Commercial support and the quandary of continuing medical education.

Commercial funding is a major source of financial support for the continued development and conduct of continuing education activities for physicians. However, we are again beginning to see challenges about who really controls these activities. Questions are being raised about the ethical behavior of physicians, sponsors, and grantors as they relate to compliance with the Standards for Commercial Support and other related documents. The Accreditation Council for Continuing Medical Education, grantors, providers, and physicians are challenged to help each other to comply with the intent, as well as the letter, of the documents. Without an immediate change, the cynicism of the public and the press may begin to drive outside forces to put into place and enforce policies that would be to the detriment of all interested parties.

Identification and expression of the Drosophila adipokinetic hormone gene.

Drosophila adipokinetic hormone (DAKH) is an eight amino acid member of a large arthropod neuropeptide family. The gene encoding the peptide precursor has been identified and sequenced providing an inferred precursor structure of 79 amino acids including a 46 amino acid carboxy-terminal fragment of unknown function. In situ hybridization identifies sites of DAKH synthesis towards the base of the third larval instar ring gland. Like other RPCH (red pigment concentrating hormone)/AKH family peptides, DAKH can act as a cardioaccelerator at least in prepupae. Peptide levels measured in wildtype and mutant flies possessing one or three copies of the DAKH gene suggest that the amount of neuropeptide per fly is tightly regulated.

The closely related neuropeptide genes encoding adipokinetic hormones I and II have very different 5'-flanking regions.

Adipokinetic hormones I and II are 10- and 8-amino-acid grasshopper neuropeptides that are derived from 63- and 61-amino-acid peptide precursors, respectively. Each precursor is encoded by a separate gene consisting of three very small exons separated by two large introns. The identical exon structure of the two genes suggests that they evolved through duplication of a common ancestral gene. Despite the precise conservation of exon structure and the similarity of the coding sequences, the two genes have very different 5'-flanking regions, suggesting that they are differentially regulated. For example, sequences similar to the vertebrate insulin enhancer elements NIR and FAR are present upstream of the promoter region of the adipokinetic hormone II gene, but not in the adipokinetic hormone I gene. Both of these insect genes contain short interspersed repetitive DNA sequences in their introns that may have facilitated a gene duplication event.

The fruitfly Drosophila melanogaster contains a novel charged adipokinetic-hormone-family peptide.

A member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of arthropod neuropeptides was identified in the fruitfly Drosophila melanogaster, and its structure was determined by automated Edman degradation and m.s. using fast-atom-bombardment ionization and a tandem hybrid instrument capable of high sensitivity. The sequence of this peptide, which we call 'DAKH', is pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 (where pGlu is pyroglutamic acid and Trp-NH2 is tryptophan carboxyamide). H.p.l.c. analyses of extracts of the three body segments revealed that more than 80% of the peptide is contained in the thorax. Although DAKH is typical of family members in its general structure and distribution in the animal, it is unique in containing a residue which is charged under physiological conditions. The evolutionary significance of this change is considered.

The structurally similar neuropeptides adipokinetic hormone I and II are derived from similar, very small mRNAs.

The grasshopper neuropeptides adipokinetic hormone (AKH) I and II were among the first of an extensive family of structurally similar arthropod hormones and neuroregulators to be isolated and sequenced. This paper reports the cloning of cDNAs derived from the unusually small mRNAs (550 bases) which code for the precursors of AKH I and II from Schistocerca nitans. Sequence analysis of the cDNAs indicates that AKH I and II are derived from small precursor proteins (63 and 61 amino acids) which are 55% identical in amino acid sequence. Each contains a 22-amino acid hydrophobic leader sequence followed by the AKH I or II sequence and an additional 28-amino acid carboxyl-terminal peptide of unknown function. Significant homology at the nucleic acid level (64% identity) is confined to the coding region of the mRNA sequences. Preliminary DNA blot analyses suggest that a single gene codes for each, and that the genes for AKH I and II may be linked. Genomic blots from various tissues fail to suggest that the high level of expression of AKH in the corpora cardiaca is due to tissue specific gene amplification.

