmonella from the following foods with a low microbial load: dried egg yolk, dry active yeast, ground black pepper, guar gum, and instant nonfat dry milk. For dry active yeast, lauryl tryptose (LT) broth, incubated at 35 degrees C, was used instead of SC broth. All of the foods were artificially inoculated with single Salmonella serovars, that had been lyophilized before inoculation, at high and low target levels of 0.4 and 0.04 colony forming units/g food, respectively. For analysis of 870 test portions, representing all of the foods except yeast, 249 Salmonella-positive test portions were detected by RV medium, 265 by TT broth (43 degrees C), 268 by TT broth (35 degrees C), and 269 by SC broth (35 degrees C). For analysis of 225 test portions of yeast, 79 Salmonella-positive test portions were detected by RV medium, 79 by TT broth (43 degrees C), 84 by TT broth (35 degrees C), and 68 by LT broth (35 degrees C). RV medium was comparable to, or even more effective than, the other selective enrichments for recovery of Salmonella from all of the foods except guar gum. It is recommended that RV (42 degrees C) and TT (35 degrees C) be used with foods that have a low microbial load, except for guar gum for which SC (35 degrees C) and TT (35 degrees C) are recommended.

Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium for the recovery of Salmonella spp. from foods with a low microbial load.

The relative effectiveness of Rappaport-Vassiliadis (RV) medium, selenite cystine (SC) broth, and tetrathionate (TT) broth for the recovery of Salmonella spp. from foods with a low microbial load was determined. RV medium made from its individual ingredients and incubated at 42 degrees C was compared with a commercial preparation of SC broth, incubated at 35 degrees C, and TT broth incubated at 35 and 43 degrees C, for the recovery of Salmonella spp. Twenty-one artificially contaminated food types that included dairy foods, spices, and egg products, as well as other low-microbial-load foods, were analyzed. The foods were inoculated with single Salmonella serovars at target levels ranging from 0.04 to 0.4 CFU/g. No significant differences (P< or =0.05) among the selective enrichment broths for the recovery of Salmonella spp. from 18 of the foods were observed. Significantly fewer Salmonella-positive test portions of gelatin, guar gum, and nonfat dry milk were recovered with RV medium than with SC broth incubated at 35 degrees C and TT broth incubated at 35 and 43 degrees C. TT broth incubated at 35 degrees C recovered the greatest number of Salmonella-positive test portions. For the recovery of Salmonella spp. from foods with a low microbial load, it is recommended that TT broth incubated at 35 degrees C and RV medium incubated at 42 degrees C be used.

Methods for the recovery of Salmonella spp. from carboxymethylcellulose gum, gum ghatti, and gelatin.

Foods analyzed for Salmonella spp. by the procedure in the U.S. Food and Drug Administration's Bacteriological Analytical Manual are preenriched at a 1:9 test portion/broth ratio. Various thickening agents preenriched at this ratio become viscous and nonpipettable after 24 h incubation at 35 degrees C. The effects of 3 factors (presence of inorganic salts, adjustment of pH, and presence of enzymes) on the viscosity of the test portion/preenrichment mixtures of various thickening agents during incubation were determined. Reduction of the viscosities of these thickening agents was accomplished as follows: carboxymethylcellulose gum, addition of cellulase to a final concentration of 0.10% in lactose broth preenrichment and incubation with no pH adjustment; gum ghatti, addition of NaCl to a final concentration of 0.10% in lactose broth preerichment and adjustment of the pH to 6.5; and gelatin, addition of papain to a final concentration of 0.10% in lactose broth preenrichment and adjustment of the pH to 6.8. With these modified preenrichments, one Salmonella spp. cell/25 g (representing an approximate most probable number value of 0.04 cell/g) was generally recovered from the thickening agents.

Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT.

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation. In addition to the human telomerase RNA component (hTR; ref. 11), TR1/TLP1 (refs 12, 13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (5pTrt1) telomerases, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Telomerase catalytic subunit homologs from fission yeast and human.

Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

Comparison of sorbitol MacConkey and hemorrhagic coli agars for recovery of Escherichia coli O157:H7 from brie, ice cream, and whole milk.

