extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
High Affinity Ca2+ Binding Sites of Calmodulin Are Critical for the Regulation of Myosin Iβ Motor Function*

We coexpressed myosin Iβ heavy chain with three different calmodulin mutants in which the two Ca2+-binding sites of the two N-terminal domain (E12Q), C-terminal domain (E34Q), or all four sites (E1234Q) are mutated in order to define the importance of these Ca2+binding sites to the regulation of myosin Iβ. The calmodulin mutated at the two Ca2+ binding sites in N-terminal domain and C-terminal domain lost its lower affinity Ca2+ binding site and higher affinity Ca2+ binding site, respectively. We found that, based upon the change in the actin-activated ATPase activities and actin translocating activities, myosin Iβ with E12Q calmodulin has the regulatory characteristics similar to myosin Iβ containing wild-type calmodulin, while myosin Iβ with E34Q or E1234Q calmodulin lose all Ca2+ regulation. While the increase in myosin Iβ ATPase activity paralleled the dissociation of 1 mol of calmodulin from myosin Iβ heavy chain for both wild type (abovepCa 5) and E12Q calmodulin (above pCa 6), the Ca2+ level required for the inhibition of actin-translocating activity of myosin Iβ was lower than that required for dissociation of calmodulin, suggesting that the conformational change induced by the binding of Ca2+ at the high affinity site but not the dissociation of calmodulin is critical for the inhibition of the motor activity. Our results suggest that the regulation of unconventional myosins by Ca2+ is directly mediated by the Ca2+ binding to calmodulin, and that the C-terminal pair of Ca2+-binding sites are critical for this regulation.

tissues with the highest expression levels in heart, lung, adrenal gland, esophagus, and stomach (6,7,9,10). Myosin I␤ localizes to actin-rich peripheral structures, such as filopodia and lamellipodia of culture cells (9), and it is thought to play a role in cytoskeleton rearrangement. Interestingly, myosin I␤ is also found in hair bundles purified from the bullfrog sacculus, suggesting that myosin I␤ may function as an adaptation motor which regulates the tip link-associated cation selective channels (11,12).
Studies from both naturally isolated and recombinant myosins I␤ have shown that calmodulin is associated with myosin I␤ heavy chain (5,13). In contrast to most calmodulindependent enzymes, the association of calmodulin with myosin I does not require Ca 2ϩ binding to calmodulin. Thus, calmodulin functions as a light chain subunit. Myosin I␤, like all the other vertebrate unconventional myosins, has several repeats of a 24 -30-amino acid sequence called the IQ motif at the neck region between the myosin head motor domain and the tail domain. This motif has been suggested to provide the binding site for EF-hand family proteins such as calmodulin (1,14). All vertebrate unconventional myosins that have been characterized so far contain calmodulin as light chains. For some unconventional myosins, other small proteins besides calmodulin have also been found to function as light chains (15). A common property of calmodulin targets that contain IQ motif, such as the unconventional myosins and neuromodulin, is that they have a higher affinity for the Ca 2ϩ -free form of calmodulin (16).
Calmodulin is one of the major intracellular Ca 2ϩ -sensor proteins, containing four EF-hand type Ca 2ϩ -binding loops. The N-terminal pair are linked to the C-terminal pair by a central flexible linker. Among the four Ca 2ϩ -binding sites, the C-terminal pair of Ca 2ϩ -binding sites have a higher Ca 2ϩbinding affinity than those of the N terminus (17,18). Ca 2ϩ binding induces structural changes in calmodulin, and it is believed that these Ca 2ϩ -induced conformational changes allow calmodulin to activate target enzymes when the cytosolic Ca 2ϩ concentration is elevated (reviewed in Ref. 19). Mutagenesis studies have shown that a conserved glutamic acid residue at the 12th position of each Ca 2ϩ -binding loop is critical for Ca 2ϩ binding, and substitution of this conserved glutamic acid with glutamine in each Ca 2ϩ -binding site abolishes its Ca 2ϩ binding ability (17,20).
Both the enzymatic and mechanical activities of vertebrate myosin I have been shown to be regulated by Ca 2ϩ (5,13,(21)(22)(23), and this is also true for other unconventional myosins, such as myosin V (15). A member of another myosin I subclass, intestinal brush-border myosin I (BBMI), 1 has been extensively characterized in terms of Ca 2ϩ effects. It was found that BBMI moved actin filaments although the velocity was quite low (ϳ0.05 m/s), and the activity was abolished at Ca 2ϩ concentrations above 5 M. On the other hand, actin-activated Mg 2ϩ -ATPase activity of BBMI increased with increasing Ca 2ϩ concentrations (24). Interestingly, partial dissociation of calmodulin from BBMI was observed at a Ca 2ϩ concentration of 10 M. Similarly, Zhu et al. (13) have shown that one of the three calmodulin molecules bound to recombinant myosin I␤ dissociated from the heavy chain at a Ca 2ϩ concentration of 10 M. While Mg 2ϩ -ATPase activity increased above pCa 6, actin sliding velocity of myosin I␤ was abolished at pCa 6 (13). These results suggest that Ca 2ϩ binding to the calmodulin light chains is critical for the regulation of vertebrate myosin I motor function. However, it is unclear whether the dissociation of calmodulin is necessary to stop myosin I motor activity since the Ca 2ϩ concentration required for the dissociation of one calmodulin from myosin I␤ heavy chain seems to be higher than the Ca 2ϩ concentration for inhibition of the actin translocating activity (13).
In the present study, we have examined the mechanism by which Ca 2ϩ and calmodulin regulate myosin I␤ motor function by coexpressing various calmodulin mutants defective in Ca 2ϩ binding with myosin I␤ heavy chain and analyzing the actinactivated ATPase activity and motor function of the expressed myosin I␤.

Expression of Bovine Myosin I␤ Together with Mutant Calmodulin in
Sf9 Cells-Expression of bovine myosin I␤ cDNA with calmodulin was performed as described previously (13). cDNA for wild-type calmodulin and calmodulin with the two N-terminal, two C-terminal, or all four Ca 2ϩ -binding sites mutated, termed E12Q, E34Q, and E1234Q, respectively (25), were subcloned into pBlueBacM baculovirus transfer vector at the EcoRI site in the polylinker region. Orientation and accuracy of the subcloning were examined by DNA sequencing (Sequenase 2.0, U. S. Biochemical Corp.). Recombinant baculoviruses containing these mutant calmodulin cDNAs were obtained by blue plaque selection and subsequent steps of purification and amplification as described in the manual from Invitrogen, MaxBac Baculovirus Expression System. Sf9 cells were coinfected with the recombinant viruses of myosin I␤ heavy chain and each calmodulin mutant.
Purification of Recombinant Myosin I␤ with Mutant Calmodulin-The purification of recombinant myosin I␤ was performed as previously with slight modifications (13). Briefly, cells were harvested after 3 days of culture at 28°C and lysed in the presence of ATP, Triton X-100, Nonidet P-40, and various protease inhibitors. The supernatant (150,000 ϫ g for 30 min) of lysed cells was incubated with 10 mM glucose and 20 units/ml hexokinase at 0°C for 30 min to completely hydrolyze residual ATP. F-actin (1 mg/ml) was added to coprecipitate the expressed myosin I␤. The pellet was resuspended with buffer containing 5 mM MgCl 2 , 100 mM KCl, 1 mM EGTA, and 25 mM Tris-HCl, pH 7.5, and 1 mM ATP was added to release myosin I␤ from the myosin I-actin complex. The sample was ultracentrifuged at 150,000 ϫ g for 30 min to remove F-actin, and the supernatant containing the expressed myosin I␤ heavy chain and calmodulin was subject to a DE52 column (1 ϫ 10 cm). The protein was eluted with a linear gradient (12 ml-12 ml) of 50 -250 mM KCl. Approximately 100 g of myosin I␤ can be obtained from 800 ml of culture.
Expression and Purification of Calmodulin Mutants-Wild-type calmodulin and mutant proteins were expressed by infecting Sf9 cells with recombinant virus. Cells were homogenized with 5 volumes of buffer A (30 mM Tris-HCl, pH 7.5, 8 M urea, 5 mM dithiothreitol, 10 g/ml leupeptin) for 5 min. After centrifugation at 35,000 ϫ g for 15 min, 5% trichloroacetic acid was added to the supernatant. The pellets were collected and suspended with 8 M urea, and the pH was adjusted to be neutral. The suspension was dialyzed against buffer B (30 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol) for 2 h. After insoluble material was removed by centrifugation, the sample was loaded to a DE52 ion exchange column (24 ϫ 3 cm) equilibrated with buffer B. Calmodulin was eluted with a 0 -0.4 M NaCl linear gradient. The fractions containing calmodulin were combined, concentrated, and loaded onto a Sephacryl S200 gel filtration column (100 ϫ 3 cm). The fractions containing calmodulin were identified by SDS-polyacrylamide gel electrophroesis analysis. They were combined and concentrated. The concentrated cal-modulin was dialyzed against buffer C (30 mM Tris-HCl, pH 7.5, 30 mM NaCl, 1 mM dithiothreitol) and stored in Ϫ80°C. Bacterially expressed wild type and mutant calmodulins were prepared as described previously (25).
Fluorimeter Titrations-Ca 2ϩ -induced conformational changes in calmodulins were determined by Ca 2ϩ titrations in the presence of 9-anthroylcholine (9-AC) according to Beckingham with some modifications (20). Purified calmodulin was dialyzed extensively against the titration buffer (0.5 mM CaCl 2 , 100 mM KCl, and 10 mM MOPS, pH 7.0). 2-ml titration buffer containing 5 M of such prepared calmodulin and 20 M of 9-AC bromide (Molecular Probes, Eugene, OR) were transferred to a quartz cuvette, and fluorescence measurements were made as sequential addition of small volume (1 to several microliters) EGTA solutions (20 mM, 200 mM, or 1 M). The amount of EGTA was added so that the concentrations of free Ca 2ϩ in the cuvette were the desired ones (26). The fluorescence intensity was measured by using a Spex Fluorolog fluorimeter (Spex Inc., Edison, NJ) with excitation at 366 nm and emission at 418 nm. The pH of the samples was examined both before and after titration.
ATPase Assays and Other Biochemical Procedures-The effect of Ca 2ϩ on actin-activated ATPase activity was assayed in 30 mM KCl, 2 mM MgCl 2 , 20 mM imidazole-HCl, pH 7.0, with or without 50 M actin, in the presence of 1 mM EGTA or various concentrations of Ca 2ϩ (26). All assays were initiated by adding 100 M [␥-32 P]ATP (Amersham Corp.) to the reaction mix. The liberated 32 P was measured as described previously (27) to determine ATPase activity.
The effect of Ca 2ϩ on the binding of calmodulin to myosin I␤ heavy chain was determined as follows: myosin I␤ was dialyzed against different Ca 2ϩ buffers in the absence of ATP, and the sample was ultracentrifuged at 150,000 ϫ g for 30 min in the presence of 1 mg/ml F-actin. Then the pellet was analyzed by SDS-polyacrylamide gel electrophroesis. The amount of the cosedimented myosin I␤ and calmodulin was determined by densitometry, using a Macintosh computer equipped with a frame grabber (LG-3, Scion Corp., Walkersville, MD) connected to a video camera (Pulnix, TM-745, Motion Analysis, Inc., Eugene, OR), and the software used was NIH Image 1.56b18 (Bethesda, MD).
All experiments were carried out at 25°C, and all the results are represented by mean Ϯ S.E.
In Vitro Motility Assay-In vitro motility assays were performed as described previously (28). A larger nitrocellulose-coated coverslip (24 ϫ 30 mm) was covered by smaller coverslip (18 ϫ 18 mm), each edge of which was applied with 0.1 g of silicon grease (Dow Corning, MI), to create a fluid-filled flow cell. After 60 l of myosin I-containing solution was applied to the flow cell, unbound myosin I was washed away with 180 l of buffer A (400 mM KCl, 25 mM HEPES, pH 7.5, 4 mM MgCl 2 , and 10 mM dithiothreitol). Then the unoccupied nitrocellulose surface was coated with 0.5 mg/ml bovine serum albumin in buffer B (30 mM KCl, 20 mM HEPES, 1 mM EGTA, pH 7.5). The flow cell was washed with buffer B, then rhodamine-phalloidin (Molecular Probes Inc., Eugene, OR) labeled actin filaments in the motility buffer (50 mM KCl, 5 mM MgCl 2 , 25 mM imidazole, 1 mM EGTA, 1% 2-mercaptoethanol, 0.5% methylcellulose, 4.5 mg/ml glucose, 216 g/ml glucose oxidase, 36 g/ml catalase) with various concentrations of Ca 2ϩ were introduced onto the myosin-coated coverslip. After 120 l of motility buffer were perfused to wash out unbound actin filaments, motility buffer containing Mg 2ϩ -ATP was applied to initiate the reaction. Movements of fluorescent actin filaments were observed using an inverted fluorescence microscope (Diaphot, Nikon) with a SIT camera (VE 1000 SIT, DAGE MTI) and a video cassette recorder. The actin sliding velocity was determined as described previously (13).