Sequence analyses of adipokinetic hormones II from corpora cardiaca of Schistocerca nitans, Schistocerca gregaria, and Locusta migratoria by fast atom bombardment mass spectrometry.

Structures of the second adipokinetic hormones (AKH II's) from three locust species have been assigned by fast atom bombardment mass spectrometry. The AKH II hormone is identical in two Schistocerca species, S. nitans and S. gregaria, but is different in Locusta migratoria. Both AKH II's are related to red pigment-concentrating hormone (RPCH) from prawns, Schistocerca AKH II being [Thr6]-RPCH and Locusta AKH II being [Ala6]-RPCH. Schistocerca AKH II is also bioactive in Locusta individuals.

Structures of two cockroach neuropeptides assigned by fast atom bombardment mass spectrometry.

Amino acid sequences have been assigned to two cockroach neuropeptides (greater than Glu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, M I, and greater than Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2, M II) by application of fast atom bombardment mass spectrometry, including high resolution and linked scan (metastable) studies. The peptides show considerable homology with two other invertebrate neuropeptides, adipokinetic hormone (AKH, from a locust) and red pigment concentrating hormone (RPCH, from a prawn), whose fast atom bombardment spectra were also studied. M I and M II are thus members of a family of structurally-related invertebrate neuropeptides.

Des-tyrosine-gamma-endorphin administration in chronic schizophrenics. A preliminary report.

The beta-lipotrophin fragment des-tyrosine-gamma-endorphin (DT gamma E) has been reported to have antipsychotic properties. We administered the compound without other psychoactive drugs to a subpopulation of schizophrenic subjects. Male patients with chronic psychotic illness and previous long-term neuroleptic therapy were given DT gamma E at a similar dose and duration of treatment that have been reported to be effective. No improvement in psychotic symptoms occurred; plasma prolactin level, a parameter characteristically altered by neuroleptic treatment, did not change. The beneficial effects of DT gamma E in schizophrenia may be specific to a diagnostic category, may be dependent on past pharmacologic treatment, or may occur only in combination with other drugs.

Treatment of Schizophrenia with ergot derivatives.

Seven neuroleptic-free schizophrenic patients received bromocriptine and eight schizophrenic patients received CF 25-397, both ergot derivatives with dopamine agonist activity. Psychosis failed to improve in response to either drug at the relatively low doses administered. Unlike the antipsychotic property of apomorphine at low dose levels, neither of these ergot drugs improved schizophrenic symptomatology.

Schizophrenic symptoms improve with apomorphine.

Eighteen chronic schizophrenic patients received subcutaneous doses of apomorphine, a dopamine receptor agonist, and of placebo in separate trials. A significant improvement in psychotic symptoms occurred after apomorphine compared to placebo. The results are interpreted as a consequence of presynaptic dopamine receptor activation by apomorphine with a subsequent decrease in dopamine-mediated neural transmission.

Aspartate transcarbamylase of Escherichia coli. Heterogeneity of binding sites for carbamyl phosphate and fluorinated analogs of carbamyl phosphate.

