Regulation of the juvenile hormone esterase gene by a composite core promoter.

Transcription from the core promoter of the juvenile hormone esterase gene (-61 to +28) requires the presence of both an AT-rich motif (TATA box) and an initiator motif for any transcription to occur, when assayed by either transcription in vitro with lepidopteran Sf9 nuclear extracts or by transient-transfection assay in Sf9 cells. Additional gel-shift experiments indicated that at least one additional binding site is essential for transcription to occur. Mutational analysis in the transcription-in vitro and cell-transfection assays demonstrated that a 14-bp region from +13 to +27 relative to the transcription start site is also essential for transcription to occur. Whereas the wild-type core promoter is highly transcriptionally active, inclusion of additional flanking sequences to position -212 reduces that activity approx. 100-fold, and inclusion of the 5' region out to position -500 reduces transcription by 200-fold. The pattern of dependence on both the AT-rich motif and the initiator for detectable transcription, and the high innate activity being repressed by 5'-binding factors, was recapitulated in mosquito C7-10 cells. This study demonstrates that the cellular juvenile hormone esterase gene is organized as a composite core promoter, dependent on both TATA-box and initiator-binding factors, an organization that has been more commonly reported for viral promoters. This highly active composite core promoter is made more complex by the absolute dependence on the presence of a third site shortly downstream from the initiator, which is distinct from the 'downstream promoter element' described from some TATA-less genes. The juvenile hormone esterase gene thus appears to be a model of a cellular composite core promoter with a multipartite, indispensible requirement for not just both the TATA box and initiator, but also for at least a third core element as well.

Transcription of the juvenile hormone esterase gene under the control of both an initiator and AT-rich motif.

The binding of transcription factors to the core promoter of the juvenile hormone esterase gene was functionally characterized using both a cell-free in vitro transcription functional assay and a cell transfection assay. A core JHE promoter (-61 to +28 bp relative to transcription start site) supported faithful transcription from the in vivo transcription start site. The nuclear extracts from the Sf9 insect cell line that provided transcription from that template also bound to that template as a probe in gel-mobility shift assays. Deletion or transversion of the initiator-binding motif (-1 to +4 bp) abolished detectable transcription either in vitro or in transfected cells. An AT-rich motif (ATATAT; -28 to -23 bp) serves another transcription factor-binding site. Mutation of the AT-rich motif to a canonical TATA-box preserved transcription, while either its deletion or complete transversion abolished or significantly reduced detectable transcriptional activity. These results indicate that, under these conditions, the functional operation of this core promoter approaches that of a composite promoter in which both the TATA- and initiator-binding protein complexes are necessary, even for basal transcription. On the other hand, these debilitating mutations to either the TATA box or initiator motif did not prevent the ability of the corresponding gel-shift competitive probes to compete with the wild-type promoter for binding by the transcription factors. Even a double transversion of both the AT-rich motif and the initiator-binding motif was able to competitively displace the protein complex that bound to the labelled wild-type probe. These data strongly indicate the presence of (an) additional core-promoter-associated transcription factor(s) (that is not the 'downstream element') that contact(s) the AT-binding complex and/or initiator-binding factor with sufficient avidity to remove them from binding to the competing wild-type promoter sequence.

Treatment of acne with a combination clindamycin/benzoyl peroxide gel compared with clindamycin gel, benzoyl peroxide gel and vehicle gel: combined results of two double-blind investigations.

