Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation.

Post-translational acetylation of the core histone amino-terminal tails has long been associated with both chromatin assembly and the regulation of gene expression. The recent identification and cloning of histone acetyltransferase genes represents a significant breakthrough in our understanding of how specific acetylation states are established. Ongoing characterization of these enzymes and their molecular cohorts supports a direct role for acetylation in a signaling pathway that modulates chromatin structure to create new patterns of transcription.

Inhibitory action of a met-enkephalin on ACTH release in man.

In order to study the mechanism of action of a Met-enkephalin (FK 33824) on the pituitary-adrenal axis eight normal male volunteers were subjected to an ACTH stimulation test. Lysine-vasopressing (LVP), 5 IU, was injected intramuscularly after pretreatment with 0.5 mg FK 33824 i.m. or a placebo. In six of the subjects the opiate was again administered preceding a single injection of 0.25 mg ACTH beta 1-24 i.m. Blood was collected at regular intervals and ACTH and cortisol concentrations analyzed in all samples. LVP induced significant plasma ACTH (P < 0.05) and cortisol (P < 0.001) increases. Pretreatment with FK 33824 completely antagonized the effect of LVP. Furthermore, the cortisol elevation after exogenous ACTH was not modified by previous administration of FK 33824. It is concluded that the Met-enkephalin derivative FK 33824 directly suppresses ACTH release from the pituitary without influencing adrenal synthesis of cortisol.

Histone acetyltransferase activity is conserved between yeast and human GCN5 and is required for complementation of growth and transcriptional activation.

Yeast and human ADA2 and GCN5 (y- and hADA2 and y- and hGCN5, respectively) have been shown to potentiate transcription in vivo and may function as adaptors to bridge physical interactions between DNA-bound activators and the basal transcriptional machinery. Recently it was shown that yGCN5 is a histone acetyltransferase (HAT), suggesting a link between enzymatic modification of nucleosomes and transcriptional activation. In this report, we demonstrate that hGCN5 is also an HAT and has the same substrate specificity as yGCN5. Since hGCN5 does not complement functional defects caused by deletion of yGCN5, we constructed a series of hGCN5-yGCN5 chimeras to identify human regions capable of activity in yeast. Interestingly, only the putative HAT domain of hGCN5, when fused to the remainder of yGCN5, complemented gcn5- cells for growth and transcriptional activation. Moreover, an amino acid substitution mutation within the HAT domain reduced both HAT activity in vitro and transcription in vivo. These findings directly link enzymatic histone acetylation and transcriptional activation and show evolutionary conservation of this potentially crucial pathway in gene regulation.

A novel H2A/H4 nucleosomal histone acetyltransferase in Tetrahymena thermophila.

Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymena macronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be approximately 80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena.

Nedd8 modification of cul-1 activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha.

Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.

Poly(2-fluoroadenylic acid). The role of basicity in the stabilization of complementary helices.

The polymerization of 2-fluoroadenosine 5'-diphosphate by polynucleotide phosphorylase to give high molecular weight poly(2-fluoroadenylic acid), poly(fl2A), is described. Both the single-stranded and double-stranded (acid) forms of poly(fl2A) exhibit strikingly similar ultraviolet and circular dichroism spectra to those of poly(A), and the enzymatic polymerization rates and thermal hyperchromicities of the two polymers are also very similar. However, the pKa of poly(fl2A) for protonation at N-1 is 2.9 compared to 5.9 for poly(A) under similar conditions. Poly(fl2A) forms a triple-stranded helix with poly(U) which has ultraviolet and cd spectra very reminiscent of poly(A) . 2 poly(U), but no conditions could be found which permitted the formation of a double helix. In the Escherichia coli ribosome system poly(fl2A) codes for the synthesis of polylysine, as does poly(A), although the rate and extent of incorporation were less in the former case. The role of basicity of adenine N-1 in these interactions is discussed.

The efficacy and tolerability of CV 205-502 (a nonergot dopaminergic drug) in macroprolactinoma patients and in prolactinoma patients intolerant to bromocriptine.

