New phases of c60 synthesized at high pressure.

The fullerene C(60) can be converted into two different structures by high pressure and temperature. They are metastable and revert to pristine C(60) on reheating to 300 degrees C at ambient pressure. For synthesis temperatures between 300 degrees and 400 degrees C and pressures of 5 gigapascals, a nominal face-centered-cubic structure is produced with a lattice parameter a(o) = 13.6 angstroms. When treated at 500 degrees to 800 degrees C at the same pressure, C(60) transforms into a rhombohedral structure with hexagonal lattice parameters of a(o) = 9.22 angstroms and c(o) = 24.6 angstroms. The intermolecular distance is small enough that a chemical bond can form, in accord with the reduced solubility of the pressure-induced phases. Infrared, Raman, and nuclear magnetic resonance studies show a drastic reduction of icosahedral symmetry, as might occur if the C(60) molecules are linked.

Mutation changing the specificity of an RNA polymerase sigma factor.

We describe a mutation that changes the fine specificity of promoter selection by a secondary form of RNA polymerase holoenzyme in Bacillus subtilis. The product of regulatory gene spo0H is an RNA polymerase sigma factor called sigma H, which directs transcription of a sporulation gene known as spoVG. We show that the spo0H mutation spo0H81, which blocks transcription from the wild-type spoVG promoter, enhances transcription from a mutant form of the spoVG promoter (spoVG249) bearing a severe down-mutation (a G.C to A.T transition) at position -13 in the "-10 region." Suppression of the spoVG249 mutation is specific in the sense that the transcription from several other spoVG mutant promoters was not restored by the mutant sigma. Evidently, spo0H81 is a change-of-specificity mutation that alters sigma H-RNA polymerase in a way that decreases its capacity to use the wild-type spoVG promoter, while increasing its capacity to use the mutant promoter. Transcription experiments in vitro using RNA polymerase containing the wild-type or mutant sigma support this interpretation. The spo0H81 mutation causes a threonine (Thr100) to isoleucine substitution in a region of sigma H that is highly homologous among sigma factors of diverse origins. We discuss the possibility that Thr100 is an amino acid-base-pair contact site and that sigma factors contact the -10 region of their cognate promoters by means of amino acid residues in this highly conserved region.

Cloning of a promoter used by sigma H RNA polymerase in Bacillus subtilis.

The secondary RNA polymerase sigma factor sigma H is essential for endospore development in Bacillus subtilis. However, only a few promoters that are used by RNA polymerase containing sigma H (E sigma H) have been identified. We used in vitro transcription of random cloned fragments of B. subtilis chromosomal DNA to identify a promoter that is used by E sigma H. This promoter is active before the onset of sporulation.

Syntheses and biological activities of a novel group of steroidal derived inhibitors for human Cdc25A protein phosphatase.

Silica gel supported pyrolysis of an azido-homo-oxa steroid led to rearrangement, presumably by a mechanism similar to that of solution phase Schmidt fragmentation, to produce a group of novel inhibitors for the oncogenic cell cycle regulator Cdc25A phosphatase. Cyano-containing acid 17, one of the best inhibitors in this group, inhibited the activity of Cdc25A protein phosphatase reversibly and noncompetitively with an IC(50) value of 2.2 microM. Structure-activity relationships revealed that a phosphate surrogate such as a carboxyl or a xanthate group is required for inhibitory activity, and a hydrophobic alkyl chain, such as the cholesteryl side chain, contributes greatly to the potency. Without the cyano group, acid 26 and xanthate 27 were found to be more selective over Cdc25A (IC(50) = 5.1 microM and 1.1 microM, respectively) than toward CD45 (IC(50) > 100 microM, in each case), a receptor protein tyrosine phosphatase. Several of these inhibitors showed antiproliferative activities in the NCI 60-human tumor cell line screen. These steroidal derived Cdc25 inhibitors provide unique leads for the development of dual-specificity protein phosphatase inhibitors.

Mycobacterium keratitis after laser in situ keratomileusis.

PURPOSE: The authors report two cases of Mycobacterium keratitis following LASIK. METHODS: The case reports are based on a retrospective review of clinical history and associated findings. RESULTS: Two patients developed infectious keratitis after undergoing laser in situ keratomileusis (LASIK). In case #1, the infection developed after manipulation of the lamellar flap to remove epithelium from the stromal bed. In case #2, prior radial keratotomy may have been a contributing factor to development of the infection. Corneal infiltrates appeared as focal, white, stromal deposits. Cultures isolated Mycobacterium fortuitum from case #1 and Mycobacterium chelonae from case #2. Topical fortified amikacin, clarithromycin, tobramycin, and ciprofloxacin eventually controlled the infection. Topical prednisolone acetate and bandage contact lenses were necessary to control inflammation and pain. Infiltrates were slow to resolve until focal necrosis eroded through the flaps leading to rapid clearing of the infiltrates; however, scarring of the cornea developed at the site of necrosis. Visual recovery was good in the first case but limited in the second. CONCLUSIONS: Mycobacterium keratitis complicating LASIK may be difficult to eradicate until the sequestered stromal infiltrate drains. Rapid recognition of the causative organism and aggressive medical and surgical management of the infection may improve the outcome.