BACKGROUND: It has previously been shown that a combination of erythromycin and benzoyl peroxide is superior to either ingredient when used alone in the treatment of acne. A clindamycin/benzoyl peroxide combination gel might have an advantage over erythromycin/benzoyl peroxide gel because the former does not require refrigeration after it is dispensed. OBJECTIVE: Our purpose was to determine the efficacy and safety of a combination clindamycin/benzoyl peroxide gel when compared with benzoyl peroxide, clindamycin, or vehicle gels. METHODS: In two double-blind, randomized, parallel, vehicle-controlled trials, patients were treated for 11 weeks with once-nightly application of one of the above preparations. Evaluations were performed at 2, 5, 8, and 11 weeks and included lesion counts and assessment of global responses and irritant effects. RESULTS: A total of 334 patients completed the study. All three active preparations were significantly superior to the vehicle in global improvement and in reducing inflammatory lesions and noninflammatory lesions. The combination gel was significantly superior to the two individual agents in global improvement and reduction of inflammatory lesions and also to the clindamycin gel in reducing noninflammatory lesions. There was no significant difference in tolerance to the active gels versus the vehicle gel. CONCLUSION: In the treatment of acne, topical clindamycin/benzoyl peroxide combination gel is well tolerated and superior to either individual ingredient.

Overview of the regulation of metamorphosis-associated genes in Trichoplusia ni.

A review is presented of our ongoing research program on the regulation of metamorphosis-associated proteins in Trichoplusia ni. Toward the identification of mechanisms by which juvenile hormone (JH) regulates expression of metamorphosis-associated proteins, we have identified a protein that is induced by JH (juvenile hormone esterase) and a related esterase that is not JH-inducible. We have also identified three hexamerins that are suppressible by JH, and one hexamerin that is not JH-suppressible. Expression of the hexamerins is regulated at the transcriptional, translational, and posttranslational levels in T. ni. The rate of transcription of the JH esterase gene increases at the time of the prepupal peak in JH, and exogenous application of JH can cause, within 3 h, the rate of transcription to be the markedly elevated above normal. Using in vitro functional transcription assay, cell line transfection functional assay, and preliminary DNase I hypersensitive site mapping we were able to identify the functional TATA box and transcription start sites of JH-sensitive genes. These methods were also observed to be powerful in the detection of regulatory DNA motifs involved in the modulation of transcriptional activity constitutively imparted by a core promoter. The experimental systems described here will also be effective in identifying those components through which JH regulatory effects are mediated. Should JH act on the genes in a primary manner, then the transcription factor mediating that action may be the (a) JH receptor. Should the JH action be in a secondary manner, then the transcription factor(s) whose activity at the JHE gene is regulated by JH will provide the tool to track back to the location and nature of the primary JH action.

Functional identification of the transcription start site and the core promoter of the juvenile hormone esterase gene in Trichoplusia ni.

The juvenile hormone (JH) esterase gene in T. ni encodes a protein that is responsible for the degradation of JH. The 5' structural characterization of this JH-sensitive gene was accomplished using reverse transcription PCR (RT-PCR) and northern analysis. The transcriptional start site of the JHE gene was biochemically determined by two methods: a 5' rapid amplification of cDNA ends (RACE) procedure which produced independent products with a sequence identical to the sequence of an exon encoded 5' to the putative first intron and by northern analysis with intronic and exonic probes. Both the transcription start site and the region containing the core promoter were also functionally identified by use of an in vitro transcription assay. The product of the in vitro transcription reaction, under the control of the putative core promoter region, initiated at the same base as identified by the RACE procedure, whether the reaction was driven by lepidopteran or by dipteran nuclear extracts. This result is the first functional identification of the core promoter region and transcription start site of any JH-sensitive gene.

Juvenile hormone action to suppress gene transcription and influence message stability.

Proteins normally expressed in high abundance only at larval-pupal metamorphosis in Trichoplusia ni were examined in a comparative analysis of the role and level of hormonal control of their expression. Some related proteins in the hemocyanin-superfamily (i.e., an acidic protein [AJHSP1] and two basic proteins [BJHSP1, BJHSP2]) were shown by nuclear run-on analysis to be specifically transcriptionally suppressed by juvenile hormone (JH), while transcription of another member of that family which is also metamorphosis-associated (arylphorin) was not specifically sensitive to JH. The stability of the mRNA for those members transcriptionally down-regulated by JH appeared to decrease under high JH conditions. While each protein was resorbed to some extent by the prepupal fat body, only the two basic proteins were quantitatively cleared from prepupal hemolymph. The JH-sensitive proteins studied appear to be encoded in single copy genes not immediately juxtaposed in the genome. These and previous studies now permit a more comprehensive understanding of the different combinations of mechanisms involving transcription, mRNA stability, translation, and protein clearance that operate to regulate these metamorphosis-associated proteins.

