Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death.

In this study, we used comparative proteomics to identify proteins that were involved in the regulation of interdigital cell death. The protein profiles of embryonic day (E) 12.5 and 13.5 mouse hindlimb interdigital tissues were compared to identify proteins that were differentially expressed. The interdigital cells are irreversibly committed to programmed cell death (PCD) at E13.5, whereas they are developmentally plastic at E12.5. We established that protein disulfide isomerase (PDI) expression was up-regulated at E13.5, while peroxiredoxin 1 (Prdx1) expression was down-regulated at this time point. Semiquantitative reverse transcriptase-polymerase chain reaction and Western blot analyses confirmed the data obtained from the two-dimensional electrophoresis gels. Furthermore, we were able to up-regulate PDI expression by manipulating the E12.5 interdigital tissues to die during culture, although this up-regulation was not possible when cell survival was promoted. In addition, we could inhibit interdigital cell death and expression of proapoptotic genes (Bmp-4 and Bambi) by treating interdigital tissues with PDI antibodies and bacitracin (a PDI enzyme inhibitor). These findings suggested that PDI was involved in the activation and maintenance of interdigital cell death. Conversely, we determined that Prdx1 expression was maintained when interdigital cultures were manipulated to survive but down-regulated when the cultures were permitted to die. The result suggested that Prdx1 was involved in maintaining interdigital cell survival. However, we were unable to induce interdigital cell death by means of RNA interference-mediated silencing of Prdx1 expression, indicating that Prdx1 down-regulation is not sufficient for PCD to occur. Proteomic analysis of the Prdx1 knock-down cells revealed that the level of NF-kappaB inhibitor epsilon (IkappaBepsilon) was dramatically reduced. Furthermore, we found an increase in NFkappaB activation and reactive oxygen species (ROS) levels in the cytoplasm as a result of Prdx1 knockdown. We also found that silencing Prdx1 made the interdigital cells more susceptible to ROS-induced cell death. Taken together, our study identifies two new players in interdigital cell death and highlights that PCD is regulated by a delicate balance of proapoptotic and survival-promoting activities.

Ubiquitin expression is up-regulated in human and rat skeletal muscles during aging.

In this study, we have used two-dimensional electrophoresis, protein sequencing, immunoblotting, and immunohistochemistry to identify proteins that were differentially expressed during aging in human and rat skeletal muscles. Ubiquitin was identified. It was expressed at high levels in old fast-twitch muscles but at low levels in young fast-twitch muscles. It was also discovered that exogenous ubiquitin could suppress the growth of C2C12 cells, in vitro. The reduction in C2C12 cell growth was not attributed to an increase in apoptosis but to an inhibition in cell cycle entry. Furthermore, it was possible to induce muscles to degenerate in vivo by injecting a high dose of exogenous ubiquitin into young healthy skeletal muscles. These results suggest that hyperactivity of the ubiquitin-proteasome pathway is involved in the aging process of fast-twitch muscles. In addition, ubiquitin-dependent growth suppression in satellite cells may be associated with the poor healing potential of old skeletal muscles.

Heart-type fatty acid binding proteins are upregulated during terminal differentiation of mouse cardiomyocytes, as revealed by proteomic analysis.

At birth, the cardiomyocytes in the mouse neonatal heart still retain their ability to proliferate. However, this lasts only a few days and then the cardiomyocytes irreversibly lose their potential to divide. It is still not fully understood what factors are involved in the cessation of cardiomyocyte proliferation. Using proliferating cell nuclear antigen (PCNA) antibodies, we established that cardiomyocytes could divide extensively in 2-day-old mouse neonatal hearts and to a lesser extent in 6-day-old hearts. By 13 days, the cardiomyocytes have mostly stopped dividing. Comparative two-dimensional gel electrophoresis (2-DE) was performed on total proteins extracted from the 2-day- and 13-day-old hearts, in order to identify peptides that might be involved in the inhibition of cardiomyocyte proliferation. Using matrix-assisted laser desorption ionization mass spectroscopy (MALDI-TOF), we identified two protein spots that have the same molecular weight (approximately 14 kDa) but different pIs (5.9 and 6.1). Mass spectra analysis determined the proteins to be isoforms of the heart-type fatty acid binding protein (H-FABP). The pI 6.1 H-FABP is also known as mammary-derived growth inhibitor (MDGI; Specht et al. 1996). MGDI is a breast tumour growth suppressor gene capable of inhibiting tumour cell proliferation (Huynh et al. 1995). Both H-FABP isoforms were expressed in 2-day-old hearts but became strongly upregulated in 13-day-old hearts. We examined whether H-FABPs and PCNA were coexpressed in 2-, 6- and 13-day-old heart histological sections, using MDGI antibodies. The antibody could detect both forms of H-FABPs. It was established that there was a correlation between an increase in H-FABP expression and a decrease in PCNA expression. Hence, we tentatively propose that H-FABP isoforms are involved in regulating cardiomyocyte growth and differentiation in mouse neonatal hearts.