The relative efficacies of hemorrhagic coli (HC) agar and several formulations of sorbitol MacConkey (SorMac) agar, with and without 0.1% (w/v) 4-methyllumbelliferyl-beta-D-glucuronide (MUG), in recovering unstressed and heat-stressed Escherichia coli O157:H7 from Brie cheese, ice cream, and whole milk were determined. Recovery of unstressed E. coli O157:H7 was determined quantitatively by spread-plating diluted samples onto different agars and performing plate counts. Recovery of stressed E. coli O157:H7 was determined qualitatively by enriching samples in modified trypticase soy broth, streaking the incubated enrichments, and isolating E. coli O157:H7 colonies from the agars. HC agar and the SorMac agar formulations did not differ significantly in their ability to recover unstressed E. coli O157:H7 from ice cream and whole milk; however, HC agar recovered significantly more unstressed E. coli O157:H7 from Brie cheese than did the SorMac agar formulations. Bacteriological Analytical Manual and Oxoid SorMac agar formulations made from individual ingredients, did not differ significantly in recovering unstressed E. coli O157:H7 from Brie cheese. The efficiency of the commercially available Oxoid SorMac agar could not be determined because of overgrowth by indigenous microflora. HC and SorMac agars did not differ significantly in recovering stressed E. coli O157:H7 from Brie cheese, ice cream, and whole milk. MUG had no apparent effect on recovery of either stressed or unstressed E. coli O157:H7 from the dairy foods examined.

Relative effectiveness of selenite cystine broth, tetrathionate broth, and rappaport-vassiliadis medium for recovery of Salmonella spp. from raw flesh, highly contaminated foods, and poultry feed: collaborative study.

A collaborative study was performed in 18 laboratories to validate use of Rappaport-Vassiliadis (RV) medium in the standard culture method for recovery of Salmonella spp. from raw, highly contaminated foods and poultry feed. RV medium made from its individual ingredients and incubated at 42 degrees C was compared with selenite cystine (SC) broth incubated at 35 degrees C and tetrathionate (TT) broth incubated at 35 degrees and 43 degrees C for effectiveness in recovery of Salmonella spp. Four artificially contaminated foods (oysters, frog legs, mushrooms, and shrimp) and poultry feed and one naturally contaminated food (chicken) were analyzed. The artificially contaminated foods were inoculated with single serovars of Salmonella at target levels of 0.04 colony-forming units (CFU)/g for the low level and 0.4 CFU/g for the high level. For analysis of 1125 test portions, RV medium (42 degrees C) recovered Salmonella from 409 test portions; TT (43 degrees C), from 368 test portions; TT (35 degrees C), from 310 test portions; and SC (35 degrees C), from 334 test portions. Overall, RV medium was comparable with or better than other selective enrichments for recovery of Salmonella from the foods in this study, except mushrooms. From mushrooms, SC broth (35 degrees C) recovered more positive test portions than did RV medium (42 degrees C) and TT broth (43 degrees C). The method for detection of Salmonella in raw, highly contaminated foods and poultry feed using RV medium has been adopted by AOAC INTERNATIONAL. AOAC Official Method 967.25, Salmonella in Foods, Preparation of Culture Media and Reagents, has been revised to include RV medium, and the applicability of AOAC Official Method 967.26, Salmonella in Foods, Detection, has been restricted to processed foods.

Usefulness of cellulase in recovery of Salmonella spp. from guar gum.

Most foods examined for Salmonella spp. by the procedure described in the U.S. Food and Drug Administration's Bacteriological Analytical Manual are preenriched at a 1:9 sample/broth ratio. However, 25 g guar gum (an emulsifying agent) is not wetted completely in 225 mL of preenrichment broth, and after a 24 h incubation at 35 degrees C, the product is transformed into a viscous, nonpipettable mass. The effects of 4 factors (inorganic salts, pH, temperature, and various enzymes) on the viscosity of the sample/preenrichment mixture during incubation were determined. Addition of various inorganic salts or adjustment of pH from 4.0 to 9.0 had no significant effect on the viscosity of the incubated mixture. Elevated incubation temperatures of 42 degrees, 44 degrees, and 46 degrees C reduced viscosity but were well above the optimal growth temperature for Salmonella, 35 degrees C. Addition of cellulose to lactose broth at a final concentration of 0.01% reduced viscosity of the mixture, making it readily pipettable. At least one Salmonella cell was consistently recovered from 25 g samples of guar gum, which represents a most probable number value of 0.04 cell per g.

Cataloging altered gene expression in young and senescent cells using enhanced differential display.

