Effect of sub-chronic treatment with Jarsin (extract of St John's wort, Hypericum perforatum) at two dose levels on evening salivary melatonin and cortisol concentrations in healthy male volunteers.

OBJECTIVE: The aim of the present study was to measure the effect of two doses of extracts from Hypericum perforatum (HP), Jarsin, on evening salivary cortisol and NA-mediated melatonin in healthy male volunteers. METHODS: Twenty healthy male volunteers were randomly given a low or high dose of Jarsin for 7 days. Saliva samples for cortisol and melatonin, and overnight urine samples were collected for cortisol and 6-sulfatoxymelatonin and measured by specific radioimmunoassays. RESULTS: Treatment significantly increased salivary cortisol throughout the whole collection period in the low dose group but had no discernable effect in the high dose group. Salivary melatonin was not increased in either dose group following treatment. CONCLUSION: Salivary cortisol was enhanced in the low dose group only and melatonin was not affected by either treatment. We suggest that HP may enhance salivary cortisol via a U-shaped dose-response relationship and that this may be mediated through a 5-HT2 mechanism.

Continuous affinity-based selection: rapid screening and simultaneous amplification of bacterial surface-display libraries.

A new method for continuous biopanning has been developed. We have combined the power of affinity chromatography with the fecundity of bacteria in a unique process that mimics clonal selection. Mixed populations of bacteria were applied to a fermenter containing the immobilized ligand of interest. Bacteria retained in this affinity fermenter were allowed to grow under continuous washout conditions, such that weakly bound organisms were selectively lost. Those initially rare founder bacteria expressing a receptor for the immobilized ligand (R+ve) were thus enriched and amplified simultaneously. From an initial culture containing 1 x 10(10) R-ve cells spiked with fewer than 30 R+ve bacteria (<1 in 10(8)), final ratios of R+ve/R-ve bacteria as high as 1 in 12 were observed, representing an enrichment factor of 55 million-fold. This technology has considerable potential for rapid screening of bacterial surface-display libraries and in facilitating directed-evolution studies.

Neuroendocrine evidence for dopaminergic actions of hypericum extract (LI 160) in healthy volunteers.

BACKGROUND: We studied the effect of a single dose of a formulation of a methanolic extract of Hypericum perforatum (HP), also known as St. John's wort, on plasma concentrations of growth hormone (GH), prolactin (PRL), and cortisol (CORT) in 12 healthy male volunteers. METHODS: Subjects received 9 tablets of the finished product Jarsin 300 and placebo in a double-blind, balanced-order, cross-over design. RESULTS: Following HP relative to placebo, there was a significant increase in plasma GH and a significant decrease in plasma PRL. Plasma CORT levels were unchanged. CONCLUSIONS: Taken together with data from animal experimental studies, the findings suggest that this dose of HP may increase some aspects of brain dopamine function in humans.

Paroxetine treatment and the prolactin response to sumatriptan.

We studied the effect of the selective serotonin re-uptake inhibitor (SSRI), paroxetine (20 mg daily for 16 days) on the neuroendocrine, cardiovascular, thermic and subjective responses to the 5-HT1D receptor agonist, sumatriptan (6 mg, SC). Compared to placebo injection, sumatriptan lowered plasma prolactin and oral temperature and increased diastolic blood pressure. While paroxetine increased baseline prolactin concentration, it had no effect on any of the responses to sumatriptan. In addition, paroxetine did not alter concentrations of sumatriptan in plasma. No adverse reactions resulted from the combination of sumatriptan and paroxetine. Our findings suggest that combined treatment with sumatriptan and paroxetine in the doses used in this study is not necessarily contra-indicated. In addition, short-term SSRI treatment may not desensitise 5-HT1D autoreceptors in humans.

Recent developments in heterologous protein production in Escherichia coli.

During the past three to four years, remarkable progress has been made in our understanding of protein folding, protein translocation across biological membranes, and the role of molecular chaperones in these processes. In conjunction with recent developments in Escherichia coli expression systems, this understanding has led to an improved capability to accumulate proteins in a soluble form, secrete proteins from the cell cytoplasm, accumulate proteins in the cytoplasmic membrane, and direct proteins to the outer membrane of the cell for surface display. These advances suggest that E. coli should now be considered seriously for applications that, only a few years ago, would have been thought beyond the scope of this organism.

Effects of hydrocortisone on brain 5-HT function and sleep.

