Diagnosis of chronic pancreatitis. Is a gold standard necessary?

The question that serves as this article's title is rhetorical. Clinicians have diagnosed and managed chronic pancreatitis without a gold standard for decades and must continue to do so in the foreseeable future. Although clinicians have a much wider array of diagnostic tools available for the diagnosis of chronic pancreatitis, a single readily applied gold standard remains elusive. Diagnostic studies are rarely compared with a true gold standard--histopathology. Furthermore, even if a safe biopsy technique were available, it might fall short of a gold standard, given the patchy nature of early-stage chronic pancreatitis. Indeed, different stages of chronic pancreatitis require not only recognition of the different clinical presentations but also different levels of intensity of diagnostic testing to establish the diagnosis confidently. The diagnosis in most patients with chronic pancreatitis can be made confidently with a good clinical history and a limited number of currently available structural and functional tests. No single diagnostic study, functional or structural, suffices for all patients. It is also axiomatic that patients with intractable abdominal pain in whom early-stage chronic pancreatitis is suspected represent a challenge for clinicians partly because of this lack of a single, dependable gold standard. Perhaps we have reached the point at which further refinements of current tests of structure or function are not beneficial because increased sensitivity is countered by loss of specificity. We suggest that a new approach to developing a gold standard for the diagnosis of chronic pancreatitis is necessary. With advances in the understanding of the mediators of the inflammatory process, it may be possible to devise a test to assess the earliest events in this disease.

Molecular biology of the peptide hormone families.

The application of recombinant molecular biology has lead to remarkable advances in our understanding of the basic mechanisms of cell function in general and of the polarized GI endocrine cell in particular. This article focuses on some of the advances made towards determining the contribution of peptide hormone gene regulation to the regulation of physiological events in the GI tract. Application of these techniques to other subcellular processes involved in peptide hormone physiology such as subcellular trafficing in the regulated secretory pathway and post-translational processing have been equally impressive. For example, many of the key enzymes in the peptide hormone processing cascade have been cloned and are being studied at a molecular level. We have focused this article on the SS and gastrin peptides because of their known physiologic importance and interactions, and the depth of analysis accomplished to date. Studies using SS and gastrin as models have established principals that cover the spectrum of luminal regulation of gene activity to the identification of a single amino acid residue responsible for cAMP induction of SS gene expression. Many genes in the GI endocrine system have been cloned and the article by Dr. Habener (elsewhere in this issue) discusses progress made in understanding the complex regulation of the glucagon gene. We anticipate similar advances in studies of cholecystokinin, secretin, motilin, VIP, pancreatic polypeptide, and neuropeptide Y, whose genes have been cloned and initially characterized. Finally, as outlined in this article, the mechanisms of regulation of a specific gene often differ between sites of expression, even within the GI tract. Direct studies of the subcellular mechanisms regulating gene expression and other processes in GI endocrine cells await novel methods to maintain and propagate these cells. These studies will almost certainly involve new and creative uses of recombinant molecular biology.

Expression of the rat carboxypeptidase-E gene in neuroendocrine and nonneuroendocrine cell lines.

To identify cis-acting elements involved with the expression of the rat carboxypeptidase-E (CPE) gene, constructs containing various regions of the 5'-flanking region of the CPE gene attached to the luciferase reporter gene were transiently expressed in cell lines derived from pituitary (AtT-20 and GH4C1), liver (SK-HEP-1), and kidney (HEK293 and COS1). Regions of the CPE gene spanning the major transcription initiation site (-12 to 47) are sufficient for low levels of transcription. Activity is enhanced 3- to 15-fold by sequences present between -12 and -395 in all cell lines examined. Sequences between -395 and -3081 influenced transcription activity up to 5-fold in some, but not all, cell lines. There was no correlation between the transcription activities of the various constructs and the level of endogenous CPE mRNA in the cell lines, indicating that the tissue-specific elements responsible for the large variations in endogenous CPE mRNA levels are not present within -3081 to 47. The region between -395 and 45 was examined in greater detail using transient expression assays and DNase-I protection analysis. Transcription activity is enhanced in GH4C1 and HEK293 cells by sequence present between -12 and -84; this region contains a potential GC box, which binds factors present in GH4C1 nuclear extracts. Other regions between -340 and 80 that bind proteins in the GH4C1 nuclear extracts include the major transcription initiation site, which has homology to the initiator sequence; the pituitary-specific transcription initiation sites (-101 and -105); and sequences with homology to NF-1, Pan-1, simian virus-40 enhancer core, and AP-2-binding sites. Taken together, these results suggest that basal expression of the CPE gene from its major transcription initiation site, which does not contain an up-stream TATA box, is primarily under the control of an initiator-like element together with an upstream GC box.

The Drosophila sloppy paired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors.

The sloppy paired locus is involved in the establishment of the metameric body plan of the Drosophila embryo. We have cloned the sloppy paired locus by P-element-mediated enhancer detection. The locus is composed of two genes, slp1 and slp2, that are structurally and functionally related. They belong to a novel class of putative transcription factors containing a fork head domain that has also been found in mammalian hepatocyte transcription factors. The spatial expression patterns of the two transcripts are very similar, suggesting common regulation of the two genes. We recovered additional sloppy paired alleles by remobilization of an enhancer detector transposon. Genetic analysis suggests that both genes contribute to the segmentation phenotype that has characteristics of both, pair-rule and segment polarity genes, and that they interact functionally. The two genes appear to share an enhancer element situated upstream of slp1 that acts on both the proximal slp1 promoter and the distal slp2 promoter.