RESULTS
Three different calmodulin Ca 2ϩ binding site mutants, termed E12Q, E34Q, and E1234Q (25), were used in this study. In each protein, particular Ca 2ϩ -binding sites were inactivated by mutation of a conserved glutamate residue (at position 12 of the Ca 2ϩ -binding loops) to glutamine. In E12Q the two Nterminal sites were mutated, in E34Q the two C-terminal sites were mutated, and in E1234Q, all four sites carried the mutation.
Wild-type calmodulin and each of the calmodulin mutants was coexpressed with myosin I␤ heavy chain in insect Sf9 cells, and the expressed myosin I␤ was isolated. All of the calmodulin mutants copurified with myosin I␤ heavy chain, suggesting that the mutations do not affect the binding of calmodulin to myosin I␤ heavy chain. The ability of Ca 2ϩ to increase electrophoretic mobility, a characteristic of wild-type calmodulin, was examined for each of these three mutants (Fig. 1). Wild type calmodulin migrated with an apparent molecular mass of 16 kDa in 5 mM Ca 2ϩ but with an apparent molecular mass of 21 kDa in the presence of 1 mM EGTA. E12Q calmodulin mutant migrated at 22 kDa in EGTA but at 19 kDa in the presence Ca 2ϩ . On the other hand, E34Q migrated at 21 and 18 kDa in the absence and presence of Ca 2ϩ , respectively. The mobility shift by Ca 2ϩ was abolished with E1234Q calmodulin mutant, which migrated at 21 kDa under both conditions. These results confirm that the calmodulin light chain associated with the myosin I␤ heavy chain in each preparation is indeed the expressed recombinant calmodulin mutant, and not endogenous calmodulin. They also suggest that the effect of mutating the two N-terminal Ca 2ϩ binding sites on the conformational change of calmodulin is different from that of mutating the two C-terminal sites.
Ca 2ϩ -induced conformational changes in the calmodulin mutants were further monitored as a function of Ca 2ϩ concentration by use of the reporter molecule 9-AC bromide. The Ca 2ϩinduced appearance of hydrophobic sites on calmodulin is revealed by the enhanced fluorescence of 9-AC upon binding to these sites, and this technique has been used previously to examine the Ca 2ϩ binding and conformational properties of Ca 2ϩ binding site mutants of calmodulin (20). The 9-AC fluorescence enhancement for wild type calmodulin as a function of Ca 2ϩ concentration is shown in Fig. 2. A single transition is detected, with midpoint at a Ca 2ϩ concentration (10 Ϫ7 ) that is lower than the dissociation constant for the high affinity sites on calmodulin. This finding probably reflects increased overall affinity for Ca 2ϩ and increased cooperativity of Ca 2ϩ binding induced by 9-AC. The hydrophobic reporter 1-anilino-8-naphtalene sulfonate has been shown previously to increase the affinity of calmodulin for Ca 2ϩ (29). The curve for the E12Q (see Fig. 2) is very similar to the wild type curve at low Ca 2ϩ concentrations but shows no increase in fluorescence enhancement at Ca 2ϩ concentrations above pCa 6. This is consistent with induction of a conformational change as a result of Ca 2ϩ binding to the two intact C-terminal sites present on this protein followed by absence of Ca 2ϩ binding and the associated conformational change in the N-terminal domain. In contrast, the E34Q mutant shows no fluorescence enhancement at low Ca 2ϩ and relatively minor enhancement of fluorescence with a midpoint at about pCa 6 as Ca 2ϩ levels are increased. Thus a major conformational change normally associated with C-terminal high affinity sites is lost in the E34Q mutant leaving a smaller conformational change associated Ca 2ϩ binding in the intact N-terminal domain. The sum of the fluorescence changes for E12Q and E34Q equals the changes for the wild type calmodulin (Fig. 2). The E1234Q mutant showed no change in 9-AC fluorescence throughout the entire pCa range tested (data not shown).
The actin-activated Mg 2ϩ -ATPase activity of myosin is coupled to actomyosin cross-bridge turnover. In order to examine the effect of Ca 2ϩ binding at the N-and C-terminal sites of calmodulin on myosin I␤ mechanoenzymatic function, actinactivated Mg 2ϩ -ATPase activity of myosin I␤ containing the mutant calmodulins was measured as a function of Ca 2ϩ (Fig.  3). For all assays, the timecourse of P i liberation was determined and the activity was estimated from the slope of the P i release timecourse. Unlike myosin I␤-containing wild-type calmodulin, for which Mg 2ϩ -ATPase activity increased in both the absence and presence of F-actin with Ca 2ϩ concentration above pCa 6 (Fig. 3A), myosin I␤-containing mutant calmodulin showed different Ca 2ϩ dependencies. The ATPase activity of E12Q myosin I␤ started to increase below pCa 6 and reached maximum at pCa 6 ( Fig. 3B). On the other hand, the ATPase activities of myosin I␤ containing both E34Q and E1234Q mutants did not show any Ca 2ϩ dependence (Fig. 3, C and D). The activity of myosin I␤ containing these two mutant calmodulins was similar to that of myosin I␤ with wild-type calmodulin in EGTA, suggesting that Ca 2ϩ binding to calmodulin in the Cterminal domain induced the enhancement of ATPase activity. These data suggest that the Ca 2ϩ -dependence of actin-activated ATPase activity of myosin I␤ is mediated through Ca 2ϩ binding to calmodulin and that the two C-terminal Ca 2ϩ -binding sites (high affinity sites) have a more important role in the Ca 2ϩ sensitivity of myosin I␤ ATPase activity.
To access the effects of mutating the N-and C-terminal Ca 2ϩ -binding sites of calmodulin on myosin I␤ motor activity, actin sliding velocity was measured by the in vitro motility assay system (Table I) with the one reported in the previous study (13). While switching motility buffer from 1 mM EGTA to 1 or 10 M Ca 2ϩ abolished the actin filament movement for wild-type and E12Q myosin I␤, it had little effect on the motility activity of E34Q and E1234Q myosin I␤. These results suggest that the Ca 2ϩ regulation of motor activity of myosin I␤ is also mediated through the binding of Ca 2ϩ to calmodulin. They further demonstrate that the two C-terminal Ca 2ϩ -binding sites but not the two N-terminal sites are critical for this regulation.
The effects of Ca 2ϩ on the binding of the various calmodulins to myosin I␤ heavy chain were also examined (Fig. 4). Purified myosin I␤ containing each calmodulin mutant was coprecipitated with F-actin at various Ca 2ϩ concentrations, and the precipitated myosin I␤ and calmodulin were subjected to SDSpolyacrylamide gel electrophroesis followed by densitometry analysis to quantify the stoichiometry of the bound calmodulin. It is known that 3 mol of calmodulin bind to 1 mol of myosin I␤ heavy chain (13). For wild-type calmodulin, one of the three molecules of bound calmodulin was dissociated from the heavy chain above pCa 5 (Fig. 4A). On the other hand, for E12Q myosin I␤, the dissociation of calmodulin was observed at lower Ca 2ϩ (i.e. pCa 6) (Fig. 4B). In contrast, Ca 2ϩ had no effect on the binding of E34Q calmodulin, i.e. all three calmodulin molecules were associated with the heavy chain even at pCa 4 (Fig.   4C). As expected, the binding of E1234Q mutant calmodulin to the heavy chain showed no Ca 2ϩ sensitivity (Fig. 4D).