The effects of hydrocortisone administration (20 mg, orally, twice daily) on the sensitivity of brain 5-HT1A receptors in healthy volunteers were studied using a buspirone challenge paradigm. The effects of hydrocortisone administration on sleep architecture were also studied. Hydrocortisone treatment significantly attenuated the hypothermic and cortisol responses to buspirone; however, the prolactin and growth hormone responses were unchanged. Hydrocortisone also decreased the amount of rapid eye movement sleep (REM). The ability of hydrocortisone to attenuate 5-HT1A receptor mediated hypothermia and decrease REM sleep is shared by certain antidepressant treatments and may be related to the effects of corticosteroids on mood.

Purification and characterization of active and stable recombinant plasminogen-activator inhibitor accumulated at high levels in Escherichia coli.

Plasminogen-activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator, is an unusual member of the serine protease inhibitor (serpin) superfamily in that it spontaneously converts to a latent form lacking activity. This latent form can be reactivated by denaturation and refolding, but the activation is usually incomplete and often leads to aggregation of the protein. In this study we have developed a high-level expression system that leads to the accumulation of PAI-1 at 30-50% total microbial protein. We have developed a single-step purification protocol which can be completed in a few hours, yielding approximately 20 mg purified recombinant PAI-1/litre culture. The purified PAI-1 was 80-100% active and was stable upon incubation at 37 degrees C with a half-life of approximately 48 h. At 20 degrees C, PAI-1 activity was stable for a week and at 4 degrees C it retained its activity completely for up to two months. Freezing caused significant loss of activity. The stability of PAI-1 activity was found to be dependent on pH and ionic strength, being most stable at pH 5.6 and at an ionic strength of 1 M salt. We show that by a combination of high-level expression and rapid purification under optimum conditions, it is possible to produce active and stable PAI-1 in high yield.

Neuroendocrine effects of sumatriptan.

The neuroendocrine effects of the 5-HT receptor agonist, sumatriptan (6 mg subcutaneously), were studied in 11 healthy male subjects using a placebo-controlled, cross-over design. Compared to placebo, sumatriptan significantly lowered levels of plasma prolactin but increased those of plasma growth hormone. There was no effect on plasma cortisol concentrations. The neuroendocrine effects of sumatriptan differ from those of previously described 5-HT-receptor agonists, and may be a consequence of selective activation of 5-HT1D or 5-HT1B receptors. However, the present data cannot exclude the possibility that the neuroendocrine changes reflect nonspecific stress responses or changes in pituitary blood flow.

Effect of lofepramine on 5-HT function and sleep.

We studied the effect of the tricyclic antidepressant lofepramine (140-210 mg daily for 16 days) on 5-hydroxytryptamine 1A (5-HT1A) receptor sensitivity in healthy volunteers, using a buspirone neuroendocrine challenge paradigm (30 mg orally). We also studied the effect of lofepramine on platelet 5-HT content and sleep architecture. Lofepramine treatment did not alter the hypothermic, endocrine or amnesic effects of buspirone but significantly lowered platelet 5-HT content and decreased rapid eye movement sleep. Our findings suggest that at clinically used doses, lofepramine inhibits the uptake of 5-HT and produces changes in sleep architecture characteristic of tricyclic antidepressants. However, lofepramine does not appear to alter the sensitivity of 5-HT1A receptors.

Pharmacological characterization of cloned human NK-2 (neurokinin A) receptor expressed in a baculovirus/Sf-21 insect cell system.