Some preparations of both native aspartate transcarbamylase from Escherichia coli and catalytic subunit have fewer tight binding sites per oligomer for carbamyl-P than the number of catalytic peptide chains. In contrast, the number of sites for the tight-binding inhibitor N-(phosphonacetyl)-L-aspartate does equal the number of catalytic chains in each case. Binding of the labile carbamyl-P was determined using rapid gel filtration, with conversion to stable carbamyl-L-aspartate during collection. Native enzyme (six catalytic chains) obtained from cells grown under the conditions of J.C. Gerhart and H. Holoubek (J. Biol. Chem. (1967) 242, 2886-2892) has 5.4 tight sites for carbamyl-P at pH 8.0 (KD = 9.9 muM), whereas native enzyme from cells grown with higher concentrations of glucose, uracil, and histidine (to yield more enzyme per unit volume of culture) has only 1.9 tight sites at pH 8.0 (KD = 4.6 muM) and only 2.3 tight sites at pH 7.0 (KD = 2.6 muM). At pH 8.0, catalytic subunit (three catalytic chains) obtained from the former native enzyme has 2.2 tight sites for carbamyl-P (KD = 2.4 muM) and the number of sites is 2.3 in the presence of 35 mM succinate, whereas catalytic subunit obtained from the latter native enzyme has 1.8 tight sites (KD = 3.6 muM) in the absence of succinate and 2.3 tight sites in its presence. The number of tight binding sites is also less than the number of subunit peptide chains in 19F nuclear magnetic resonance experiments performed with catalytic subunit and two fluorinated analogs of carbamyl-P at comparable concentrations of analogs and active sites. A model is proposed in which incomplete removal of formylmethionine from the NH2 termini of the enzyme under conditions of extreme depression affects affinity for ligands.

Evidence from 13C NMR for protonation of carbamyl-P and N-(phosphonacetyl)-L-aspartate in the active site of aspartate transcarbamylase.

Nuclear magnetic resonance has been used to study the binding of [13C]carbamyl-P (90% enriched) to the catalytic subunit of Escherichia coli aspartate transcarbamylase. Upon forming a binary complex, there is a small change in the chemical shift of the carbonyl carbon resonance, 2 Hz upfield at pH 7.0, indicating that the environments of the carbonyl group in the active site and in water are similar. When succinate, an analog of L-aspartate, is added to form a ternary complex, there is a large downfield change in the chemical shift for carbamyl-P, consistent with interaction between the carbonyl group and a proton donor of the enzyme. The change might also be caused by a ring current froma nearby aromatic amino acid residue. From the pH dependence of this downfield change and from the effects of L-aspartate analogs other than succinate, the form of the enzyme involved is proposed to be an isomerized ternary complex, previously observed in temperature jump and proton NMR studies. The downfield change to chemical shift for carbamyl-P bound to the isomerized complex is 17.7 +/- 1.0 Hz. Using this value, the relative ability of other four-carbon dicarboxylic acids to form isomerized ternary complexes with the enzyme and carbamyl-P has been evaluated quantitatively. The 13C peak for the transition state analog N-(phosphonacetyl)-L-aspartate (PALA), 90% enriched specifically at the amide carbonyl group, is shifted 20 Hz downfield of the peak for free PALA upon binding to the catalytic subunit at pH 7.0. In contrast, the peak for [1-13C] phosphonaceatmide shifts upfield by about 6 Hz upon binding. Since PALA induces isomerization of the enzyme and phosphonacetamide does not, these data provide further evidence consistent with protonation of the carbonyl group only upon isomerization. The degrees of protonation is strong acids of the carbonyl groups of PALA, phosphonacetamide and urethan (a model for the labile carbamyl-P) have been determined, as have the chemical shifts for these compounds upon full protonation. From these data it is calculated that the amide carbonyl groups of carbamyl-P and PALA might be protonated to a maximum of about 20% in the isomerized complexes at pH 7.0. The change in conformation of the enzyme-carbamyl-P complex upon binding L-aspartate, previously proposed to aid catalysis by compressing the two substrates together in the active site, may be accompanied by polarization of the C=O bond, making this ordinarily unreactive group a much better electrophile. A keto analog of PALA, 4,5-dicarboxy-2-ketopentyl phosphonate, also binds tightly to the catalytic subunit and induces a very similar conformational change, whereas an alcohol analog, 4,5-dicarboxy-2-hydroxypentyl phosphonate, does not bind tightly, indicating the critical importance of an unhindered carbonyl group with trigonal geometry.