DISCUSSION
In this study, we have coexpressed myosin I␤ heavy chain with three different calmodulin mutants, in which the conserved critical glutamic acid residue at the 12th position of the two N-terminal, two C-terminal, or all four of the Ca 2ϩ binding loops were substituted by glutamine. The importance of the carbonyl side chain of this glutamic acid to the Ca 2ϩ coordination system was revealed by crystallographic studies of calmodulin (30). As expected, the Ca 2ϩ -binding abilities of the mutated sites are completely abolished, based upon the Ca 2ϩ binding induced conformational changes probed by the fluorescent hydrophobic reporter molecule, 9-AC. The results are consistent with the earlier study in which the conserved glutamic acid residue in each individual Ca 2ϩ -binding site was mutated (20).
The fluorescence titration results show that the two C-terminal Ca 2ϩ -binding sites have a higher affinity for Ca 2ϩ than the two N-terminal sites. The different electrophoretic mobility of calmodulin in the presence of EGTA as compared with Ca 2ϩ also reflects conformational changes upon Ca 2ϩ occupation. The obtained results suggest that the conformational change caused by Ca 2ϩ binding to the two N-terminal sites (E34Q) is different from that caused by Ca 2ϩ binding to the two Cterminal sites (E12Q); occupation of the two C-terminal Ca 2ϩbinding sites has a greater impact on the overall conformational change. This is consistent with the previous findings that the Ca 2ϩ binding at the C-terminal sites of calmodulin induces a larger conformational change (31). Recent structural studies suggest that the C-terminal domain of calmodulin exists in a semi-open conformation in contrast to the close conformation of the N-terminal domain in the absence of Ca 2ϩ , and it changes to an open conformation upon Ca 2ϩ binding (32).   concentration are mediated through calmodulin, and that its two high affinity C-terminal Ca 2ϩ -binding sites are critical for the regulatory effect of Ca 2ϩ .
For myosin I␤ associated with E12Q, the effects of Ca 2ϩ on actin-activated ATPase activity, motor activity, and the calmodulin dissociation are similar to those for myosin I␤ associated with wild-type calmodulin. On the other hand, the disruption of the Ca 2ϩ -binding sites in the C-terminal domain of calmodulin abolishes the Ca 2ϩ dependence of the ATPase activity, the motor activity of myosin I␤, and the dissociation of 1 mol of calmodulin from the heavy chain at above pCa 6. These results suggest that Ca 2ϩ binding to the C-terminal domain of calmodulin, i.e. high affinity Ca 2ϩ binding sites, is responsible for the dissociation of one calmodulin molecule from myosin I␤ heavy chain. The increase in ATPase activity parallels the dissociation of one molecule of calmodulin according to the Ca 2ϩ dependence data (Fig. 4), thus it is reasonable to conclude that the dissociation of calmodulin increases myosin I␤ ATPase activity. It should be noted, however, actin independent ATPase activity but not actin dependent activity increases with Ca 2ϩ . Similar result has also been found for conventional myosin in which the dissociation of regulatory light chain increases basal myosin ATPase activity (33). It should be noted that while the deletion of the Ca 2ϩ binding at the N-terminal domain of calmodulin did not prevent the dissociation of 1 mol of calmodulin from the heavy chain, it shifted the Ca 2ϩ required for calmodulin dissociation to lower concentration. This result suggests that there is a cross-talk between the N-terminal and C-terminal domains and that deletion of the Ca 2ϩ binding ability at the N-terminal domain of calmodulin affects the conformational change at the C-terminal domain of calmodulin. This is consistent with the earlier finding that conformation of the Tyr-138 in the C-terminal domain of calmodulin is significantly influenced by a change in the Ca 2ϩ binding properties of the N-terminal domain (25).
Clearly, the binding of Ca 2ϩ to calmodulin at the C-terminal sites is critical for the inhibition of actin translocating activity of myosin I␤ by Ca 2ϩ , since this Ca 2ϩ -induced inhibition of the motility was not observed with myosin I␤ containing E34Q or E1234Q calmodulin (Table I). However, it is more complicated to determine whether or not the dissociation of the calmodulin molecule from myosin I␤ heavy chain is critical for the inhibition of the motor activity. Thus, although the wild-type myosin I␤ still binds all three calmodulin light chains at pCa 6, its motor activity is completely inhibited at this Ca 2ϩ concentration. One possible explanation is that although the C-terminal domain of the one calmodulin molecule is dissociated from myosin I␤ heavy chain at pCa 6, the N-terminal domain is still associated with the heavy chain at this Ca 2ϩ concentration, and further conformational change induced by Ca 2ϩ binding at the N-terminal low affinity sites of this molecule is necessary for the complete dissociation (Fig. 5). Presumably, this incomplete association of calmodulin with myosin I␤ is no longer able to support motor activity. For the E12Q mutant, the conformational change induced by the binding of Ca 2ϩ to the higher affinity sites may be sufficient to dissociate calmodulin from myosin I␤ heavy chain. It should be noted, however, Ca 2ϩ binding to calmodulin dissociates only one of the three bound calmodulin from myosin I␤ heavy chain. According to the amino acid sequence, myosin I␤ has three IQ motifs, one of which is not a completely matched IQ motif, IQXXXRGXXXR (one-letter amino acid code; X is any amino acid residue) (6). It is plausible that the calmodulin bound to the incomplete IQ motif is dissociated from myosin I␤ when Ca 2ϩ binds to the C-terminal domain. Alternatively, the conformational change in all three calmodulin upon Ca 2ϩ binding to the C-terminal domain results in the inhibition of motility and the additional conformational change upon the Ca 2ϩ binding at the N-terminal low affinity sites destabilizes the association of one of the bound calmodulin to the heavy chain presumably due to steric hindrance. Further studies are needed to clarify the reason why only one molecule of calmodulin is dissociated from myosin I␤.
Studies reported here with mutant calmodulin show that motor function of myosin I␤ is regulated by Ca 2ϩ binding to the high affinity sites of calmodulin light chains. This regulatory mechanism may also apply to those of other unconventional myosins which contain calmodulin as their light chains.


extfieldsofstudy: Biology Medicine

text:
Cloning and characterization of human eIF4E genes.

Two human eukaryotic initiation factor 4E (eIF4E) genes were isolated and characterized from placental and chromosome 4-specific genomic libraries. One of the genes (EIF4E1) contained six introns, but the other gene (EIF4E2) was intronless, flanked by Alu sequences and 14-base pair (bp) direct repeats, and terminated by a short poly(A) stretch, all characteristics of retrotransposons. Numerous additional intronless eIF4E pseudogenes were found, but unlike EIF4E2, all contained premature in-frame stop codons. The entire EIF4E1 gene spanned >50 kilobase pairs. The coding regions of these two genes differed in four nucleotide residues, resulting in two amino acid differences in the predicted proteins. The promoter of EIF4E1 has been characterized previously. The putative promoter of EIF4E2 contained no TATA box but did contain a transcription initiator region (Inr) and numerous other sequence motifs characteristic of regulated promoters. EIF4E2 contained only two of the three polyadenylation signals present in EIF4E1. Evidence for transcription of both genes was obtained from primer extension, S1 mapping, ribonuclease protection, and reverse transcriptase-polymerase chain reaction experiments. Transcription was found to initiate 19 bp upstream of the translational initiation codon in the case of EIF4E1 and 80 bp in the case of EIF4E2. The two genes were differentially expressed in four human cell lines, Wish, Chang, K562, and HeLa.

The best understood mechanisms for the regulation of protein synthesis involve modifications in the levels or activities of the initiation factors (1)(2). Changes involving eukaryotic initiation factor (eIF) 1 2 and associated proteins affect binding of the initiator tRNA to the 40 S ribosomal subunit and occur in response to heat shock, virus infection, deprivation of nutrients, and other conditions. Changes in the eIF4 factors affect binding of mRNA to the 43 S initiation complex and occur in response to mitogens, fertilization, and other conditions. The eIF4 factors consist of the ATP-dependent RNA helicase eIF4A (3,4), the RNA-binding protein eIF4B (5,6), the cap-binding protein eIF4E (7), and eIF4G, which has specific binding sites for eIF3, eIF4A, eIF4E, and the poly(A)-binding protein (8 -10).
Mammalian eIF4E is a 25-kDa protein of known three-dimensional structure (11) which binds to the mRNA cap (7), to eIF4G (3,4), to eIF4A (12), and to the eIF4E-binding proteins (13). Its ability to function in protein synthesis is regulated by at least three processes. First, the phosphorylation of eIF4E correlates positively with the rate of translation in a large number of systems (1) and increases the protein's affinity for cap analogues by 3-to 4-fold (14). Second, eIF4E availability is regulated by eIF4E-binding proteins, the phosphorylation of which, in response to insulin and other mitogens, releases them from eIF4E and permits eIF4E binding to eIF4G (13). Third, eIF4E levels are regulated at the transcriptional level. eIF4E mRNA is increased by overexpression of c-Myc as well as transformation of cells by v-Src and v-Abl (15). eIF4E mRNA levels are also elevated in a variety of cells that have been oncogenically transformed by in vivo transfection, viral infection, or chemical mutagenesis (16).
Changes in the intracellular levels of eIF4E have a profound effect on cellular growth control. Ectopic overexpression of eIF4E leads to accelerated cell growth (17), transformation in culture and tumorigenesis in nude mice (18), prevention of apoptosis in growth factor-restricted fibroblasts (19), and elevated intracellular levels of growth-regulated proteins such as cyclin D1, c-Myc, ornithine decarboxylase, ornithine aminotransferase, P23, vascular endothelial growth factor, and fibroblast growth factor (20 -26). Reduction in intracellular eIF4E levels by expression of antisense RNA (27) results in phenotypic reversal of ras-transformed fibroblasts (28 -29). Naturally occurring breast (30 -31) and head-and-neck (32) tumors express elevated levels of eIF4E. The eIF4E gene is increasingly being referred to as a proto-oncogene (30 -34).
Previous studies have resulted in the cloning and sequencing of human eIF4E cDNA (35) and a 1.4-kb fragment from the 5Ј-end of the eIF4E gene (36). To shed more light on the expression of eIF4E, especially at the transcriptional level, we have determined the entire gene structure for human eIF4E. Surprisingly two genes were found, one containing introns and the other not. Furthermore, both genes appear to be expressed in human cells, at least at the transcriptional level.  1 The abbreviations used are: eIF, eukaryotic initiation factor; PCR, polymerase chain reaction; kb, kilobase pair; bp, base pair; nt, nucleotide; RT-PCR, reverse transcriptase-polymerase chain reaction; PIPES, 1,4-piperazinediethanesulfonic acid; Inr, initiator region. cells from conjunctiva, K562 cells from chronic myelogenous leukemia, and HeLa cells from epithelioid carcinoma of the cervix were purchased from the ATCC. All cell lines were grown in Dulbecco's modified Eagle's medium with 10% calf serum in 5% CO 2 at 37°C and harvested after 2-3 days.

Materials-Two
Screening of Human Genomic DNA Libraries-Recombinant phage were propagated in the host bacterial strains NM538 and LE392 using standard protocols (37). Plaques were screened by colony hybridization and/or PCR using primers that spanned intron/exon junctions. For screening by colony hybridization, a plasmid (pTCEEC) containing human eIF4E cDNA (38) and PCR fragments derived from it were labeled with [␣-32 P]dCTP by nick translation. Prehybridization was performed in 50% formamide at 42°C for 2 h, radiolabeled probes were added, and hybridization was carried out for Ն16 h. Membranes were washed twice in 2 ϫ SSC, 0.2% SDS at room temperature for 30 min each and once in 0.1 ϫ SSC, 0.2% SDS at 65°C for 30 min, where 1 ϫ SSC is 0.15 M sodium chloride and 15 mM sodium citrate. The filters were exposed to Kodak x-ray film or analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For screening by PCR, the library phage lysates were divided into 50 pools (10 6 plaque-forming units/each), and 1 l of phage lysate from each pool was used as a PCR template. The positive pools were plated and allowed to grow 8 h, and the agar was again subdivided into 50 pools and screened by PCR. This process was performed a total of three to four times until a single positive plaque was obtained. DNA from selected recombinant phage was prepared from bacterial lysates (37). DNA inserts were mapped with restriction enzymes, and those containing exons were identified by Southern blotting. Exon-containing restriction fragments were excised from agarose gels, subcloned into pBluescript II (Ϫ) (Stratagene), and sequenced to identify exon/intron junctions.
Screening of the EMBL3-SP6/T7 library using radiolabeled human eIF4E cDNA yielded positive plaques corresponding to an intronless form of the eIF4E gene, here named EIF4E2. Screening of the same library with a hybridization probe synthesized with primers 9 and 10 (Table I) yielded the recombinant phage 4E1-A. Two human chromosome-specific genomic libraries in Charon 21 were screened using radiolabeled human eIF4E cDNA, one from chromosome 4 and one from chromosome 20. No positive plaques were obtained from the chromosome 20-specific library, but a number of positive plaques were obtained from the chromosome 4-specific genomic library and were termed 4E1-B, 4E1-C, and 4E1-D. A second chromosome 4-specific Charon 21 library was screened with cDNA probes and yielded 4E1-E. The LAO4NLO1 library was screened by PCR as described above using primers 3 and 7 and yielded 4E1-F. The same strategy was applied to this library but with primers 11 and 12, leading to the isolation of 4E1-G, and to the FIX II library with primers 1 and 8 leading to 4E1-H.
Southern Blot Analysis-DNA from purified positive plaques (1 g) was separated on agarose gels and transferred to nitrocellulose (37). DNA probes were labeled with 32 P by nick translation. Prehybridization and hybridization were performed as described above for colony hybridization.
DNA Sequence Analysis-DNA fragments were subcloned into pBluescript II vectors, and sequences were determined by dideoxy chain termination (39) using SK and KS primers (Stratagene) as well as exon-specific oligonucleotide primers. For both EIF4E1 and EIF4E2, the numbering system is based on human eIF4E cDNA (35), i.e. the location of the first ATG in the coding region is designated ϩ1, with upstream nucleotides having negative numbers and downstream nucleotides positive numbers. Nucleotides in introns are not numbered.
RNA Isolation-Total RNA was prepared from either human placental tissue or human cell lines by the guanidine thiocyanate method (40). In the latter case, ϳ10 7 cells were washed with phosphate-buffered saline before homogenization.
Primer Extension Analysis-Oligonucleotide primers complementary to eIF4E mRNA were end-labeled with T4 polynucleotide kinase (Promega) and [␥-32 P]ATP (37). Primer (300,000 cpm) and total RNA (ϳ100 g) were heated at 90°C for 5 min in 30 l of Hybridization Buffer (80% formamide, 40 mM PIPES, pH 6.4, 400 mM NaCl, and 1 mM EDTA) and then incubated at 30°C for 16 h. Hybrids were precipitated in ethanol and dissolved in 30 l of 50 mM Tris-HCl, pH 8.3, 7.5 mM MgCl 2 , 0.5 mM dNTP, 2 mM dithiothreitol, and 800 units/ml RNasin. Extension was produced by 400 units/ml Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) for 1 h at 42°C. After ethanol precipitation, products were separated on sequencing gels. S1 Analysis-A single-stranded antisense DNA probe corresponding to the sequence between Ϫ1000 and ϩ25 of EIF4E1 was produced by asymmetric PCR (41) from the recombinant phage 4E1-H using primers 1 and 4 ( Table I). The DNA was electrophoretically separated on 1.2% agarose gels, purified using a Geneclean kit, and then end-labeled with T4 polynucleotide kinase (Promega) and [␥-32 P]ATP (37). Total RNA (ϳ100 g) was hybridized with the probe (50,000 -100,000 cpm) at 30°C for 16 h in 30 l of Hybridization Buffer and then digested with 150 -200 units of S1 nuclease (Sigma) for 1 h at 30°C. Protected DNA fragments were separated on 8% sequencing gels (37).
Ribonuclease Protection Analysis-The sequence between Ϫ312 and ϩ184 of EIF4E2 was amplified from phage 4E2 by PCR using primers 2 and 3 (Table I). PCR was performed using the following program: after an initial heating step (95°C, 10 min), 2 units of Taq DNA polymerase were added to the 100-l reaction, after which 35 cycles of PCR were carried out (95°C, 30 s; 57°C, 30 s; 72°C, 2 min). The amplified PCR product was purified by agarose gel electrophoresis followed by treatment with Geneclean and then subcloned into the pGEM-T vector (Promega). RNA was transcribed in vitro from pGEM-T linearized by EcoRI and radiolabeled with [ 32 P]UTP with a riboprobe transcription system (Promega). A control RNA was transcribed in vitro from Hin-dIII-linearized pTCEEC. Ribonuclease protection assays were performed as described previously (40).
RT-PCR-Total RNA from human placental tissue (100 g) was treated with RNase-free DNase (Promega) at 37°C for 30 min. After inactivation of the DNase by heating at 70°C for 15 min, the RNA was hybridized with primer 3 (Table I) in Hybridization Buffer at 30°C overnight. Primer extension was performed as described above. RNA was removed by DNase-free RNase (Promega), and the remaining DNA from 1 l of the primer extension reaction mixture was amplified by PCR as described above, except that the annealing temperature was 65°C.