Recently, a novel PCR-based technique, differential display (DD), has facilitated the study of differentially expressed genes at the mRNA level. We report here an improved version of DD, which we call Enhanced Differential Display (EDD). We have modified the technique to enhance reproducibility and to facilitate sequencing and cloning. Using EDD, we have generated and verified a catalog of genes that are differentially expressed between young and senescent human diploid fibroblasts (HDF). From 168 genetags that were identified initially, 84 could be sequenced directly from PCR amplified bands. These sequences represent 27 known genes and 37 novel genes. By Northern blot analysis we have confirmed the differential expression of a total of 23 genes (12 known, 11 novel), while 19 (seven known, 12 novel) did not show differential expression. Several of the known genes were previously observed by others to be differentially expressed between young and senescent fibroblasts, thereby validating the technique.

Relative effectiveness of selective plating agars for recovery of Salmonella species from selected high-moisture foods.

The relative effectiveness of 6 selective plating media were compared for effectiveness in recovery of Salmonella spp. from selected high-moisture foods. Three new plating agars (EF-18, Rambach, and xylose lysine Tergitol-4) and 3 selective plating agars (bismuth sulfite, Hektoen enteric, and xylose lysine desoxycholate) recommended by AOAC INTERNATIONAL and the Bacteriological Analytical Manual (BAM) were compared. The agars were streaked from cultures selectively enriched in selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium. The high-moisture foods studied were naturally contaminated pork sausage, chicken parts, turkey parts, and frog legs and artificially contaminated shrimp, oysters, egg yolks, and lettuce. The relative effectiveness of each selective plating agar was determined by recovery of Salmonella spp. and enumeration of false-positive and false-negative reactions. Although the new selective plating agars compared favorably with the AOAC/BAM-recommended agars, they offered no advantage. Incubation of selective enrichment broths at elevated temperatures decreased the numbers of false-positive and false-negative reactions for all 6 selective plating agars.

Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium for the recovery of Salmonella from raw flesh and other highly contaminated foods: precollaborative study.

The effectiveness of selenite cystine (SC) broth, tetrathionate (TT) broth, and Rappaport-Vassiliadis (RV) medium for recovery of Salmonella spp. from 8 highly contaminated foods was determined. RV medium prepared from individual ingredients and incubated at 42 degrees and 43 degrees C was compared with 2 commercial (Difco and Oxoid) media incubated at 42 degrees C. Naturally and artificially contaminated foods were tested under 2 protocols. For Protocol 1, each food was preenriched in the appropriate medium. After incubation, serial 10 fold dilutions of the preenriched foods were inoculated into selective enrichment media and incubated at 35 degrees, 42 degrees, or 43 degrees C. Effectiveness of these conditions was evaluated by most probable number determination of Salmonella spp. recovered. Productivity of selective enrichments did not differ significantly with this protocol, except that with Oxoid RV medium the number of Salmonella cells recovered from most of the foods was significantly reduced. For Protocol 2, twenty 25 g test portions from artificially inoculated foods were examined qualitatively for Salmonella spp. The effectiveness of the broth/temperature combinations was determined by the number of positive tests under each condition. RV medium prepared from individual ingredients and TT broth incubated at 43 degrees C yielded significantly more Salmonella-positive tests from frog legs and lettuce than did SC and TT broths incubated at 35 degrees C or commercial RV medium incubated at 42 degrees C. With pork sausage and ground beef, significantly fewer Salmonella-positive tests were found with Oxoid RV medium incubated at 42 degrees C and SC incubated at 35 degrees C than from other selective enrichments.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of extracytoplasmic proteins from Serpulina hyodysenteriae B204 and molecular cloning of the flaB1 gene encoding a 38-kilodalton flagellar protein.