Growth arrest-specific 2 gene expression during patellar tendon healing.

We examined the cellular and molecular processes involved in patellar tendon healing following induced injury. A wound was surgically created at the center of the patellar tendon of adult rats. The wound site was examined at selected time intervals by immunohistochemical and in situ hybridization techniques. It was found that, between the 2nd and 7th day postoperation, fibroblast-like cells invaded the wound site. DiI-labelling experiments suggested that the majority of cells that occupied the wound originated from the edges of the wound. Furthermore, immunohistochemical studies revealed that at the wound site a meshwork of fibronectin developed that can support the migration of the DiI-labelled cells. We also examined the spatial and temporal expression patterns of the growth arrest specific 2 (GAS2) gene during patellar tendon healing. GAS2 was found strongly expressed in the tenocytes of unoperated patellar tendons. The gene was also expressed in the intact regions of operated tendons but not in the fibroblast-like cells that occupied the wound site, when examined 2 days postoperation. In addition the strip of intact tendon directly opposite the wound site also did not express GAS2. Examination of the experimental tendon at the 3rd month, when cells had completely occupied the wound site, revealed that Gas2 was expressed by all cells found in the wound. Bromodeoxyuridine (BrdU) incorporation analysis revealed that the presence of Brdu-positive cells in the wound indirectly correlated with the absence of Gas2 expression. We speculate that the GAS2 gene might play a role in regulating tenocyte proliferation during tendon healing.

Staphylococcus aureus in the oral cavity: a three-year retrospective analysis of clinical laboratory data.

OBJECTIVE: A retrospective analysis of laboratory data to investigate the isolation of Staphylococcus aureus from the oral cavity and facial area in specimens submitted to a regional diagnostic oral microbiology laboratory. METHODS: A hand search of laboratory records for a three-year period (1998-2000) was performed for specimens submitted to the regional diagnostic oral microbiology laboratory based at Glasgow Dental Hospital and School. Data were collected from forms where S. aureus was isolated. These data included demographics, referral source, specimen type, methicillin susceptibility and clinical details. RESULTS: For the period 1998-2000, there were 5,005 specimens submitted to the laboratory. S. aureus was isolated from 1,017 specimens, of which 967 (95%) were sensitive to methicillin (MSSA) and 50 (5%) were resistant to methicillin (MRSA). The 1,017 specimens were provided from 615 patients. MRSA was isolated from 37 (6%) of patients. There was an increasing incidence of S. aureus with age, particularly in the >70 years age group. The most common specimen from which MSSA was isolated was an oral rinse (38%) whilst for MRSA isolates this was a tongue swab (28%). The clinical condition most commonly reported for MSSA isolates was angular cheilitis (22%). Erythema, swelling, pain or burning of the oral mucosa was the clinical condition most commonly reported for MRSA isolates (16%). Patients from whom the MSSA isolates were recovered were most commonly (55%) seen in the oral medicine clinic at the dental hospital, whilst patients with MRSA were more commonly seen in primary care settings such as nursing homes, hospices and general dental practice (51%). CONCLUSION: In line with more recent surveys, this retrospective study suggests that S. aureus may be a more frequent isolate from the oral cavity than hitherto suspected. A small proportion of the S. aureus isolates were MRSA. There were insufficient data available to determine whether the S. aureus isolates were colonising or infecting the oral cavity. However, the role of S. aureus in several diseases of the oral mucosa merits further investigation.

Salvianolic acid B inhibits fibril formation and neurotoxicity of amyloid beta-protein in vitro.