RESULTS AND DISCUSSION
Screening of human genomic libraries yielded two genes capable of encoding eIF4E, one of which (EIF4E1) contained introns and one of which (EIF4E2) did not (Fig. 1). Although a previous study reported eIF4E-like genes on chromosome 4 and 20 (42), the fact that most of the fragments in Fig. 1A were cloned from chromosome 4-specific libraries strongly argues that the EIF4E1 gene is on chromosome 4. The entire length of cloned DNA comprising the EIF4E1 gene was 50 kb. The EIF4E2 gene was flanked by three Alu sequences and two 14-bp direct repeats and contained a 3Ј-terminal poly(A) stretch (Fig. 2). All of these features are characteristic of retrotransposons (43). Additional screening yielded DNA fragments representing intronless genes with more substantial differences from the cDNA and containing additional in-frame stop codons. Intron-Exon Structure of EIF4E1-Comparison of the sequence of EIF4E1 with that of the cDNA (35) indicated that the gene is organized into seven exons and six introns (Fig. 1A). The regions in and around the exons were sequenced (Fig. 2). Exon 1 is the smallest (37 bp) and exon 7 is the largest (1.3 kb) ( Table II). The introns of this gene range from 1.2 to more than 10 kb and include only two of the three possible types (44), types 0 and 2 (Table III). All the exon/intron junction sequences conform to the GT/AG rule (44) (Table IV).
The exon structure of EIF4E1 can be compared with the recently published three-dimensional structure of eIF4E (11) with respect to the theory that protein-coding genes evolved by assembly of exons encoding functional domains (45)(46). Results of mass spectrometry and x-ray diffraction suggested that amino acids 1-35 are disordered in the absence of other proteins. These are encoded by exons 1 and 2 (Table II) which may have been added after the evolution of a core cap-binding protein for the purpose of binding other proteins (see Introduction). Exon 3 encodes most of ␤ strand 1 (S1), the 1-2 loop, ␤ strand 2 (S2), and most of ␣ helix 1 (H1). These structurally contiguous elements make up a region of the polypeptide chain that constitutes one side of the protein and include one of the two conserved Trp residues that "sandwich" the 7-methylguanine moiety, Trp-56. Exon 4 encodes essentially only one element, S3. Exon 5 encodes the 3-4 loop, S4, and most of H2. This collection of elements contains the other conserved sandwich Trp residue, Trp-102, as well as Glu-103, which forms hydrogen bonds to the N-1 and amino groups of 7-methylguanine. A similar set of interactions occurs between m 7 GMP and a Type 0 introns occur between two codons; type 1 introns interrupt a codon between the first and second nucleotide, and type 2 introns interrupt a codon between the second and third nucleotides (44).   2. Alignment of the nucleotide sequences of EIF4E1 and EIF4E2. A, the EIF4E1 gene. B, the EIF4E2 gene. The entire sequence for EIF4E2 from Ϫ712 to ϩ1894 is given (see "Experimental Procedures" for numbering system), but the introns have been omitted from the EIF4E1 sequence. Exon-exon junctions in EIF4E1 are marked by vertical lines. The proposed major transcriptional initiation sites of the two genes at Ϫ80 and Ϫ19 are indicated by arrows. The transcriptional initiator region (Inr), translational initiation codons, and polyadenylation signals are boxed. Other consensus motifs within the putative promoter region of EIF4E2 are underlined. Three Alu repeats in EIF4E2 are indicated in boldface, and a 14-bp direct repeat is doubly underlined. The sequence of a 2.7-kb portion of 4E2 has been deposited in GenBank TM /EMBL Data Bank (accession number, M77222).