We studied the effect of CV 205-502 in 12 patients with macroprolactinomas and 8 patients with PRL-secreting tumors, who were selected because of previous repeatedly shown intolerance to bromocriptine in even small doses. We also investigated serum insulin-like growth factor-I (IGF-I) levels before and during CV 205-502 therapy. In 12 macroprolactinoma patients followed for 1 yr, 0.075-0.450 mg CV 205-502 lowered PRL levels by 91.2 +/- 5.4%. Only 3 of the patients had transient side-effects of nausea, dizziness, or fatigue. In eight patients with PRL-secreting tumors who were bromocriptine intolerant, CV 205-502 (0.075-0.300 mg daily) lowered PRL levels by 80.2 +/- 6.3%. Four of these patients showed transient side-effects (nausea, fatigue, and/or tachycardia). None of the patients discontinued therapy. There was a close correlation between pretreatment circulating PRL levels and tumor size, expressed in cubic millimeters. The decrease in pituitary tumor size after 52 weeks of CV 205-502 therapy (-74 +/- 6%) was also correlated with the decrease in PRL levels (P less than 0.01). In four patients with hypopituitarism, lowered IGF-I levels did not change during CV 205-502 therapy. However, in seven previously untreated patients with macroprolactinoma and normal CV 205-502 is a potent dopaminergic drug, which effectively controls PRL secretion and induces tumor shrinkage. At the doses used in our study, it causes only mild and transient side-effects in a minority of patients and can also be used to treat hyperprolactinemic patients who have shown intolerance to bromocriptine therapy.

Specific effect of CV 205-502, a potent nonergot dopamine agonist, during a combined anterior pituitary function test.

CV 205-502, an octahydrobenzo[g]quinoline, is a dopamine agonist compound that is not an ergot or ergoline derivative. To investigate the site of action of CV 205-502, three groups of five men were given single daily doses of CV 205-502 (0.04, 0.06, or 0.08 mg/day, doses that suppress plasma PRL by 60-80% for 24 h) for 5 days; on day 6 a combined anterior pituitary function test using iv administration of four hypothalamic releasing hormones (TRH, 200 micrograms; GHRH, 100 micrograms; CRH, 100 micrograms; LHRH, 100 micrograms) was performed. One month later the challenge tests were repeated to obtain control values. The following hormones were measured by RIA in plasma: TSH, ACTH, cortisol, PRL, GH, LH, FSH, and testosterone. With the exception of plasma PRL levels, basal and releasing hormone-stimulated values were similar after CV 205-502 administration and after the 1-month washout period. Basal plasma PRL was lower after CV 205-502 administration, and the response to TRH was attenuated by all three doses of CV 205-502 (the mean percent inhibition values were 76%, 93%, and 94%, respectively). All three doses of CV 205-502 were well tolerated, and another group of men well tolerated 0.1 mg daily. The results confirm that CV 205-502 is a potent dopamine agonist, which directly inhibits lactotropic cells but has no effect on other pituitary cell types.

Effects of the dopamine agonist CV 205-502 in human prolactinomas resistant to bromocriptine.

In 21 patients with prolactinomas resistant to bromocriptine, we studied the effects of CV 205-502 on PRL hypersecretion and tumor mass, as assessed by consecutive computed tomography examinations. Cell culture studies were performed in 9 of such tumors. In 11 patients (group I; 52%) with a mean baseline plasma PRL level of 468 +/- 160 micrograms/L (+/- SE), normal PRL values were achieved after 1-6 months of treatment with 0.1-0.5 mg/day CV 205-502. Tumor size was reduced by 25% or more in 6 of 11 patients. In group II (n = 10), PRL levels (948 +/- 538 micrograms/L at baseline) were reduced by 48% after treatment with 0.1 mg/day CV 205-502. A progressive increase in the daily dose up to 0.5 mg did not further improve the partial reduction of PRL. No reduction in tumor size was observed in this group. The cell culture studies showed that 1) a brief exposure to both drugs provoked PRL suppression lasting 3 days; 2) in group I, CV 205-502 suppressed PRL release more efficiently than bromocriptine, with a maximal inhibition of 72% at 10(-9) mol/L; and 3) in group II, CV 205-502 only achieved a 26% inhibition of PRL release at 10(-8) mol/L, superimposable to that of bromocriptine. These data indicate that in at least half of such adenomas resistant to bromocriptine, CV 205-502, probably due to its higher affinity toward the D2 dopamine receptor, can overcome such resistance.