Using the novel ligand [4,5-3H-Leu9]neurokinin A ([4,5-3H-Leu9] NKA) in a receptor binding assay, we characterized the pharmacology of a cloned neurokinin NK-2 receptor from human lung (hNK-2R), expressed in baculovirus-infected Sf-21 insect cells. Functional hNK-2R cDNA clones were isolated from human lung using a polymerase chain reaction-based methodology. hNK-2R was cloned into pAcYM1, a vector designed to couple expression to the polyhedrin promoter, and the recombinant baculovirus was isolated and used to infect Sf-21 insect cells. hNK-2R expression levels were monitored by Northern blots and 125-I-NKA binding assays. Isolates demonstrating the highest specific binding of 125-I-NKA were grown and membrane preparations from high-speed centrifugations were prepared from both hNK-2R-expressing and wild-type virus-infected cells. [3H]NKA bound in a protein-dependent, saturable (Bmax = 820 +/- 167 fmol/mg of protein), and highly specific (88 +/- 5%) manner to hNK-2R, but not to membranes from cells infected with wild-type virus (14 +/- 8%, 7 +/- 10 fmol/mg of protein). [3H]NKA binding was rapid (k1 = 0.085 nM-1 x min-1) and reversible (t1/2 = 4-5 min). Equilibrium binding experiments demonstrated binding to a mixture of receptors in high and low affinity states (Kd1 = 2.28 +/- 0.26 nM and Kd2 = 266 +/- 91 nM). Binding to hNK-2R was greatly enhanced (400%-600%) by Ca2+ and Mg2+ (EC50 values of 30 microM and 140 microM, respectively), whereas guanosine-5'-O-(3'-thio)triphosphate and guanosine-5'-(beta, gamma-imido)diphosphate were inhibitory. Competition experiments with agonists also demonstrated binding to high and low affinity states, with the following order of potency: NKA > [Nle10]NKA(4-10) > [beta-Ala8]NKA(4-10) >> substance P; Senktide and the NK-1 antagonist CP96,345 (10 microM) did not inhibit binding. Inhibition of binding by selective NK-2 antagonists was consistent with a single affinity state and demonstrated the following order of affinity: SR48,968 >> MEN10,376 > L659,877 > R396. These data suggest that infection of Sf-21 cells with baculovirus expression vector harboring the cDNA of hNK-2R resulted in expression of high affinity, G protein-coupled hNK-2R, with pharmacological selectivity compatible with the NK-2A receptor subtype.

Neuroendocrine and temperature effects of nefazodone in healthy volunteers.

The effects of a novel antidepressant, nefazodone (50 mg, 100 mg, and 200 mg orally) on neuroendocrine function and temperature were assessed using a single-blind, crossover design in eight healthy male volunteers. Nefazodone significantly increased plasma levels of prolactin (PRL) and raised oral temperature. There was also a trend towards an increase in plasma cortisol. These results are consistent with an acute facilitatory effect of some aspects of 5-HT neurotransmission, perhaps mediated through nefazodone's metabolism to its major metabolite, m-chlorophenylpiperazine (mCPP).

Functionally important conserved amino-acids in interferon-alpha 2 identified with analogues produced from synthetic genes.

A gene was chemically synthesised and expressed in Escherichia coli to produce [Ala30,32,33]IFN-alpha 2, an analogue of human alpha 2-interferon (IFN-alpha 2) which is devoid of activity on human cells. Eight additional analogues provided single changes in IFN-alpha 2 at each of these three conserved positions. No one residue is essential for activity, but both antiviral and anti-proliferative activity are particularly sensitive to changes in the side-chain of Arg33.

The isolation and characterization of three types of vitamin B6 auxotrophs of Escherichia coli K12.

Approximately 500 vitamin B6 auxotrophs were isolated from 18 independent cultures of Escherichia coli strain CR63. None grew in minimal medium supplemented with 2'-hydroxypyridoxine. Eighteen auxotrophs which had arisen independently were further characterized. All of them were defective in vitamin B6 synthesis rather than in an aminotransferase involved in vitamin B6 utilization. Two different phenotypes were recognized: 'Oxidase' mutants which grew only when supplied with pyridoxal or pyridoxal 5'-phosphate and 'Pre Pn' mutants which would also grow with pyridoxine or pyridoxine phosphate. "Oxidase' mutants were confined to a single linkage group, but data from interrupted mating experiments established that 'Pre Pn' mutants fall into two linkage groups which are possibly identical to pdxA and pdxB. All mutations in the in the pdxA region were allelic rather than located in two closely linked genes.

Synthesis of vitamin B6 by a mutant of Escherichia coli K12 and the action of 4'-deoxypyridoxine.

Mutants of Escherichia coli K12 blocked in the oxidation of pyridoxine 5'-phosphate ('Oxidase' mutants) excreted pyridoxine at an initial rate of 19 pmol h-1 (10(8) bacteria)-1, i.e.0.6 nmol h-1 (mg dry wt)-1, when starved for pyridoxal. Glycolaldehyde, L-phosphoserine, DL-serine and, to a lesser extent, L-leucine stimulated the rate of pyridoxine excretion, but there was no significant stimulation by 2'-hydroxypyridoxine. 4'-Deoxypyridoxine inhibited or stimulated growth of the "Oxidase' mutant, depending on the relative concentrations of added pyridoxal and 4'-deoxypyridoxine. It was concluded that stimulation of growth by 4'-deoxypyridoxine was due to its conversion to pyridoxal.