Comparison of the EIF4E1 and EIF4E2
Genes-Sequence alignment of the exonic portions of the EIF4E1 gene with the entire EIF4E2 gene indicates no differences in the 13-nt region immediately upstream of the ATG, four single base differences in the 651-nt coding region, and five single base differences in the 980-nt 3Ј-untranslated region up to nt 1641 (Fig. 2). In the 3Ј-untranslated region of the EIF4E2 gene there is also a 10-nt insertion of T residues after nt 847 and the absence of a 9-nt stretch from nt 1238 to 1246. Upstream of nt Ϫ13 and downstream of nt 1641 there is no similarity between the two genes. As previously reported (36), the promoter of EIF4E1 lacks a canonical TATA box but includes two consensus sites for c-Myc. The putative promoter of EIF4E2 also lacks a consensus TATA box but, importantly, contains a consensus initiator region (Inr), TCATACC. A strong match to the Inr consensus sequence is commonly seen in TATA-less promoters (48) and may serve as a binding site for the YY1 protein (49). Other consensus motifs within this region include GATA1, AP1, AP4, GFI1, NF-B, STAT, c-Myb, SRY, SREBP1, and HFH2 (Fig. 2). The EIF4E2 gene contains only two of the three polyadenylation signals present in the eIF4E cDNA (35).
Both genes encode proteins of 217 amino acids with only two amino acid differences, Glu versus Lys at position 19 and Arg versus Trp at position 61. The location of the first of these sites in the three-dimensional structure of the protein is not known, but the second occurs at the beginning of S1. Amino acid residues at positions 19 and 61 do not make contact with m 7 GDP. Also, neither falls into the group of amino acid residues previously shown, by site-directed mutagenesis, to be important for cap binding (50 -54). Finally, neither Glu-19 nor Arg-61 is universally conserved in all eIF4E sequences. For these reasons, we predict that eIF4E-2 would be a functional protein.
Transcription Initiation Sites-Previous studies have indicated that transcription for mammalian eIF4E mRNA begins between Ϫ7 and Ϫ27; (i) the cloned human eIF4E cDNA begins at Ϫ18, although since this is a C residue, the mRNA is more likely to begin at the G at Ϫ19 (35); (ii) the cloned mouse eIF4E cDNA begins at Ϫ19 (55); and (iii) primer extension of mRNA from one mouse and one human cell line indicates a major start site at Ϫ16 and minor start sites at Ϫ7, Ϫ24, and Ϫ27 (55). The putative promoter of EIF4E2 bears no similarity to that of EIF4E1 and hence would be unlikely to initiate transcription in the same region. To determine whether EIF4E2 was transcribed, we performed primer extension with a primer complementary to the 5Ј-coding region of eIF4E mRNA, which should therefore produce extension products from both EIF4E1 and EIF4E2 transcripts. Primer extension using human placental RNA and primer 4 (Table I) produced major bands corresponding to initiation sites at Ϫ80 and Ϫ76 as well as minor bands at Ϫ19 and Ϫ20 (Fig. 3A). Primer extension with RNAs isolated from several human cell lines produced similar results (Fig.  3B). HeLa cell RNA (H) gave an initiation site at Ϫ80, the same as placental RNA, but Wish cell RNA (W) also gave minor products corresponding to initiation of transcription at Ϫ56 and Ϫ19. Chang cell RNA (C) yielded much more product corresponding to Ϫ19, less product from Ϫ80, and a small amount of product corresponding to Ϫ46 and Ϫ56. Finally, K562 cell RNA (K) produced even less of the Ϫ80 product, more of the Ϫ46 product, and a great deal of the Ϫ19 product.
To determine the origins of the various primer extension products, we performed S1 and RNase protection analysis using probes based on EIF4E1 and EIF4E2 sequences. S1 mapping with RNA from Wish cells using a probe spanning from Ϫ1000 to ϩ25 of the EIF4E1 gene produced a cluster of fragments corresponding to protection from Ϫ11 to Ϫ14 (Fig. 4A). As the EIF4E1 and EIF4E2 genes have the same sequences immediately downstream of Ϫ13 (Fig. 2), fragments of this size could be derived from either EIF4E1 transcripts initiated in the region of Ϫ11 to Ϫ14 or EIF4E2 transcripts initiated at this point or upstream. This results does, however, indicate that there are no unspliced transcripts from EIF4E1 beginning upstream of this point. It is possible, however, that a transcript is initiated upstream of Ϫ11 to Ϫ14 in EIF4E1 and then spliced, with the 3Ј splice site being located at Ϫ11 to Ϫ14. To test this possibility, we performed Southern analysis of restriction fragments of 4E1-H (Fig. 1A), which contains ϳ20 kb upstream of the coding region, using a probe consisting of the Ϫ80 primer extension product eluted from the polyacrylamide gel. However, no positive hybridization was observed, indicating the lack of an additional upstream exon (data not shown).
We tested directly for transcripts from EIF4E2 by ribonuclease protection analysis of human placental RNA using a radiolabeled RNA probe from Ϫ113 to ϩ184 of the EIF4E2 gene (Fig.  4B, lane 1). A control RNA, transcribed in vitro from the cloned eIF4E cDNA contained in plasmid pTCEEC (38), produced a protected fragment at Ϫ13 as expected (lane 2). Placental RNA  produced a number of protected fragments, the major ones corresponding to the probe terminating at Ϫ80 and Ϫ13 (lane 3). The Ϫ80 fragment is likely to be the same as the Ϫ80 primer extension product (Fig. 3), suggesting that transcription from EIF4E2 begins at Ϫ80. The Ϫ13 protection fragment is likely to have been produced by EIF4E1 transcripts, since the sequence of EIF4E1 and EIF4E2 is common only downstream of Ϫ13.
To confirm expression of the EIF4E2 gene, we performed RT-PCR using primers 3-6 (Table I). Primer 3 was used to make eIF4E-specific cDNA from human placental RNA. The sequence from Ϫ80 to ϩ25 of the EIF4E1 gene can be specifically amplified by PCR using primers 4 and 6, and the sequence from Ϫ66 to ϩ25 of the EIF4E2 gene can be specifically amplified using primers 4 and 5 (Fig. 5A). A product was obtained with primers 4 and 5 (Fig. 5B, lane 3) but not with primers 4 and 6 (lane 6), indicating that an mRNA is present in placental RNA that is initiated at or upstream of the Ϫ66 position of EIF4E2 but not at or upstream of position Ϫ80 of EIF4E1. This experiment was repeated using the Ϫ80 primer extension product of Wish cell RNA as template instead of placental RNA (Fig.  5C). Primers 4 and 5 allowed synthesis of a DNA product of the correct size (lane 2) but primers 4 and 6 did not (lane 4), providing direct proof that the Ϫ80 primer extension product of Fig. 4 is derived from EIF4E2. Finally, the PCR product in Fig.  5C, lane 2, was purified from the agarose gels and sequenced. The sequence matched the 5Ј-region of the EIF4E2 gene (Fig.  2). All of these results are evidence that mRNA transcribed from position Ϫ80 of EIF4E2 is present in human cells.
Additional evidence that these upstream sequences represent bona fide promoter regions came from an analysis of their chromatin structure. Micrococcal nuclease digestion products derived from the upstream regions of EIF4E1 and EIF4E2 were found to be heterogeneous by ligation-mediated PCR, despite the fact that bulk chromatin in the same digests gave rise to typical nucleosome-protected ladders (data not shown). This result is consistent with these regions being sites of nucleosome disruption, a hallmark of transcriptional regulatory regions (56).
The evidence presented here supports the view that both EIF4E1 and EIF4E2 are expressed at the level of mRNA. Two major primer extension products were detected, corresponding to transcription starts at Ϫ80 and Ϫ19 (Fig. 3). The data indicate that EIF4E1 is initiated at Ϫ19 and that EIF4E2 is initiated at Ϫ80. Other examples have been described in which both intron-containing and intronless genes code for the same protein (43,(57)(58)(59), and in at least two cases, both genes yield functional proteins (43,59). It is unclear, however, why human cells would express two functional genes for eIF4E. The fact that the promoter of EIF4E1 contains c-Myc-binding elements (36) whereas that of EIF4E2 does not suggests that the former may be inducibly expressed, e.g. during rapid cell growth, while the latter may be consititutively expressed. The differences in the relative amounts of the Ϫ80 and Ϫ19 primer extension products in various cultured human cell lines (Fig. 3B) suggests that the two genes may be differentially transcribed in response to factors such as growth rate or cell lineage. The differential expression of EIF4E1 and EIF4E2 may also explain why EIF4E2 cDNA was not initially cloned (35); fibroblast and lymphocytes, the sources for mRNA used in the cloning of human eIF4E cDNA, may express EIF4E1 predominantly. Similarly, the cultured cells chosen for primer extension analysis in the previously published study (55) may express EIF4E1 predominantly.
FIG. 4. S1 and RNase protection analysis of EIF4E1 and EIF4E2 transcripts. A, S1 nuclease analysis performed with no RNA (N) or total RNA (R) from Wish cells using a single-stranded DNA probe from the 5Ј-region of the EIF4E1 gene. The numbers refer to nucleotide positions (Fig. 2). C, T, A, and G are a sequencing ladder derived from EIF4E1. B, RNase protection performed with an RNA probe from the 5Ј-region of the EIF4E2 gene and human placental RNA. pTCEEC RNA was transcribed in vitro from a linearized plasmid containing eIF4E cDNA (38) and corresponds to an EIF4E1 transcript. Numbers on the left correspond to nucleotide positions (Fig. 2). Numbers on the right give the sizes in nucleotides of RNA markers (M).  Table I. B, PCR was performed using the indicated primers and templates consisting of human placental total RNA, reverse-transcribed with primer 3 (R), 4E1-H (1), and 4E2 (2). The products were separated on 2% agarose gels. M, DNA markers. C, PCR was performed as in B except that in the indicated lanes (P) the template was the primer extension product terminating at Ϫ80 from Wish cell RNA, excised from a sequencing gel similar to that shown in Fig. 3B. The products were separated on 6% polyacrylamide gels.


text:
Absorbability of calcium from a new calcium supplement prepared from bovine marrow-free bone in rats.

Powdered bovine marrow-free bone was completely solubilized with lactic and citric acids under reduced pressure. The resulting solution was lyophilized to obtain a stable powder form (total bone extract, TBE), and the calcium (Ca) absorbability of TBE from intestine was investigated in normal rats. Each animal perorally received 10 mg of Ca in 1 mL of distilled water as extrinsic 45Ca-labeled TBE, intrinsic 45Ca-labeled Ca lactate, or intrinsic 45Ca-labeled Ca carbonate. The amount of radioactivity in plasma was measured periodically up to 34 h after dosing, and pharmacokinetic parameters were calculated from the radioactivity in plasma. The time taken to reach the maximal 45Ca level (Tmax) did not differ among the three groups. The area under the plasma 45Ca level/time curve (AUC infinity) and the radioactivity at Tmax (Cmax) values for the TBE group were significantly higher than those of the Ca carbonate group. Similar results were observed between the Ca lactate and the Ca carbonate groups. No significant difference was observed in the AUC infinity and the Cmax values between the TBE and the Ca lactate groups. Radioactivity in a femur 34 h after dosing was highest in the Ca lactate group and lowest in the Ca carbonate group among the three groups. Both the TBE and the Ca lactate groups showed significant higher whole-body 45Ca retention than the Ca carbonate group did, although no significant difference was found between the TBE and the Ca lactate groups. These findings indicate that the Ca absorbability of TBE is almost comparable with that of Ca lactate and higher than that of Ca carbonate. Therefore TBE would be useful as a Ca supplement with relatively high absorbability from intestine.

Summary
Powdered bovine marrow-free bone was completely sol ubilized with lactic and citric acids under reduced pressure. The resulting solution was lyophilized to obtain a stable powder form (total bone extract, TBE), and the calcium (Ca) absorbability of TBE from intestine was investigated in normal rats. Each animal perorally received 10mg of Ca in 1mL of distilled water as extrinsic 45Ca-labeled TBE, intrinsic 45Ca -labeled Ca lactate , or intrinsic 45Ca-labeled Ca carbonate. The amount of radioactivity in plasma was measured periodically up to 34 h after dosing, and pharmacokinetic parameters were calculated from the radioactivity in plasma. The time taken to reach the maximal 45Ca level (Tmax) did not differ among the three groups. The area under the plasma 45Ca level/time curve (AUCoo) and the radioactivity at Tmax (Cmax) values for the TBE group were significantly higher than those of the Ca carbonate group. Similar results were observed between the Ca lactate and the Ca carbonate groups. No significant difference was observed in the AUC co and the Cmax values between the TBE and the Ca lactate groups. Radioactivity in a femur 34h after dosing was highest in the Ca lactate group and lowest in the Ca carbonate group among the three groups. Both the TBE and the Ca lactate groups showed significant higher whole-body 45Ca retention than the Ca carbonate group did, although no significant difference was found between the TBE and the Ca lactate groups. These findings indicate that the Ca absorbability of TBE is almost comparable with that of Ca lactate and higher than that of Ca carbonate. Therefore TBE would be useful as a Ca supplement with relatively high absorbability from intestine.
It is generally believed that bovine bone, which consists mainly of hy droxyapatite, a crystal form of Ca phosphate, is a relatively inferior calcium source compared with other Ca sources, such as Ca carbonate, Ca lactate , Ca citrate, Ca gluconate, milk, and other dairy products. This belief results from the hypothesis that the absorbability of various Ca salts is greatly dependent on their solubility in aqueous (1) solution because Ca is believed to be absorbed as its free or ionized form from intestine (2). Therefore Ca salts must be dissolved in water before absorption. Hydroxyapatite is well known to be insoluble in water. However, Heaney et al, using a double-isotope method with 45Ca and 47Ca, reported that the absorbability of food Ca was not clearly related to the solubility of the dominant chemical form in the food concerned (3). They also suggested that even under controlled, chemically defined conditions, the solubility of a Ca source has very little influence on its absorbability and that the absorbability of Ca from food sources is mainly determined by other food components. Therefore the absorbability of Ca from bovine bone may not markedly be inferior to that from other Ca salts, regardless of poor solubility. However, only a few studies have been carried out on the in vivo absorption of Ca from bovine bone in humans and animals (3,4). Ohtani et al recently developed a new Ca supplement prepared from bovine bone , namely, total bone extract (TBE) (5). TBE is prepared by solubilizing powdered bovine marrow-free bone with lactic and citric acids in water under reduced pressure . The resulting solution is lyophilized to obtain a stable powder form, TBE. Thus TBE mainly consists of hydroxyapatite, lactic acid, and citric acid and contains some minerals and proteins originally present in bone. In this study we compared the Ca absorbability of TBE with that of Ca carbonate and of Ca lactate, the solubility of which in water is respectively very low and relatively high (6) . TBE was extrinsically labeled with 45CaC12, and Ca carbonate and Ca lactate were intrinsically labeled with the same radioisotope. Isotopic tracer procedures for measuring the absorption of Ca from Ca supplements are based on the assumption that the tracer is absorbed with the same efficiency as the intrinsic nonradioactive Ca in a supplement (7,8).