Long term treatment with CV 205-502 in patients with prolactin-secreting pituitary macroadenomas.

CV 205-502, a new long-acting nonergot dopamine agonist, was given to 15 patients (6 women and 9 men) with PRL-secreting pituitary macroadenomas. The compound was administered in a single daily dose for a period of 6-12 months. The treatment resulted in normalization of plasma PRL levels (less than or equal to 20 micrograms/L) in 5 of 6 women at a mean dose of 135 micrograms (range, 75-300 micrograms) and in 6 of 9 men at a mean dose of 192 micrograms (range, 75-300 micrograms). Among patients for whom computed tomographic scans were available before and after at least 6 months of therapy, definite tumor shrinkage occurred in 6 of 7 patients. Libido was improved in 5 of 6 women and in 6 of 8 men, galactorrhea disappeared in all cases (3 women and 1 man) and menses resumed in 3 of 5 women. Plasma testosterone rose to normal levels in 3 of 6 men who were not receiving testosterone injections. The PRL response to TRH was blunted in 4 of 6 patients with normalized basal PRL. Serum total cholesterol was reduced by CV 205-502 treatment in women from 5.35 +/- 0.49 to 4.63 +/- 0.51 mmol/L (P = 0.031) and in men from 5.93 +/- 0.89 to 5.28 +/- 0.82 mmol/L (P = 0.045). Side-effects included mainly headache, nausea, and dizziness. One side-effect or more occurred transiently and with mild intensity in 14 patients. No patient discontinued the therapy because of side-effects. In conclusion, CV 205-502 appears to be a safe and valuable compound in the treatment of patients with PRL-secreting macroadenomas.

Carbocyclic analogues of xylofuranosylpurine nucleosides. Synthesis and antitumor activity.

(+/-)-4 alpha-Amino-2 alpha,3 beta-dihydroxy-1 alpha-cyclopentanemethanol (6), the carbocyclic analogue of xylofuranosylamine, was synthesized from the previously reported 4 alpha-acetamido-2 alpha,3 alpha-epoxycyclopentane-1 alpha-methanol. Amine 6 was converted to (+/-)-4 alpha-[(5-amino-6-chloro-4-pyrimidinyl)amino]-2 alpha,3 beta-dihydroxy-1 alpha-cyclopentanemethanol (7) by condensation with 5-amino-4,6-dichloropyrimidine. From 7, the carbocyclic analogues of xylofuranosyladenine and xylofuranosyl-8-azaadenine were prepared. In contrast to 9-beta-D-xylofuranosyladenine and its 8-aza analogue, the corresponding carbocyclic nucleosides were resistant to deamination by adenosine deaminase. The carbocyclic 8-aza derivative 10 exhibited significant in vivo antitumor activity which varied according to treatment schedule.

Carbocyclic puromycin: synthesis and inhibition of protein biosynthesis.

The carbocyclic analogue of puromycin was prepared by the coupling of N-(benzyloxycarbonyl)-p-methoxy-L-phenylalanine to the racemic aminonucleoside (+/-)-9-[3 beta-amino-2 beta-hydroxy-4 alpha-(hydroxymethyl)cyclopent-1 alpha-yl]-6-(dimethylamino)purine, followed by separation of the diastereomers and subsequent removal of the Cbz blocking group. Kinetic studies indicate that carbocyclic puromycin is an excellent substrate for the peptidyltransferase reaction with both prokaryotic and eukaryotic ribosomes. A comparison of carbocyclic puromycin with previously synthesized analogues indicate that the furanosyl ring oxygen and the hydroxymethyl group of puromycin do not contribute to ribosomal binding, but both moieties contribute to the rate of product formation from the enzyme-substrate complex. Carbocyclic puromycin was equal to puromycin when evaluated for cytotoxicity using P-388 mouse lymphoid leukemia cells in culture.

Synthesis and biological evaluation of 5'-sulfamoylated purinyl carbocyclic nucleosides.