Extracytoplasmic proteins were released from Serpulina (Treponema) hyodysenteriae (strain B204) by treatment of whole cells with a nonionic detergent (Tween 20). Centrifugation of the Tween 20-released proteins at 100,000 x g sedimented 10 major extracytoplasmic proteins with approximate molecular masses of 44, 43.5, 42, 39, 38, 34, 33.5, 33, 31, and 29 kDa. Treatment of the sedimented fraction with 6 M urea solubilized all of the proteins except the 39-kDa protein. Peptide sequences were obtained for the purified 42-, 39-, 38-, 34-, 31-, and 29-kDa proteins. The peptide sequences of the 42-, 38-, and 31-kDa proteins indicate that they likely are components of the periplasmic flagella. The amino-terminal peptide sequence of the 38-kDa protein was used to design an oligonucleotide probe and to clone an S. hyodysenteriae DNA fragment containing the gene encoding this protein. The predicted 290-amino-acid protein sequence derived from the cloned gene was highly homologous to those of several other bacterial flagellar proteins and is preceded by consensus sigma D nucleotide sequences found upstream of other flagellar genes. On the basis of its similarity to the FlaB proteins of other spirochetes, we propose to designate the cloned S. hyodysenteriae gene flaB1 and its encoded protein FlaB1. Vaccination of pigs with FlaB1 or its recombinant counterpart did not protect them from an experimental challenge.

Effectiveness of a one-day procedure for recovery of Salmonella from milk powders.

A rapid procedure for enumerating Salmonella in milk powders was evaluated. Dry whole milk and instant nonfat dry milk were rehydrated, artificially inoculated with various numbers of Salmonella cells, and stomached. Test portions were then treated with Tween 80 and pancreatic trypsin, and incubated for 1 h at 30 degrees C. The incubated test portions were centrifuged at 10,000 x g for 15 min at 5 degrees C, and the resuspended pellets were plated on xylose lysine desoxycholate agar. The effectiveness of the procedure was expressed in terms of percentage recovery of the inoculum. The procedure, which was evaluated in 76 trials using 7 Salmonella serovars, recovered < or = 73% of the inoculum for half of the trials conducted. Its effectiveness was dependent on the serovar, level of inoculation, and type of milk powder used.

Modulation of glycosaminoglycan addition in naturally expressed and recombinant human thrombomodulin.

The two major glycoforms of full-length human thrombomodulin (TM), one with (TM(CS+)) and one without (TM(CS-)) chondroitin sulfate (CS) were analyzed on Western blots of primary and transformed cells and in cells expressing recombinant TM. TM on the surface of Chinese hamster ovary and COS-7 cells is solely TM(CS-). Primary arterial endothelial cells (HAEC and HPAEC) express a greater fraction of TM with CS attached than venous cells (HUVEC). Human lung carcinoma cells (A549) express more TM(CS+) than primary cells and recombinant TM on human melanoma cells (CHL-1) occurs in two very high molecular weight forms of TM(CS+). We explored this variation in TM(CS+) with soluble recombinant TM in several cell lines and analyzed the ambiguous CS addition site in human TM by site-directed mutagenesis. Mutation of Ser474 to Ala blocks CS addition in Chinese hamster ovary and COS-7 cells but not CHL-1 cells which add CS to Ser472 and Ser474. Structure of the O-link domain affects partitioning into TM(CS+) since substituting with the decorin CS addition sequence, substituting all Ser and Thr except Ser474 with Ala, and deleting around the potential beta-turn all increase the ratio of TM(CS+) to TM(CS-). A combination of the decorin substitution and deletion of the remaining O-link domain yields the most TM(CS+).

Human formyl peptide receptor ligand binding domain(s). Studies using an improved mutagenesis/expression vector reveal a novel mechanism for the regulation of receptor occupancy.

Recently, we reported the domain requirements for the binding of formyl peptide to its specific receptor. Based on experiments using receptor chimeras, we also postulated an importance for the amino-terminal domain of the receptor in ligand binding (Perez, H. D., Holmes, R., Vilander, L., Adams, R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295). We have begun to perform a detailed analysis of the regions within the formyl peptide receptor involved in ligand binding. To address the importance of the receptor amino-terminal domain, we substituted (or inserted) hydrophilic sequences within the amino-terminal domain, expressed the receptors, and determined their ability to bind ligand. A stretch of nine amino acids next to the initial methionine was identified as crucial for receptor occupancy. A peptide containing such a sequence specifically completed binding of the ligand to the receptor. Alanine screen mutagenesis of the second extracellular domain also identified amino acids involved in ligand binding as well as a disulfide bond (Cys98 to Cys176) crucial for maintaining the binding pocket. These studies provide evidence for a novel mechanism involved in regulation of receptor occupancy. Binding of the ligand induces conformational changes in the receptor that result in the apposition of the amino-terminal domain over the ligand, providing a lid to the binding pocket.