Multiple-dose pharmacokinetics and safety of oral amifloxacin in healthy volunteers.

The multiple-dose pharmacokinetics and safety of amifloxacin, a new fluoroquinolone antibacterial agent, were evaluated in healthy male volunteers. Amifloxacin was administered orally at 200, 400, or 600 mg every 12 h (q12h) and 400, 600, or 800 mg every 8 h (q8h) for 10 days. An additional dose was administered on day 11. Concentrations of amifloxacin in plasma and urine were measured on days 1, 5, and 11 by high-performance liquid chromatography. Steady-state amifloxacin concentrations were reached by day 5. Mean +/- standard deviation maximum observed amifloxacin concentrations in plasma were 2.52 +/- 1.12, 4.98 +/- 1.44, 5.40 +/- 2.02, 4.59 +/- 2.17, 6.53 +/- 2.44, and 8.01 +/- 3.00 micrograms/ml after the initial dose and 2.30 +/- 0.98, 5.41 +/- 0.74, 8.05 +/- 1.68, 6.87 +/- 2.81, 9.53 +/- 0.50, and 11.9 +/- 1.92 micrograms/ml on day 11 of the study for the 200-, 400-, and 600-mg q12h and 400-, 600-, and 800-mg q8h regimens, respectively. Amifloxacin was rapidly absorbed, as evidenced by the mean time to the maximum observed amifloxacin concentration of 0.98 h. Mean values for the terminal amifloxacin half-life in plasma ranged from 3.58 to 5.78 h. Mean amifloxacin concentrations in urine on day 11 in samples collected 0 to 2 h after dosing were 105, 417, 376, 336, 518, and 464 micrograms/ml for the 200-, 400-, and 600-mg q12h and 400-, 600-, and 800-mg q8h regimens, respectively. The mean amount of the dose excreted in the urine as amifloxacin was 53.9%. Amifloxacin was generally well tolerated, although there was a tendency for the subjects who received amifloxacin to experience more gastrointestinal, central nervous system, and cutaneous complaints than did those who received placebo. Clinically significant adverse reactions, including pruritus and transaminase elevations, occurred only at doses of 1,200 mg/day or above. Clinical and pharmacokinetic data suggest that orally administered amifloxacin may have utility in the treatment of urinary tract infections.

Randomized, controlled trial of a 10-day course of amifloxacin versus trimethoprim-sulfamethoxazole in the treatment of acute, uncomplicated urinary tract infection. Amifloxacin Multi-Center Trial Group.

We conducted a randomized controlled trial of orally administered amifloxacin versus trimethoprimsulfamethoxazole (TMP-SMX) as treatments of acute uncomplicated urinary tract infection in women. Amifloxacin at a dosage of 200 mg twice a day appeared as safe and effective as TMP-SMX, but amifloxacin at 400 mg twice a day tended to cause adverse events more frequently than did TMP-SMX.

Identification of protein phosphatase 2A as the major tyrosine hydroxylase phosphatase in adrenal medulla and corpus striatum: evidence from the effects of okadaic acid.

(i) The major sites on bovine adrenal tyrosine hydroxylase (TH) phosphorylated by calmodulin-dependent multiprotein kinase (CaM-MPK) and cyclic AMP-dependent protein kinase were shown to be Ser-19 and Ser-40, respectively, while Ser-40 was also phosphorylated slowly by CaM-MPK. (ii) Type 2A and type 2C phosphatases accounted for approximately 90% and approximately 10% of TH phosphatase activity, respectively, in extracts of adrenal medulla and corpus striatum assayed at near physiological free Mg2+ (1 mM), while type 1 and type 2B phosphatases had negligible activity towards TH. (iii) Incubation of adrenal chromaffin cells with okadaic acid increased TH phosphorylation by 206% and activity by 77%, establishing that type 2A phosphatases play a major role in regulating TH in vivo.