AIM: To observe the effect of salvianolic acid-B (SalB) on amyloid beta-protein (A-beta) fibril formation and its toxicity towards PC12 cells. METHODS: A-beta (1 - 40) was incubated with or without SalB at 25 degrees C for 30 h, 48 h, and 100 h. Fibril formation was then viewed under an electron microscope. Toxicity of the A-beta (1 - 40) towards PC12 cells was measured with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). A-beta (25 - 35) was aged by incubating at 25 degrees C for 7 d, then the peptide was incubated with PC12 cells with or without SalB. Toxicity of A-beta (25-35) towards PC12 cells was observed with MTT. RESULTS: Following incubation at 25 degrees C for 30 h, A-beta (1 - 40) (100 mg/L) aggregated and formed fibrils. SalB 10-100 nmol/L completely prevented the fibril formation within 30 h. Extension of amyloid fibrils increased with prolonging the incubation time. SalB inhibited the fibril formation process during this period. In the MTT assay A-beta (1 - 40) incubated with SalB manifested significantly lower toxicity to PC12 cells compared with that without SalB. Besides, SalB 1 micromol/L significantly attenuated the toxicity of aged A-beta (25 - 35) to PC12 cells. CONCLUSION: SalB could inhibit A-beta aggregation and fibril formation, as well as directly inhibit the cellular toxicity of aged A-beta towards PC12 cells.

Fibroblast growth factor-8b-stimulated myogenic cell proliferation is suppressed by the promyelocytic leukemia gene.

Muscle cell growth is regulated by growth-promoting and -inhibiting factors. In this study, the physiological effects of fibroblast growth factor (FGF)-8b and the promyelocytic leukemia (PML) gene on G8 myogenic cells were examined. FGF-8b was found to strongly stimulate myogenic cell proliferation. Signal transduction assays using AP-1/SEAP and E-box/SEAP reporters revealed that the transcriptional factors junB/c-fos and c-myc were involved in FGF-8b-stimulated G8 cell growth. Besides examining factors that positively stimulate myogenic cell growth, we also examined genes that negatively affect cell growth. PML is a growth suppressor gene and we studied its expression in G8 cells under different growth conditions. Immunohistochemical staining revealed that in the presence of low serum, PML was expressed in approximately 23.2% of all cultured G8 cells. However, under normal culture conditions (10% serum), PML expression dropped to about 2.6%. We found that the PML gene acted antagonistically to FGF-8b, as the overexpression of PML in G8 cells significantly inhibited FGF-8b-stimulated cell proliferation. It also inhibited AP-1 and E-box transactivation. However, we believe that PML functions as a stress-response gene in G8 cells rather than as a gene normally involved in regulating muscle development.

Functions of the growth arrest specific 1 gene in the development of the mouse embryo.

The growth arrest specific 1 (gas1) gene is highly expressed in quiescent mammalian cells (Schneider et al., 1988, Cell 54, 787-793). Overexpression of gas1 in normal and some cancer cell lines could inhibit G(0)/G(1) transition. Presently, we have examined the functions of this gene in the developing mouse embryo. The spatial-temporal expression patterns for gas1 were established in 8.5- to 14.5-day-old embryos by immunohistochemical staining and in situ hybridization. Gas1 was found heterogeneously expressed in most organ systems including the brain, heart, kidney, limb, lung, and gonad. The antiproliferative effects of gas1 on 10.5 and 12.5 day limb cells were investigated by flow cytometry. In 10.5 day limbs cells, gas1 overexpression could not prevent G(0)/G(1) progression. It was determined that gas1 could only induce growth arrest if p53 was also coexpressed. In contrast, gas1 overexpression alone was able to induce growth arrest in 12.5 day limb cells. We also examined the cell cycle profile of gas1-expressing and nonexpressing cells by immunochemistry and flow cytometry. For 10.5 day Gas1-expressing heart and limb cells, we did not find these cells preferentially distributed at G0/G1, as compared with Gas1-negative cells. However, in the 12.5 day heart and limb, we did find significantly more Gas1-expressing cells distributed at G0/G1 phase than Gas1-negative cells. These results implied that Gas1 alone, during the early stages of development, could not inhibit cell growth. This inhibition was only established when the embryo grew older. We have overexpressed gas1 in subconfluent embryonic limb cells to determine the ability of gas1 to cross-talk with various response elements of important transduction pathways. Specifically, we have examined the interaction of gas1 with Ap-1, NFkappaB, and c-myc responsive elements tagged with a SEAP reporter. In 10.5 day limb cells, gas1 overexpression had little effect on Ap-1, NFkappaB, and c-myc activities. In contrast, gas1 overexpression in 12.5 day limb cells enhanced AP-1 response while it inhibited NFkappaB and c-myc activities. These responses were directly associated with the ability of gas1 to induce growth arrest in embryonic limb cells. In the 12.5 day hindlimb, gas1 was found strongly expressed in the interdigital tissues. We overexpressed gas1 in these tissues and discovered that it promoted interdigital cell death. Our in situ hybridization studies of limb sections and micromass cultures revealed that, during the early stages of chondrogenesis, only cells surrounding the chondrogenic condensations expressed gas1. The gene was only expressed by chondrocytes after the cartilage started to differentiate. To understand the function of gas1 in chondrogenesis, we overexpressed the gene in limb micromass cultures. It was found that cells overexpressing gas1/GFP could not participate in cartilage formation, unlike cells that just express the GFP reporter. We speculated that the reason gas1 was expressed outside the chondrogenic nodules was to restrict cells from being recruited into the nodules and thereby defining the boundary between chondrogenic and nonchondrogenic forming regions.