The first series of 5'-sulfamoylated carbocyclic purinyl nucleosides was synthesized and tested for antitumor and antibacterial activities. The target compounds were formed by reacting the 2',3'-acetonide-protected carbocyclic nucleosides with sulfamoyl chloride, followed by deprotection. The agents were tested for cytotoxic activity against P388 mouse leukemia cells. Two compounds, 5'-sulfamoyl carbocyclic adenosine (2) and 5-sulfamoyl-8-aza carbocyclic adenosine (6) exhibited IC50 values as low as 62 and 15 nM, respectively. These analogs inhibited protein biosynthesis and slowed down DNA and RNA biosyntheses in the P388 cells. None of the target molecules were as potent against Escherichia coli as they were against the tumor cells. Also, in cell-free systems, agents 2 and 6 were more effective inhibitors of protein synthesis in rabbit reticulocyte lysate than in E. coli. These new carbocyclic derivatives appear to be somewhat selective for eukaryotic over prokaryotic cells in affecting translation.

Characterization of ergot and non-ergot serotonin antagonists by prolactin and growth hormone profiles during wakefulness and sleep.

In order to further clarify the involvement of serotonin in the control of secretion of pituitary lactogens, diurnal and sleep prolactin (PRL) and growth hormone (GH) profiles were investigated in healthy subjects treated with ergot and non-ergot serotonin antagonists. A group of 10 subjects received a single oral dose of 2 mg each of pizotifen, methysergide, and the dopaminergic drug bromocriptine as reference substance, in comparison with placebo. Blood was collected at hourly intervals for 6 h. Another group of 10 individuals received either a placebo, 2.5 mg bromocriptine (n = 6) twice daily for 4 days or pizotifen (n = 10) 0.5 mg 3 times daily for 12 days before undergoing EEG recording and blood sampling during night sleep. The diurnal plasma profiles of PRL and GH were not modified by pizotifen, a non-ergot drug. Methysergide and bromocriptine, two ergot derivatives, significantly (P < 0.01 and P < 0.001 respectively) suppressed the basal secretion of PRL throughout the trial and increased plasma GH significantly (P < 0.01). The sleep profile of PRL was not modified by pizotifen but there was a moderate reduction in GH reaching the level of significance (P < 0.02) between hours 1 and 2 of sleep. Bromocriptine suppressed completely PRL secretion throughout the entire sleep period and significantly (P < 0.05) prolonged the secretory profile of GH. The results indicate the presence in the ergot molecule of a dopaminergic moiety responsible for PRL inhibition and GH stimulation. This effect is independent of the serotonin active component of the drug.

The effect of two dopaminergic drugs on menstrual function and psychological state in hyperprolactinemia.

OBJECTIVE: To investigate the effect of dopaminergic drugs on the well being in hyperprolactinemic patients. DESIGN: A psychometric test for well being, the SCL-90, was applied at baseline and in the 24th week of a double-blind randomized prospective study comparing the effectiveness and safety of the new dopamine d2 agonist CV 205-502 with bromocriptine. SETTING: Outpatient department of a university clinic for obstetrics and gynecology. PATIENTS: Twenty-four women with hyperprolactinemia, 9 of whom had a prolactinoma. Twenty had been treated before, and 11 were known to react unfavorably to bromocriptine. RESULTS: The effectiveness of CV 205-502 was identical to bromocriptine: its tolerability appeared to be better, especially in the initial phase of treatment. At baseline, the mean scores of the SCL-90 were significantly elevated over the reference values. Sixteen patients had normal scores. The elevations were caused by 8 patients with scores in the range found in psychiatric disease (211 +/- 30 [SD] [CV 205-502] and 182 +/- 32 [bromocriptine]). They were depressed, anxious, and hostile. At 24 weeks, the patients treated with CV 205-502 scored better (130 +/- 23.5) in the SCL-90 than the patients treated with bromocriptine (149.5 +/- 20). CONCLUSION: The markedly increased well being in patients treated with CV 205-502 cannot be explained by its better tolerability and is probably caused by a specific central activity of CV 205-502. Further research into the antidepressive properties of this compound is warranted.

The effectiveness, safety, and tolerability of CV 205-502 in hyperprolactinemic women: a 12-month study.