An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues.

The type 2A protein phosphatases in mammalian tissue extracts are inhibited completely and specifically by 1-2 nM okadaic acid. In contrast, type 1 protein phosphatases are hardly affected at these concentrations, complete inhibition requiring 1 microM okadaic acid. These observations have been exploited to develop an improved procedure for the identification and quantitation of type 1, type 2A and type 2C protein phosphatases in tissue extracts.

Remarkable similarities between yeast and mammalian protein phosphatases.

Protein phosphatase activities in extracts of the yeast Saccharomyces cerevisiae showed remarkable similarities to the mammalian type 1, type 2A and type 2C enzymes. Similarities included their substrate specificities, including selectivity for the alpha-and beta-subunits of muscle phosphorylase kinase, sensitivity to okadaic acid and to mammalian inhibitor 1 and inhibitor 2, and requirement for divalent cations. The results suggest that the function and regulation of these enzymes has been highly conserved during evolution and indicate that the improved procedure for identifying and quantitating protein phosphatases [(1989) FEBS Lett. 250,000,000] may be applicable to all eukaryotic cells.

The major type-1 protein phosphatase catalytic subunits are the same gene products in rabbit skeletal muscle and rabbit liver.

The catalytic subunit of protein phosphatase-1 (PP1) isolated from rabbit liver had the same electrophoretic mobility as, and yielded peptide maps identical to those of the 33 kDa form of rabbit skeletal muscle PP1. The predicted amino-acid sequences of PP1 obtained from three rabbit liver cDNA clones were identical to that of PP1 alpha from rabbit skeletal muscle. These findings suggest that the distinctive substrate specificities and regulatory properties of hepatic and skeletal muscle type-1 protein phosphatases are not conferred by the catalytic subunits themselves, but by regulatory subunits that are complexed to the catalytic subunits in vivo.

Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes.

The type-1 protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase a and glycogen synthase (labelled in sites-3) as substrates) was 50-fold higher than that of the glycogen-bound enzyme. (2) The microsomal enzyme had a greater sensitivity to inhibitors-1 and 2. (3) Release of the catalytic subunit from the microsomal type-1 phosphatase by tryptic digestion was accompanied by a 2-fold increase in synthase phosphatase activity, whereas release of the catalytic subunit from the glycogen-bound enzyme decreased synthase phosphatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the microsomes with 0.3 M NaCl, whereas little activity could be released from the glycogen fraction with salt. (5) The type-1 phosphatase separated from glycogen by anion-exchange chromatography could be rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCl) could not. These findings indicate that the synthase phosphatase activity of the microsomal enzyme is not explained by contamination with glycogen-bound enzyme. The microsomal and glycogen-associated enzymes may contain a common catalytic subunit complexed to microsomal and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inhibitor of the dephosphorylation of ribosomal protein S6, HMG-CoA reductase and glycogen synthase, by the glycogen-associated type-1 protein phosphatase. By contrast, thiophosphorylase a did not inhibit the dephosphorylation of S6 or HMG-CoA reductase by the microsomal enzyme, although the dephosphorylation of glycogen synthase was inhibited. The I50 for inhibition of synthase phosphatase activity by thiophosphorylase a catalysed by either the glycogen-associated or microsomal type-1 phosphatases, or for inhibition of S6 phosphatase activity catalysed by the glycogen-associated enzyme, was decreased 20-fold to 5-10 nM in the presence of glycogen. The results suggest that the physiologically relevant inhibitor of the glycogen-associated type-1 phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsomal type-1 phosphatase by phosphorylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-1 appears to be the principal S6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.