[Experimental study on protective effective of congsheng capsule in acute cerebral ischemia and improvement of cerebral blood flow and energy metabolism].

OBJECTIVE: To investigate the protective effect of Congsheng capsule (CSC) on acute cerebral ischemia in mice and rats. METHODS: Cerebral ischemia model was established by adding FeCl3 to block the blood flow of unilateral middle cerebral artery (MCA) for 30 mins. Acute incomplete cerebral ischemia was induced by ligation of bilateral carotid arteries in rats. Bilateral carotid arteries of old mice were repeatedly ischemia and reperfusion combined with caudal bloodletting to build the cerebral ischemia model. The effect of CSC on neurologic symptom, cerebral infarction size, cerebral water content, cerebral blood flow and cerebral tissue energy metabolism were observed. RESULTS: CSC 1, 3, 6 g/kg obviously reduce the size of cerebral infarction and cerebral water content, markedly improve the neurological symptoms and cerebral blood flow; 3, 6 g/kg significantly ameliorate the energy burden. CONCLUSION: CSC has anti-cerebral ischemia function through increasing blood supply of cerebral tissue and improve the energy metabolism.

Bmp-4 requires the presence of the digits to initiate programmed cell death in limb interdigital tissues.

The effects of Bmp-4 on interdigital cell death were investigated in the mouse. Affi-Gel beads, loaded with recombinant Bmp-4 protein, were transplanted into the interdigital tissues of day 12.5 hindlimb, ex utero. It was established that Bmp-4 could induce precocious interdigital cell death. Using in situ hybridization, the expression patterns of bmp-4 and alk-6 receptor were established. Both genes were found coexpressed in the interdigital region of 12.5- and 13. 5-day hindlimbs. This suggests that Bmp-4 may act in an autocrine fashion. We have also studied the effects of Bmp-4 on 12.5-day interdigital tissue cultures. In all specimens examined, the interdigital tissues produced cartilage instead of participating in cell death. The addition of exogenous Bmp-4 to the interdigital cultures did not induce apoptosis but instead enhanced chondrogenesis. The discrepancy between the effects of Bmp-4 in vitro and ex utero was attributed to the presence of digits. When a flanking digit was left attached to the interdigital tissues, in vitro, Bmp-4 promoted apoptosis instead of chondrogenesis. In sum, the results suggest that Bmp-4 is a multifunctional protein and its effect on the interdigital tissues is dependent on the modulating influence of the digits.

The use of optical sensors to understand cellular interactions with renal cells.