MATERIALS AND METHODS
Preparation of total bone extract (TBE). TBE was prepared from fresh bovine marrow-free cortical bone. The bone was frozen in liquid nitrogen and crushed into a fine powder. The resulting bone powder was mixed with lactic acid in distilled water (DW) at a weight ratio of bone powder: lactic acid: DW of 1:1.2:7.8 for 1h under reduced pressure. The residue was subsequently mixed with citric acid at a weight ratio of bone powder (initial weight): citric acid of 1:0.028 under the above condition. The resulting clear solution was lyophilized to obtain a stable powder form, TBE.
Preparation of radiolabeled calcium sources. TBE was extrinsically labeled with radioactive Ca (45Ca) using 45Ca chloride (45CaC12) (9, 10). Both Ca carbonate and Ca lactate were intrinsically labeled with the same radioisotope according to the method of Kanerva et al (9,10 groups. Figure 2 shows the amount of radioactivity in the femur of rats 34h after the administration of 45Ca. The radioactivity level was highest in the Ca lactate group and lowest in the Ca carbonate group among the three groups. Figure 3 shows the amount of radioactivity in kidney. No significant difference was observed among the three groups. The percent of whole-body 45Ca retention was calculated as the difference between the amount of 45Ca ingested and the amount of 45Ca excreted in the feces and urine over a 34h period. The TBE and the Ca lactate groups both showed significantly higher whole-body 45Ca retention than the Ca carbonate group did, though no significant difference was noted between the I BE and the Ca lactate groups (Fig. 4). DISCUSSION The results reported here provide evidence that Ca absorption from TBE, which was prepared by solubilizing powdered bovine marrow-free bone with lactic acid and citric acid in distilled water, is comparable to and higher than that from Ca lactate and Ca carbonate, respectively. Furthermore, Ca from TBE is incor porated into bone more effectively than that from Ca carbonate without inducing high Ca accumulation in kidney. It is generally believed that Ca is largely absorbed in its free or ionized form in intestine, and therefore it is likely that differences in solubility correlate directly with differences in the availability of Ca for absorption. Thus it is widely believed that Ca from bovine and fish bone may not be absorbed easily because these bones consist mainly of hydroxyapatite, a water-insoluble form of Ca phosphate. The results obtained here, however, dem onstrate clearly that the absorbability of Ca from TBE was almost comparable to that of Ca from Ca lactate. However, TBE does not yield a clear solution in water; thus it is difficult to accurately measure the solubility of TBE in water. Several possibilities may explain the relatively high absorbability of Ca from TBE. First, TBE may be more soluble in the intestine than Ca carbonate and Ca phosphate are. To confirm this, the solubility of Ca in TBE in a luminal fluid must be determined. Second, the inhibitory effect of phosphate on Ca absorption may not occur for TBE. Ca balance studies in humans have demonstrated that phosphate has little effect on Ca absorption unless the Ca and phosphate intakes are both high (12). In this study, the amount of Ca present in the TBE ingested was 10.0mg. In our preliminary experiment using Ca carbonate, it was confirmed that the dose-dependent absorption of Ca takes place in the range from 5.0 to 20.0mg Ca under the same conditions. Therefore it is likely that the absorption of 10.0mg of Ca from the intestine is nutritionally sufficient and that the phosphate present in TBE does not affect Ca absorption. Third, Ca chelators, such as lactic acid and citric acid, may improve Ca absorption either by keeping absorbable Ca in solution as the pH rises or by favoring Ca absorption as a Ca-chelate complex (13)(14)(15). The latter possibility is supported by the fact that Ca in the form of a Ca-phosphopeptide complex (CPP) isolated from milk is highly absorbable (16). Furthermore, it has been reported that calcium-citrate-malate (CCM), a mixture of Ca carbonate, citric acid, and malic acid, exhibits approximately six times the solubility of either Ca citrate or Ca malate, both of which are much more soluble in water than Ca carbonate is (17). It is also well known that Ca from CCM is better absorbed in humans and animals than Ca from Ca carbonate is (18)(19)(20)(21). Similar to CCM, TBE is a composite salt of Ca phosphate derived from bovine bone, citric acid, and malic acid. Therefore it can be assumed that a Ca phosphate-citric acid-malic acid complex is more soluble and absorbable than Ca from Ca carbonate and bovine bone. At present we cannot confirm this, but investigations are under way.
In this study we used a single isotope extrinsic labeling method for TBE . Marshall and Nordin (11) compared several methods for estimating Ca absorption from various Ca salts and concluded that a dual isotope labeling method was not more effective than a single isotope labeling method. Miller et al (18) reported that Ca absorption from CaC12, milk, or other dairy products was similar whether an intrinsic or extrinsic radiolabeling method was used . Further experiments should be designed to elucidate the mechanism for the rapid and significant increased absorption of Ca from TBE. These studies should include the analysis of a chemical form of Ca from TBE and the contributions of organic acids, such as lactic acid and citric acid. Further studies are needed to determine the effects of TBE on skeletal development in animals.
This study indicates that the bioavailability of Ca from TBE is almost comparable to that from Ca lactate and much higher than that from Ca carbonate.
We conclude that TBE would be a useful Ca supplement with relatively high absorbability from intestine.


extfieldsofstudy: Medicine Biology

text:
Lactosylceramide Mediates Tumor Necrosis Factor- a -induced Intercellular Adhesion Molecule-1 (ICAM-1) Expression and the Adhesion of Neutrophil in Human Umbilical Vein Endothelial Cells*

The endothelial expression of adhesion molecules by proinflammatory cytokines such as tumor necrosis factor- a (TNF- a ) has been suggested to contribute to the initiation of atherosclerotic plaque formation. Since lactosylceramide (LacCer) accumulates in large quantities in human atherosclerotic plaque, we have explored its role in TNF- a -induced expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells and their consequent adhesion to polymorphonuclear leukocytes (PMNs). We found that TNF- a increased LacCer synthesis by way of stimulating the activity of UDP-galactose:glucosylceramide b (1 3 4)-galactosyltransferase in a time-dependent fashion. The TNF- a -induced expression of ICAM-1 was abrogated by D -1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( D -PDMP), an inhibitor of UDP-galactose:glucosylceram-ide b (1 3 4)-galactosyltransferase. However, the addition of LacCer reversed the D -PDMP effect on TNF- a -induced ICAM-1 expression in human umbilical vein endothelial cells. Northern hybridization analysis of mRNA levels and enzyme-linked immunosorbent assays revealed that

The endothelial expression of adhesion molecules by proinflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣) has been suggested to contribute to the initiation of atherosclerotic plaque formation. Since lactosylceramide (LacCer) accumulates in large quantities in human atherosclerotic plaque, we have explored its role in TNF-␣-induced expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells and their consequent adhesion to polymorphonuclear leukocytes (PMNs). We found that TNF-␣ increased
One of the critical events in the pathogenesis of atherosclerotic lesion formation is the focal accumulation of lipid-laden foam cells beneath an intact arterial endothelial lining (1,2). Localized attachment of circulating monocytes and lymphocytes to the arterial endothelium appears to precede the formation of early foam cell lesions (1,2). Various studies have demonstrated that the intercellular cell adhesion molecule-1 (ICAM-1), 1 E-selectin (endothelial leukocyte adhesion molecule; ELAM-1), and vascular cell adhesion molecule (VCAM-1) are all inducible on the endothelial surface following stimulation with TNF-␣ (3)(4)(5). ELAM-1 serves as a ligand for neutrophils (5), eosinophils (6), monocytes (7), and subpopulation of circulating lymphocytes (8). VCAM-1, which is induced on endothelial cells by IL-1, TNF-␣, or IL-4 (9) serves as a ligand for very late antigen-4 on lymphocytes, monocytes, and eosinophils (10). ICAM-1 is a specific ligand for lymphocyte function-associated antigen-1 (11) and Mac-1 (CD11/CD18), which is expressed on neutrophils and monocytes (12). In vivo studies have suggested that both ICAM-1 and VCAM-1, which are inducible by TNF-␣, are expressed in human atherosclerotic lesions (13). It is well documented that TNF-␣ induces the expression of cell adhesion molecules by activating an oxidantsensitive signal transduction pathway (14,15). However, to the best of our knowledge, the role of glycosphingolipids (GSLs), particularly lactosylceramide (LacCer), in this phenomenon has not been explored.
LacCer, a ubiquitous GSL, plays a pivotal role in the biosynthesis of complex glycosphingolipids (16). Moreover, the level of this GSL is elevated in several proliferative diseases including human polycystic kidney disease (17), familial hypercholesterolemia (18,19), and atherosclerosis (20,21). In addition, recently, there has been a surge in reports suggesting that sphingolipids may be implicated as second messengers in mediating diverse molecular events, including cell proliferation (22)(23)(24) and programmed cell death (apoptosis) (25), and as adhesive molecules (16). In this paper, we demonstrate that TNF-␣ stimulates the synthesis of LacCer by stimulating the activity of a UDP-galactose:glucosylceramide ␤(134)-galactosyltransferase (GalT-2). In turn, LacCer, via the generation of superoxide, up-regulates the expression of ICAM-1 on the surface of 1  Glycosphingolipids and all other chemicals were purchased from Sigma. The purity of glycosphingolipids was assessed by high pressure liquid chromatography and/or high performance thin layer chromatography to be Ͼ99%. Anti-ICAM-1 and anti-SOD antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-VCAM-1 and anti-Eselectin were obtained from Pharmingen (San Diego, CA). Cell Tracker dye green and Cell Tracker dye orange were obtained from Molecular Probes, Inc. (Eugene, OR). Diphenylene iodonium (DPI) was from Calbiochem. SOD virus carrying cDNA for superoxide dismutase and control virus carrying the ␤-galactosidase (no SOD) gene was a generous gift from Dr. Ronald G. Crystal (National Institutes of Health). cDNA for ICAM-1 was a gift from Dr. Mark Stipetic (Emory University School of Medicine).
Cells-HUVECs were purchased from Clonetics. Eahy 926 cells, a hybridoma permanent endothelial cell line, derived from HUVECs and human epithelial cell line A549 (26), was a generous gift from Dr. Roger Harrison (University of Bath, United Kingdom). HUVECs were maintained in endothelial cell growth medium supplemented with bovine brain extract (27). Eahy 926 cells were maintained in RPMI 1640 medium (Life Technologies, Inc.) containing 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (0.1 mg/ml).
Vehicle for Glycosphingolipids-Stock solutions of LacCer and other glycosphingolipids were prepared in Me 2 SO as described (23) and added to culture medium to achieve the desired concentrations of LacCer. The final Me 2 SO concentration exposed to cells was 0.01%. DPI stock solutions were also prepared in Me 2 SO and stored at Ϫ20°C until use. Cells incubated with 0.01% Me 2 SO served as a control. An aqueous solution of NAC was prepared in the culture medium.
Cell Surface ICAM-1, VCAM-1, and E-selectin Expression Assays-The quantitative expression of ICAM-1, VCAM-1, and E-selectin on the surface of the endothelial cell monolayers was determined by a modified ELISA in 96-well plates (28). Following incubation with antagonists and agonists, endothelial cell monolayers were fixed with 3.7% formaldehyde (pH 7.4) containing 0.1 M L-lysine monohydrochloride and 0.01 M sodium m-periodate for 20 min at 4°C and then blocked with PBS containing 1% bovine serum albumin and 0.1 M glycine overnight at 4°C. The fixed monolayer was then incubated with mouse monoclonal anti-human ICAM-1 or anti-human VCAM-1 or anti-human E-selectin for 1 h at 37°C. Next it was incubated with peroxidase-conjugated anti-mouse IgG F(abЈ) 2 for 1 h at 37°C. After washing, a developing substrate (0.2% H 2 O 2 , 0.4 mg/ml O-phenylenediamine) was added for 5 min, and the reaction was stopped with 2 N H 2 SO 4 . The plates were read on a spectrophotometric plate reader at 520 nm (28). ICAM-1 expression was assessed qualitatively in Eahy 926 cells by immunofluorescence staining using fluorescein isothiocyanate (FITC)-conjugated IgG following fixing of cells incubated with or without LacCer with 3.7% formaldehyde as described above.
Measurement of GalT-2 Activity-The activity of GalT-2 in cells incubated with TNF-␣ was measured employing UDP-[ 14 C]galactose as a nucleotide sugar donor and glucosylceramide as an acceptor as described previously (29). Briefly, the GalT-2 assay mixture contained 100 g of enzyme preparation, 20 M of cacodylate buffer (pH 6.8), 1.0 mM Mn 2ϩ /Mg 2ϩ , 0.2 mg/ml Triton X-100-cutscum (1:2, v/v), 30 nmol of GlcCer, and 0.1 mmol of UDP-galactose. Assays without exogenous GlcCer served as blanks and were subtracted from all corresponding data points. The assay was terminated by the addition of 25 mol of EDTA plus 2.5 mol of KCl. Chloroform/methanol (2:1, v/v) and 5 g of human kidney GSL were added, and the products were isolated and separated by Whatman SG-81 paper chromatography by developing in chloroform/methanol/water (60:17:2, v/v/v). Chromatogram areas corresponding in migration with standard LacCer were cut, and radioactivity was measured in a Beckman LS-3800 scintillation spectrometer using the background subtract setting and automatic quench setting.
Measurement of LacCer Synthesis-Cultures of HUVECs were incubated in growth medium containing [ 3 H]galactose (5 Ci/ml) for 24 h. Next, the medium was removed, and the monolayer was washed with sterile PBS to remove nonspecifically bound radioactivity. Fresh serumfree medium (5 ml/plate) was added. One set of dishes was incubated with D-PDMP (20 M) in PBS for 2 h. Next, TNF-␣ (300 units/ml) was added to cells incubated with or without D-PDMP. At various time points, cells were harvested and washed with PBS and centrifuged. The cell pellet was subjected to extraction of lipids with organic solvents as described (20). The total lipid extract was then subjected to silicic acid column chromatography. The mixture of glycosphingolipids was eluted with acetone-methanol (9:1, v/v), and carrier LacCer (10 g) was added and dried under N 2 atmosphere. Glycosphingolipids were separated onto high performance thin layer chromatography plates using chloroform/methanol/water (100:42:6, v/v/v) as the developing solvent. The chromatographic plate was dried in air and stained with iodine vapor. The gel area corresponding to LacCer was scraped, and radioactivity was measured employing "liquiscint" (NEN Life Science Products) as a scintillating fluid.
Northern Blot Analysis of ICAM-1 mRNA Expression in HUVECs-Total cellular RNA was isolated (30) from HUVECs that were preincubated with 15 mM NAC for 30 min, 5 M diphenylene iodonium for 30 min, followed by stimulation with the indicated concentrations of Lac-Cer/TNF-␣ for 2 h. Twenty micrograms of total RNA were separated by electrophoresis on 1% agarose gel (25 mM MOPS, pH 7.8, 1 mM EDTA, 1% (w/v) formaldehyde), transferred to a -probe blotting membrane (Bio-Rad) and hybridized with a 32 P-labeled ICAM-1 cDNA probe as described. The transfer of RNA to membrane and equal amount RNA loading was confirmed by ethidium bromide staining.
Measurement of Superoxide Generation in Intact HUVECs-Lucigenin, an acridylium compound that emits light upon interaction with O 2 . , was used to measure O 2 . production by chemiluminescence (23).
Confluently grown endothelial cells were harvested, and O 2 . generation from the intact endothelial cell suspension was measured using a 96well plate containing dark-adapted lucigenin (250 M) in balanced salt solution, as described by Bhunia et al. (23). The viability of the suspended cells was Ͼ90% as determined by trypan blue exclusion. LacCer or other GSL solutions were added as putative agonists, and the resulting increases in photon emission were measured every 20 s thereafter for 10 min in a scintillation counter (Packard TOP counter). The net increase in O 2 . generation at each time point was calculated by compar-