Forty-one hyperprolactinemic women (prolactin [PRL] greater than 2,000 mU/L) were treated between 12 and 52 weeks with the new nonergot, long-acting dopamine agonist octahydrobenzo [g] quinoline (CV 205-502). The treatment resulted in normalization of PRL secretion in 71% of the women at a once-daily dose of less than or equal to 0.100 mg. All women responded with a significant decrease in serum PRL. Menstrual function normalized in all women except 1, whereas galactorrhea disappeared in all but 2 patients. During the observation period, four pregnancies were recorded with an additional three in the immediate posttreatment period. Until now, four healthy children have been born. Regarding tolerability, women with fair or poor response bromocriptine (Parlodel) in the past tolerated CV 205-502 better. Two women with no PRL decrease while on Parlodel responded with a decrease while on CV 205-502. All safety parameters remained normal while on treatment, and no significant changes were observed in blood pressure (supine and standing), pulse rate, or electrocardiogram (ECG) recordings. CV 205-502 therefore seems to be a valuable new compound in the management of patients with hyperprolactinemia.

Hormonal effects of CV 205-502, a novel octahydrobenzo [g] quinoline with potent dopamine agonist properties.

Following the success of ergot alkaloids and their synthetic derivatives in treating a variety of pathophysiological disturbances, efforts have been concentrated on the synthesis of new derivatives and partial structures with the aim of dissecting out a specifically dopaminomimetic pharmacophore. Accordingly CV 205-502, a structure which superposes the linear benzo [g] quinoline segment of apomorphine on the substituted pyrrolo [3,4- g] quinoline moiety of the ergolines was designed. This study was performed in normal young volunteers to investigate the effect of single oral doses of CV 205-502 on plasma prolactin levels and on other endocrine parameters (GH, LH, FSH, TSH and cortisol) as well as on tolerability. 10 volunteers participated in a dose-ranging study. They received single oral doses of 0.01 to 0.09 mg CV 205-502, in order to assess the prolactin suppressant action. 6 volunteers were given a dose of 0.06 mg CV 205-502 in order to determine the endocrine profile of the compound. The duration of action of 0.06 mg CV 205-502 was investigated in 6 subjects by measuring plasma PRL and GH levels for 48 h. The results show strong dose-dependent suppression of PRL appearing following doses between 0.04 and 0.09 mg of CV 205-502. PRL was markedly suppressed for more than 24h. and the peaks of the normal sleep profile were abolished. Intermittent transient GH increases occurred during the first 6 hours; sleep profiles were normal. Compared with placebo values, no changes were seen in the levels of any other hormone except TSH, which decreased. Tolerability was good and no drug attributable changes in safety measures occurred. This study demonstrates that CV 205-502 is a potent and long acting PRL suppressant compound and suggest that this novel octahydrobenzo [g] quinoline will prove to be a therapeutically useful dopaminomimetic compound.

WIN 64821, a novel neurokinin antagonist produced by an Aspergillus sp. I. Fermentation and isolation.

WIN 64821, a nonpeptide neurokinin antagonist, was isolated from a strain of Aspergillus sp., SC319. The compound was produced in different fermentation media with greatest yields observed when the culture was grown in a synthetic medium supplemented with L-tryptophan and L-phenylalanine. After 6 days fermentation, yields greater than 600 mg/liter were obtained. Two analogs of WIN 64821 were also identified in the culture extracts and subsequently tested for biological activity. WIN 64821 was the most potent compound isolated from this culture and exhibited activity as a substance P-binding inhibitor with submicromolar potency against the human neurokinin 1 receptor.

Isolation and structure elucidation of two new calpain inhibitors from Streptomyces griseus.

Two new peptides, a diketopiperazine of N-methyltyrosine (1) and a tetrapeptide containing N-methyltyrosine (2), were isolated from an actinomycete strain Streptomyces griseus. These compounds inhibit the enzyme calpain in the micromolar range and were characterized on the basis of spectroscopic analysis, amino acid analysis and sequencing. The structure of the tetrapeptide N-methyltyrosyl-N-methyltyrosyl-leucyl-alanine (2), was also confirmed by total synthesis.

High-order Cerenkov laser gain.

Cerenkov free-electron lasers have primarily operated on the fundamental guided mode of the dielectric waveguide. Higher-order generation would allow short wavelength emission in a relatively large scale resonator. In comparison to the fundamental mode, we find that gain on higher-order modes can be significant in a planar geometry. This analysis is presented with a discussion of practical limits.