Optical biosensor technology has revolutionized the assessment of receptor binding, enabling the characterization of low affinity interactions in real time. We report the application of the LAsys Optical Biosensor to the investigation of the affinity and specificity of the putative proximal tubular scavenging receptor for protein reabsorption and the specificity of AGE-modified protein interactions with primary human mesangial cells. Using the LLCPK cell line, the carboxy-methyl dextran cuvette surface and five different proteins (ranging in size and charge), we have shown that there is evidence to support the existence of a single scavenging receptor for all the proteins tested. The proteins competed with each other differing only in their relative binding affinity for the common receptor. We have also shown that human mesangial cells can bind to AGE-modified human serum albumin (AGE-HSA) immobilized onto the carboxylate surfaced planar cuvette and that binding can be inhibited using increasing concentrations of soluble AGE-HSA. However, increasing concentrations of soluble Non-AGE modified HSA can also inhibit binding to a similar extent which implies that there is relatively little AGE-receptor (RAGE) expression on cultured primary human mesangial cells. These results demonstrate the exciting potential of this technology as a tool to explore cellular interactions with renal cells.

gas2 is a multifunctional gene involved in the regulation of apoptosis and chondrogenesis in the developing mouse limb.

The growth-arrest-specific 2 (gas2) gene was initially identified on account of its high level of expression in murine fibroblasts under growth arrest conditions, followed by downregulation upon reentry into the cell cycle (Schneider et al., Cell 54, 787-793, 1988). In this study, the expression patterns of the gas2 gene and the Gas2 peptide were established in the developing limbs of 11.5- to 14. 5-day mouse embryos. It was found that gas2 was expressed in the interdigital tissues, the chondrogenic regions, and the myogenic regions. Low-density limb culture and Brdu incorporation assays revealed that gas2 might play an important role in regulating chondrocyte proliferation and differentiation. Moreover, it might play a similar role during limb myogenesis. In addition to chondrogenesis and myogeneis, gas2 is involved in the execution of the apoptotic program in hindlimb interdigital tissues-by acting as a death substrate for caspase enzymes. TUNEL analysis demonstrated that the interdigital tissues underwent apoptosis between 13.5 and 15.5 days. Exactly at these time points, the C-terminal domain of the Gas2 peptide was cleaved as revealed by Western blot analysis. Moreover, pro-caspase-3 (an enzyme that can process Gas2) was cleaved into its active form in the interdigital tissues. The addition of zVAD-fmk, a caspase enzyme inhibitor, to 12.5-day-old hindlimbs maintained in organ culture revealed that the treatment inhibited interdigital cell death. This inhibition correlated with the absence of the Gas2 peptide and pro-caspase-3 cleavage. The data suggest that Gas2 might be involved in the execution of the apoptotic process.

Hepatocyte growth factor stimulates chemotactic response in mouse embryonic limb myogenic cells in vitro.

In this study we investigate the influence of Hepatocyte Growth Factor (HGF) on the motility of embryonic forelimb myoblasts. Using Blindwell chemotactic chambers, it was found that HGF at concentrations of 1-50 ng/ml dramatically enhanced the ability of myogenic cells to migrate. This stimulatory effect was elicited in a dose-dependent fashion and the effect was reversed with the addition of HGF neutralizing antibodies. A checkerboard analysis was performed and it revealed that HGF's effect on limb myoblast motility was through both chemokinesis and chemotaxis. HGF was also examined for its ability to stimulate myogenic cell proliferation, using MF20 antibody as the myogenic marker. At all concentrations tested, HGF did not stimulate an overall increase in the numbers of MF20-positive myoblasts in culture. To examine the chemokinetic effect of HGF on cell migration in the limb, cells were isolated from the proximal regions of the limb (areas rich in myogenic cells), exposed to HGF, labeled with DiI and transplanted into 11.5 day mouse forelimbs. After 36 h of culture, it was found that DiI-labeled limb cells, pretreated with HGF, migrated significantly further in the limb than labeled cells that have not been exposed to HGF. The chemotactic effect of HGF was also investigated by implanting beads loaded with and without HGF into the 11.5 day limb. Proximal to the beads, DiI-labeled limb cells were also transplanted. It was found that HGF was able to chemotactically attract and direct the migration of DiI-labeled limb cells. Immunohistological staining was performed with HGF antibodies to determine the distribution of HGF in the 11.5 day mouse forelimb. It was found that HGF was strongly expressed by the apical ectodermal ridge (AER), the ectoderm and the mesenchyme directly beneath the AER. Positive staining was also obtained for the myogenic regions. However, the pattern was heterogeneous--punctuated with myogenic cells expressing and not expressing HGF.