FIG. 1. Effects of TNF-␣ on the activity of GalT-2 and LacCer synthesis in HUVECs.
A, stimulation of GalT-2 activity by TNF-␣. Cells were incubated with TNF-␣. At various time intervals, cells were harvested in phosphate-buffered saline and lysed, and GalT-2 activity was measured as described under "Materials and Methods." E, control; q, TNF-␣ (100 units/ml); OE, TNF-␣ (300 units/ml). B, effects of TNF-␣ and D-PDMP on the incorporation of [ 3 H]galactose into lactosylceramide in cultured HUVECs. Cells were metabolically labeled with [ 3 H]galactose (5 Ci/ml) for 24 h at 37°C. Next, one set of dishes was incubated with fresh medium with and without D-PDMP (20 M) for 2 h at 37°C. Incubation was continued for the indicated time period following the addition of TNF-␣ (300 units/ml). Next, cells were harvested, total lipids were extracted with organic solvents, and glycosphingolipids were purified as described under "Materials and Methods." The radiolabeled glycosphingolipids were separated by high performance thin layer chromatography using chloroform/methanol/water (100:42:6, v/v/v) as the developing solvent. The chromatography plate was stained with iodine vapor, plate area corresponding to LacCer was scraped, and radioactivity was measured using a scintillation counter. The control specific activity was 805 Ϯ 52 cpm/mg of protein. ison with a standard curve generated using xanthine/xanthine oxidase.
Cell Fractionation and NADH/NADPH Oxidase Assay-Confluently grown HUVECs were incubated with or without 5 M LacCer. After various time intervals, the cells were harvested and homogenized, and the membrane and cytosolic fractions were separated by differential centrifugation, as described previously (23). NADH and NADPH oxidase activities were measured in both the cytosolic and the membrane fractions by lucigenin chemiluminescence as described above. In some experiments, NADPH oxidase activity was measured in the membrane fraction in the presence of 1 mM rotenone (a mitochondrial poison) (31). Protein content was measured by the method of Lowry et al. (32) with bovine serum albumin serving as a standard.
Adenovirus Vector-mediated Overexpression of SOD in Endothelial Cells-A replication-incompetent adenovirus (Ad-SOD) carrying the cDNA for human CuZn-SOD (33) and a control virus (Ad-␤-galactosidase) without the SOD gene but encoding only the Escherichia coli lacZ gene (34) were amplified in 293 cells and purified as described by Crawford et al. (33). Confluently grown HUVECs in 96-well plates (1.3 ϫ 10 4 cells/well) were infected with virus containing CuZn-SOD gene in multiplicities of infection (MOI) ranging from 0 to 75. Other plates of endothelial cells were infected with the control virus (Ad-␤galactosidase) at corresponding dilutions. Twenty-four hours later, the cells were washed with PBS to remove uninfecting virus particles. The endothelial cells were then incubated for an additional 48 h in fresh medium. The cells were then fixed with 3.7% formaldehyde in PBS, and the expression of intracellular SOD was measured by ELISA after permeabilization with 0.2% Triton X-100 in PBS for 20 min. The permeabilized endothelial cells were probed with monoclonal anti-human SOD and then with FITC-conjugated goat anti-mouse IgG. FITC fluorescence was measured on a fluorescence plate reader (CytoFluor 2300) at 480 nm (excitation) and 530 nm (emission). The SOD activities in the cell extracts were measured by the method of McCord and Fridovich (35).
Confocal Microscopic Visualization of Adhesion of Human Neutrophils to HUVECs-For the isolation of polymorphonuclear leukocytes (PMNs), human blood was collected in 10 units/ml heparin from laboratory volunteers by a protocol approved by the Johns Hopkins Joint Committee for Clinical Investigations and centrifuged at 1300 ϫ g for 10 min at 4°C. The white blood cell layer was removed and layered over cold Accu-prep gradient and centrifuged at 600 ϫ g for 30 min for the separation of leukocytes. The red blood cell/PMN layer was then resuspended in red blood cell lysing buffer. After incubation at room temperature, the preparation was centrifuged at 1300 ϫ g for 2 min, and this step was repeated until the PMN pellet was free of red blood cells. The PMN pellet was then washed and was found to contain Ͼ96% PMNs following modified Wright-Giemsa staining. The PMNs were labeled by incubation with 5 M fluorescent Cell Tracker dye (green) for 30 min at 37°C. At the same time, LacCer-stimulated (5 M, 4 h, 37°C)/control (0.01% Me 2 SO) endothelial cell monolayers grown in glass chamber slides were also labeled with 10 M Cell Tracker dye (orange). The labeled PMNs were incubated with confluently grown fluorescencelabeled endothelial cell monolayers in the glass chamber slides for 30 min at 37°C. The nonadherent PMNs were then removed by gentle washing with PBS. A drop of 4% glycerol in PBS was added, and a glass coverslip was mounted on the slide and visualized and photographed using confocal microscopy.
Quantitative Measurement of PMN-Endothelial Cell Adhesion and Blockade by ICAM-1 Antibody-Endothelial cells, grown in 24-well plates were incubated with various concentrations (0 -10 M) of LacCer for 4 h at 37°C and washed with PBS. Next, human PMNs labeled with Cell Tracker dye (green) as described above were incubated with the LacCer-stimulated endothelial cells for 30 min. Nonadherent PMNs were removed by gentle washing (three times) with PBS. The residual adherent PMNs were quantified on a fluorescence plate reader (Millipore Corp.) at 480 nm (excitation)/530 nm (emission) as described by Arai et al. (36). In a parallel experiment, following stimulation with LacCer, endothelial cells were preincubated with ICAM-1 antibody (1:2000 dilution) for 30 min before incubation with labeled PMNs as described above. The number of adherent PMNs was expressed as the number of PMNs/mm 2 of endothelial cell monolayer, based on the mean fluorescent intensity of each PMN, determined from a standard curve. To rule out the possibility of nonspecific adhesion stimulated by HU-VEC reactivity in some PMN adhesion assays, endothelial monolayers in one half of each 24-well plate were fixed first with 2% buffered formalin for 5 min and then washed three times with PBS. The endothelial cells on the other half of the plate were left unfixed but were similarly washed before use in adhesion assay.

TNF␣ Stimulates LacCer Synthesis in a Time-and Concentration-dependent Fashion in HUVECs-
We found that TNF-␣ (both 100 and 300 units/ml) stimulated GalT-2 activity in a time-dependent manner (Fig. 1A). Within 5 min, about 1.5-fold stimulation of GalT-2 activity was observed compared with the control. Thereafter, the activity of GalT-2 continued to rise in cells stimulated with TNF-␣. This phenomenon was accompanied by a time-dependent increase in the synthesis of LacCer as evidenced by an increase in the incorporation of [ 3 H]galactose into LacCer in cells stimulated with TNF-␣ (Fig. 1B). For example, 5 min after the stimulation with TNF-␣, a 1.5-fold increase in LacCer synthesis was observed and continued to increase 60 min after stimulation. Preincubation of cells with D-PDMP decreased the synthesis of LacCer ϳ0.4-fold compared with the control. D-PDMP effect was not altered significantly after stimulation of cells with TNF-␣.  Fig. 2A). Moreover, this inhibition by D-PDMP was reversed upon incubation with exogeneous LacCer but not by exogeneous ceramide or glucosylceramide (Fig. 2B), suggesting the involvement of LacCer in TNF-␣induced ICAM-1 expression.
LacCer Stimulates the Expression of ICAM-1 mRNA Levels in HUVECs-As shown in Fig. 4, LacCer exerted a concentrationdependent increase in the mRNA levels of ICAM-1 in HUVECs. A maximum stimulation of ICAM-1 mRNA level occurred following incubation of cells with 5 M LacCer for 2 h. Moreover, this phenomenon was abrogated by preincubation of cells with N-acetyl-L-cysteine, an antioxidant and a scavenger of free oxygen radicals, as well as DPI, an inhibitor of NADPH oxidase. Interestingly, the magnitude of stimulation of ICAM-1 gene transcription by LacCer was comparable with TNF-␣. . Generation in HUVECs-At various time points following stimulation with or without (5 M) LacCer, NADPH oxidase activity was measured in the membrane preparations (Fig. 6). Three-fold stimulation of NADPH oxidase activity was observed at 5 M LacCer, as compared with nonstimulated cell membrane preparations (Fig. 6A). Preincubation of LacCerstimulated/nonstimulated cell membrane preparations with DPI, a potent NADPH oxidase inhibitor (23), attenuated the LacCer induced increase in NADPH-dependent oxidase activity in the membrane preparations (Fig. 6A). In contrast, LacCer did not alter the activity of NADH oxidase activity in stimulated/nonstimulated membrane preparations (Fig. 6B). Neither NADPH oxidase nor NADH oxidase activity was observed in the LacCer-stimulated/unstimulated cell cytosol (data not shown). On the other hand, allopurinol, a specific inhibitor of xanthine oxidase (23), failed to inhibit LacCer-induced O 2 . generation (data not shown). Since rotenone also failed to inhibit NADPH oxidase activity in the membrane fraction, it appears that plasma membrane-associated NADPH oxidase is the source of LacCer-induced O 2 . generation in endothelial cells. . as well as ICAM-1 expression (Fig. 7, A and B). However, exogeneously added SOD did not inhibit the LacCer-induced O 2 . levels in endothelial cells and ICAM-1 expression (Fig. 7, A and B). These findings suggest that the NADPH oxidase-dependent endogenous generation of O 2 . is necessary to mediate LacCer-induced ICAM-1 expression.