New RNA polymerase sigma factor under spo0 control in Bacillus subtilis.

In Bacillus subtilis transcription of spoVG is activated within minutes after the initiation of sporulation. Mutations in several spo0 genes prevent the activation of spoVG transcription. We have found a sigma-like protein that is capable of directing core RNA polymerase to use the spoVG promoter in an in vitro run-off transcription assay. This sigma-like protein was not found to be associated with RNA polymerase in a spo0A or spo0B mutant but was present in a spo0H mutant. We suggest that one role of the spo0A gene product in transcription of spoVG is the modulation of RNA polymerase activity by this sigma-like protein.

Sigma H-directed transcription of citG in Bacillus subtilis.

The RNA polymerase sigma factor sigma H is essential for the onset of endospore formation in Bacillus subtilis. sigma H also is required for several additional stationary-phase-specific responses, including the normal expression of several genes that are required for the development of competence for DNA uptake. It is necessary to identify the genes that are transcribed by sigma H RNA polymerase (E sigma H) in order to understand the role of this sigma factor during the transition from exponential growth to stationary phase. Feavers et al. (Mol. Gen. Genet. 211:465-471, 1988) proposed that citG, the structural gene for fumarase, is transcribed from two promoters, one of which (citGp2 [P2]) may be used by E sigma H. It is likely that the citGp2 promoter is used by E sigma H because we found that this promoter was used accurately in vitro by E sigma H and directed expression of xylE in vivo. This xylE expression was dependent on spo0H, the structural gene for sigma H, and was independent of the citGp1 promoter. Comparison of the nucleotide sequences of several sigma H-dependent promoters showed that these sequences were similar at two regions approximately 10 and 35 base pairs upstream from the start points of transcription. These sequences may signal recognition of these promoters by E sigma H. Primer extension analyses were used to examine transcription from three sigma H-dependent promoters during growth and sporulation. The citGp2 promoter appeared to be active during the middle and late stages of exponential growth, whereas activation of the spoIIA promoter was delayed until after the end of exponential growth. Evidently, promoters used by E sigma H can display different temporal patterns of expression.

rpoD operon promoter used by sigma H-RNA polymerase in Bacillus subtilis.

Three promoters direct transcription of the sigA (rpoD) operon in Bacillus subtilis. Promoters P1 and P2 are used during the exponential growth phase, whereas P3 is used only during the stationary phase. We examined the use of these promoters in promoter-probe plasmids and found that expression from P3 was prevented by a mutation in spoOH, which encodes the secondary RNA polymerase sigma factor sigma H. Moreover, we found that sigma H-containing RNA polymerase efficiently and accurately used the P3 promoter in vitro. Evidently, this operon, which is essential for exponential growth, is transcribed during the early phase of sporulation by this secondary form of RNA polymerase. Comparison of the nucleotide sequences of the P3 promoter and the spoVG promoter, which also is used by sigma H-RNA polymerase, revealed sequences at the -10 and -35 regions of these promoters that may signal recognition of promoters by sigma H-RNA polymerase.

Mutations that affect utilization of a promoter in stationary-phase Bacillus subtilis.

Transcription of the ctc gene in Bacillus subtilis is activated only after exponentially growing cells enter stationary phase. The promoter of the ctc gene is utilized in vitro by two minor forms of RNA polymerase, E sigma 37 and E sigma 32, but not by the most abundant form of RNA polymerase, E sigma 55. We have used the ctc promoter to direct transcription of the xylE gene on plasmid pLC4 and observed that xylE was expressed only in stationary-phase B. subtilis. We also have constructed a series of homologous plasmids that differ only by specific base substitutions in the ctc promoter. We observed that the base substitutions that affected utilization of the ctc promoter in vivo (xylE expression) were the same as those that we had previously shown to affect utilization of the promoter in vitro by E sigma 37 and E sigma 32. We conclude that it is likely that the ctc promoter is utilized in vivo by E sigma 37 or E sigma 32.

Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library.

The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed random hexapeptide library containing 2 x 10(8) independent recombinants. A total of 35 phage clones that express a peptide sequence that can be hydrolyzed by the recombinant catalytic domain of human collagenase 3 are identified. The translated DNA sequence of these clones reveals highly conserved putative P1, P2, P3 and P1', P2', and P3' subsites of the peptide substrates. Kinetic analysis of synthetic peptide substrates made from human collagenase 3 selected phage clones reveals that some of the substrates are highly active and selective. The most active substrate, 2, 4-dinitrophenyl-GPLGMRGL-NH(2) (CP), has a k(cat)/K(m) value of 4.22 x 10(6) m(-)(1) s(-)(1) for hydrolysis by collagenase 3. CP was synthesized as a consensus sequence deduced from the preferred subsites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-1, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MMP-processing site in aggrecan. Phage display screening also selected five substrate sequences that share sequence homology with a major MMP cleavage sequence in aggrecan and seven substrate sequences that share sequence homology with the primary collagenase cleavage site of human type II collagen. In addition, putative cleavage sites similar to the consensus sequence are found in human type IV collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type II and type IV collagens.

Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE).

The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.

Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha.

Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.