Fibroblast growth factors 2 and 4 stimulate migration of mouse embryonic limb myogenic cells.

Fibroblast growth factors (FGFs) are believed to be vital for limb outgrowth and patterning during embryonic development. Although the effect of FGFs on the formation of the skeletal elements has been studied in detail, their effect on the development of the limb musculature is still uncertain. In this study, we used Blindwell chemotactic chambers to examine the effect of FGF-2 and FGF-4 on the motility of myogenic cells obtained from the proximal region of the day 11.5 mouse forelimbs. The limb myogenic cells were found to be chemotactically attracted to FGF-2 and FGF-4 at 10-50 ng/ml. Both FGFs increased myogenic cell migration in a dose-dependent manner, with maximal responses attained at 1-50 ng/ml for FGF-2 and at 10 ng/ml for FGF-4; however, FGF-2 was found to be a more potent chemoattractant than FGF-4. It was possible to inhibit the myogenic cells' response to FGF-2 and FGF-4 by the addition of the appropriate neutralizing antibody. The effects of FGF-2 on cell migration were further investigated by loading this cytokine into Affi-Gel blue beads and transplanting them into day 11.5 forelimb buds. The results showed that FGF-2 attracted DiI-labelled proximal cells to migrate toward the implanted beads and that the migration was more extensive than that observed in the absence of FGF-2. A checkerboard assay was performed in which various concentrations of FGF-2 and FGF-4 were introduced to both the upper and lower wells of the Blindwell chambers. The results indicated that both FGF isoforms can stimulate chemokinesis as well as chemotaxis in myogenic cells. In addition, the effect of FGF-2 at 0.1-10 ng/ml stimulated a significant increase in the number of myocytes expressing sarcomeric myosin on examination after 48 hr in culture, but the effect of FGF-4 was negligible at all concentrations analyzed; however, both FGF-2 and FGF-4 inhibited myocyte fusion compared with the spontaneous fusion observed in control cultures. Finally, we used in situ hybridization and immunohistochemical techniques to determine the distribution of myogenic cells and FGF-2 protein in the day 11.5 mouse forelimbs.

Induction of metallothionein in the Reuber H-35 rat hepatoma cell.

The feasibility of using the Reuber H-35 rat hepatoma cell (RH-35 cells) as model for studying metallothionein induction was examined. The RH-35 cells were treated with Cd, a toxic metal which is known to induce metallothionein. The LC50 after a 3-h treatment was 70 microM. The value was significantly higher (P < 0.05) if the cells were pre-treated with a sublethal dose of CdCl2 (5 microM) for 2 days, indicating that pre-treatment with a low dose of Cd can protect against a subsequent higher dose of the same metal. Both the mRNA and the gene product metallothionein can be identified in the cells 2 days after treatment with 5 microM Cd. In addition to Cd, Zn and Cu were also able to induce the expression of metallothionein to various degrees. The results indicate that the MT gene is present in RH-35 cells and is responsive to treatment with various metals. Thus, this cell line can be used as a model to study metallothionein induction.

Adherence to human colonocytes of an Escherichia coli strain isolated from severe infantile enteritis: molecular and ultrastructural studies of a fibrillar adhesin.

Escherichia coli 469-3 (O21:H-) was isolated from a child with severe enteritis. Ultrastructural analysis of the surface of the strain indicated the presence of very fine fimbriae which mediated mannose-resistant hemagglutination of human blood and caused the bacteria to adhere to human epithelial cell lines and to brush borders of isolated human colonic, but not duodenal, enterocytes. A cosmid library of total DNA of the strain, expressed in laboratory strains of E. coli, was screened by a rapid hemadsorption method, and a number of positive clones were identified. Restriction endonuclease fragments specifying mannose-resistant adherence were subcloned from the cosmid DNA of a strongly hemagglutinating clone in a plasmid vector. The identity of the adhesin was confirmed by biochemical, electron-microscopic, and immunological comparisons with the adhesin synthesized by the clinical isolate. It comprised a high-molecular-weight aggregate of a 14,000-dalton subunit protein which bound antiserum raised against the mannose-resistant adhesin of strain 469-3. The adhesin was synthesized by both the clone and the parental strain at growth temperatures above 18 degrees C but by only a fraction of the cells in a pure culture, although all the bacteria which adhered to human cells expressed the protein.