Overexpression of Intracellular SOD Inhibits LacCer-induced ICAM-1 Expression-Since exogeneous SOD did not inhibit LacCer-induced O 2
. levels and ICAM-1 expression, we overexpressed SOD via adenovirus-mediated gene transfer. The expression of SOD in Ad-SOD-infected endothelial cells was 2.5-fold higher at 50 MOI compared with that of Ad-␤galactosidase-infected cells, as measured by immunofluorescence using monoclonal anti-SOD (Fig. 8A). The increase in SOD activity in the Ad-SOD-infected endothelial cell lysate was paralleled with an increase in MOI (Fig. 8B). SOD enzymatic activity in Ad-SOD-infected endothelial cells was also increased, dose-dependently, compared with that of the control (Ad-␤-galactosidase-infected) cells (Fig. 8B). In the Ad-␤-galactosidase-infected control cells, LacCer stimulated ICAM-1 ex- q, LacCer. D, effects of TNF-␣ and LacCer on the expression of VCAM-1 in HUVECs. Following incubation with LacCer (5 M) or TNF-␣ (300 units/ml), cells were fixed, and VCAM-1 expression was measured by ELISA using anti-human VCAM-1 antibody. E, effects of TNF-␣ and LacCer on the expression of E-selectin in HUVECs. The protocol in this experiment was similar to that described for D except that the level of E-selectin expression was measured employing anti-human E-selectin antibody. Each point is the mean Ϯ S.D. of five separate experiments. F, immunofluorescence assay of ICAM-1. Eahy 926 cells (endothelial cells) were confluently grown in glass chamber slides and incubated with or without 5 M LacCer for 8 h. Next, cells were fixed in 3.7% formaldehyde, and ICAM-1 expression was monitored by immunofluorescence using ICAM-1 monoclonal antibody followed by incubation with FITC-conjugated anti-mouse IgG and photographed employing fluorescence microscopy. Data presented here are from one of the two separate experiments with identical results. pression (Fig. 8C). However, in cells infected with Ad-SOD, LacCer-induced ICAM-1 expression was proportionally decreased with an increase in MOI and SOD expression. This finding suggests that the LacCer-induced stimulation of ICAM-1 expression was mediated by the intracellular generation of O 2 . .

LacCer Stimulates the Adhesion of Neutrophils to
HUVECs-Confocal microscopic visualization revealed that in control endothelial cells, incubated with vehicle only (0.01% Me 2 SO), neutrophil adhesion was not observed. In contrast, LacCer (5 M) markedly stimulated the adhesion of green fluorescencestained neutrophils to the orange fluorescence-stained endothelial cell monolayer (Fig. 9). Endothelial cells preincubated with NAC or NADPH oxidase inhibitor, DPI, attenuated the LacCer-induced neutrophil adhesion (Fig. 9). This qualitative observation was further substantiated by the quantitative measurement of adhesion. As shown in Fig. 10A, LacCer markedly stimulated the adhesion of PMN to endothelial cells. This phenomenon was abrogated by anti-human ICAM-1 antibody in a concentration-dependent fashion (Fig. 10B), suggesting the involvement of ICAM-1 in LacCer-induced PMN adhesion to HUVECs. DISCUSSION Significant information has been generated supporting the concept that TNF-␣ plays an important role in the adhesion of monocytes and neutrophils to the endothelial cells (3)(4)(5). This phenomenon precedes the production of superoxides, activation of NF-B, and an increase in the expression of cell adhesion molecules such as ICAM-1 in endothelial cells (14,15). This oxidant-sensitive transcriptional pathway plays an important role in vascular lesion formation (1,2). In the present study, we have extended the mechanism over previous observations by demonstrating that TNF-␣ increases the synthesis of LacCer via activating GalT-2. In turn, LacCer induced ICAM-1 expression and PMN adhesion. This may be an early process in the TNF-␣-mediated atherosclerotic lesion formation. Our current hypothesis describing how LacCer serves as a lipid second messenger in the activation of an oxidant-sensitive signal transduction pathway that leads to the adhesion of neutrophil to endothelial cells is summarized in Fig. 11 LacCer is a ubiquitous glycosphingolipid and plays a pivotal role in the biosynthesis of complex glycosphingolipids (16). Moreover, LacCer may contribute to proliferative diseases such as polycystic kidney disease, atherosclerosis (17), and familial hypercholesterolemia (18,19). The biological function of Lac-Cer was illustrated previously with two examples. First, in aortic smooth muscle cells, LacCer stimulated a redox-regulated signaling pathway via activating p21 ras GTP loading, p44 MAPK , and the expression of c-fos (23,24). Second, LacCer stimulated the expression of Mac-1(CD8/CD11) in human neutrophils. In turn, Mac-1 is recognized by ICAM-1 expressed on the surface of endothelial cells, and that leads to the adhesion of PMN (36).
Our earlier studies also revealed that in aortic smooth mus-

FIG. 5. Effects of LacCer concentration and time of incubation on the generation of superoxide in HUVECs.
Confluently grown endothelial cell monolayers were harvested and suspended in balanced salt solution. Next, the rate of generation of superoxide was measured by lucigenin chemiluminescence as described under "Materials and Methods." cle cells, oxidized low density lipoprotein stimulated GalT-2 activity and LacCer production. LacCer in turn served as a surrogate for oxidized low density lipoprotein to induce cell proliferation (37,38). In the present study with endothelial cells, a parallel observation was made in which TNF-␣ also activated GalT-2 and produced LacCer. Accordingly, we focused our studies on the upstream step in this signaling pathway. We employed D-PDMP as an inhibitor of glycosyltrans- ferases (37)(38)(39)(40) to assess whether the inhibition of LacCer synthesis could abrogate ICAM-1 expression. Indeed, as shown in Figs. 1 and 2, in endothelial cells D-PDMP not only inhibited the activity of GalT-2 and LacCer synthesis but also abrogated ICAM-1 expression. Since D-PDMP, an analog of glucosylceramide (GlcCer), also inhibits the synthesis of GlcCer from ceramide, it was not clear whether GlcCer or LacCer was required for the TNF-␣-induced expression of ICAM-1. To address this issue, the following experiment was performed. First, we preincubated cells with D-PDMP followed by stimulation with TNF-␣, GlcCer, and ceramide. LacCer was added next, and ICAM-1 expression was measured. Only LacCer bypassed the inhibitory effect of D-PDMP on ICAM-1 expression, while GlcCer or Cer did not. These observations suggest that LacCer may serve as a lipid second messenger in TNF-␣-induced ICAM-1 expression.
In agreement with previous studies, we found that TNF-␣ up-regulates the expression of VCAM-1 and E-selectin in HU-VECs. However, LacCer specifically stimulates the expression of ICAM-1 in these cells both at transcriptional and translational levels. At present, we cannot explain the biochemical basis of this observation. We can speculate, however, that TNF-␣-mediated expression of VCAM-1 or E-selectin may require additional signaling molecules other than LacCer.
Previous studies have shown that TNF-␣ induces the expression of adhesion molecules via the generation of ROS (14,15). We found that LacCer-induced ICAM-1 expression is also dependent on the generation of ROS. This tenet is supported by the following observations. First, direct measurement by lucigenin chemiluminescence showed that LacCer induces the generation of superoxide in a time-and concentration-dependent manner. We employed lucigenin chemiluminescence to assay LacCer-induced O 2 . generation, since this method detects both intracellular and extracellular O 2 . accurately even at a very low concentration (23,31). Second, antioxidants NAC and pyrrolidone dithiocarbamate (41) and overexpression of superoxide dismutase gene abrogated ICAM-1 expression. Third, DPI, an inhibitor of the NADPH oxidase-dependent ROS-generating system (23), completely abrogated LacCer-induced ICAM-1 expression both at the transcriptional and translational level. Finally, TNF-␣-induced O 2 . generation in HUVECs was abrogated by preincubation of cells with D-PDMP (data not shown). These findings suggest that TNF-␣-induced LacCer production is necessary for the generation of O 2 . , and that in turn induces ICAM-1 expression. Increased expression of ICAM-1 in atherosclerotic plaque has been previously thought to be due to increased O 2 . levels (15,16,(42)(43)(44). Moreover, endothelial cells produce higher amounts of O 2 . in hypercholesterolemia (45). Since the level of LacCer is also elevated in atherosclerotic plaque intima (20,21) and in patients with familial hypercholesterolemia (18,19), it is tempting to speculate that this GSL may serve as a lipid second messenger that may be required to stimulate O 2 . generation and ICAM-1 expression in atherosclerosis. One of the key transcription factors in ROS-regulated gene expression is NF-B (14, 46). We found that LacCer/TNF-␣ stimulated NF-B expression as measured by the binding activity of NF-B to the consensus oligonucleotide sequence spe- cific for NF-B (data not shown). Previous studies have shown that the cytosolic NF-B migrates to the nucleus and participates in nuclear events that ultimately lead to the expression of ICAM-1 (14). Clearly, further studies are required to explore in detail the LacCer-induced phenomenon described above.
The localized attachment of circulating leukocytes and monocytes to the endothelium of inflamed vessels is an important event in the initiation and progression of atherosclerotic lesion formation (47). As a response to injury, the endothelium secretes cytokines, for example, TNF-␣, IL-8, platelet activation factor, and monocyte chemotactic factor (2). Employing confocal microscopy, we found that LacCer facilitated the adhesion of PMNs to endothelial cells. These qualitative observations were substantiated by quantitative analysis of the number of PMNs adhering to the endothelial cells. Thus, LacCer may be implicated in the initiation of atherosclerotic plaque formation.
In summary, the adhesion of leukocytes to endothelial cells is a complex process, important in the pathogenesis of atherosclerosis and inflammatory phenomenon. Our findings indicate that TNF-␣ and oxidized low density lipoprotein implicated in atherosclerosis can activate GalT-2. TNF-␣-mediated activation of GalT-2 can generate LacCer. LacCer, in turn, generates O 2 . and stimulates ICAM-1 expression in HUVECs. By virtue of stimulating the expression of the ligand Mac-1 (CD11/CD18) for ICAM-1 (36), LacCer facilitates the adhesion of leukocytes to activated endothelial cells that overexpress ICAM-1. This constitutes a novel biochemical mechanism in regard to the potential role of LacCer and ROS in TNF-␣-induced initiation and progression of atherosclerosis.