Structural characterization of the rat carboxypeptidase-E gene.

Several genomic clones encoding carboxypeptidase-E (CPE) have been isolated and partially sequenced. Southern blot analysis indicates that a single copy of this gene is present in the rat genome. The entire gene spans approximately 50 kilobases and consists of nine exons, each of which contains protein-coding regions. Only one of the exon/intron junctions of the rat CPE gene is present in a comparable position within the genes for carboxypeptidase-A and -B, both of which are only 17-21% homologous to CPE at the amino acid level. Nuclease protection analysis shows that alternative splicing of exons 7, 8, and 9 does not occur, indicating that the heterogeneity of the C-terminal region of CPE is due to posttranslational processing. Primer extension and nuclease protection analyses have identified the 5' end of CPE mRNA to be 105 nucleotides up-stream from the ATG used for protein translation. The 5' flanking region does not contain TATA and/or CCAAT boxes in the near vicinity of the transcription initiation site. The 5' flanking region is GC rich, containing 70% GC residues over nucleotides -1 to -150 (relative to the transcription initiation site). Putative consensus sites for the enhancer elements SP-1, NF-1, Pan-1, and AP-2 are present in the region from -60 to -330. Since this report describes the first neuropeptide-processing enzyme gene to be partially sequenced, it is not possible to compare the sequence with those of other processing enzymes that show similar tissue-specific expression. However, comparison of the CPE sequence with 5' flanking regions of other neuroendocrine genes has revealed a short region (12-18 nucleotides) that is highly conserved among CPE, neuropeptide-Y, oxytocin, insulin, and tyrosine hydroxylase genes.

P-element-mediated enhancer detection allows rapid identification of developmentally regulated genes and cell specific markers in Drosophila.

We have employed a new technique in Drosophila that allows in vivo detection of genomic regulatory elements using a beta-galactosidase reporter gene. A translational fusion of the reporter gene to the P-transposase gene, which is encoded by the P-transposon of Drosophila, places the expression of beta-galactosidase under the control of the weak P-transposase promoter. Flies carrying single insertions of this P-element construct at different locations in the Drosophila genome frequently stain for beta-galactosidase activity in a temporally and spatially restricted fashion in embryos, larvae and adult ovaries, reflecting the influence of nearby genomic regulatory elements on the P-transposase promoter. This technique is a powerful tool as it can be used to produce very many different cell markers and to isolate developmentally regulated genes in Drosophila. We discuss the implications of our results and the applications of the technique to further the study of Drosophila development.

P-element-mediated enhancer detection: a versatile method to study development in Drosophila.

We generated and characterized greater than 500 Drosophila strains that carry single copies of a novel P-element enhancer detector. In the majority of the strains, the beta-galactosidase reporter gene in the P-transposon responds to nearby transcriptional regulatory sequences in the genome. A remarkable diversity of spatially and temporally regulated staining patterns is observed in embryos carrying different insertions. We selected numerous strains as markers for different embryonic organs, tissues, and cells. Many of these strains should allow the study of complex developmental processes, such as nervous system development, which have not been convenient to analyze previously. Also, we present genetic evidence that some of the detected regulatory elements control nearby Drosophila genes. In light of our results, we discuss the diversity and complexity of cis-acting regulatory elements in the genome and the general applications of the enhancer detector method for the study of Drosophila development.

P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila.

We describe a new approach for identifying and studying genes involved in Drosophila development. Single copies of an enhancer detector transposon, P[1ArB], have been introduced into flies at many different genomic locations. The beta-galactosidase reporter gene in this construct is influenced by a wide range of genomic transcriptional regulatory elements in its vicinity. Our results suggest that a significant proportion of these regulatory sequences are control elements of nearby Drosophila genes. These genes need not be disrupted for their regulatory elements to be identified by P[1ArB]. The P[1ArB] transposon has been designed to facilitate both rapid cloning and deletion analysis of genomic sequences into which it inserts. Therefore, the enhancer detection system is an efficient method of screening for genes primarily on the basis of their expression pattern and then rapidly analyzing those of particular interest at the molecular and genetic levels.

Structural analysis of a distinct subtype of CCK receptor on human gastric smooth muscle tumors.

Human gastric smooth muscle tumors (leiomyosarcomas) have been shown to express cholecystokinin (CCK) binding sites that are functionally similar to physiologically important receptors present on their cells of origin. In this work, we have applied affinity-labeling techniques using 125I-D-Tyr-Gly-[Nle28,31]CCK-(26-33) to attempt to define the ligand-binding subunit of this receptor, and we have used the receptor antagonist L364,718 and deglycosylating enzymes to compare this molecule with well-defined CCK receptors on the classical peripheral targets (pancreas and gallbladder) of this hormone. To validate the use of 125I-D-Tyr-Gly-[Nle28,31]CCK-(26-33) for this tissue, we demonstrated that it bound to leiomyosarcoma membranes in a rapid, reversible, saturable, specific, and high-affinity manner (Kd = 0.8 nM). Although previous affinity labeling of this tissue with a CCK-33-based probe identified multiple bands, only one of those candidate proteins was predominantly labeled in the present work (Mr 100,000) by using a probe that is cross-linked through a site in greater proximity to the receptor-binding domain. Labeling was inhibited in a concentration-dependent manner by CCK-8 but not by structurally unrelated ligands. Although endo-beta-N-acetyl-glucosaminidase F digestion shifted this band by Mr 5,000, demonstrating that it was a glycoprotein, the deglycosylation product was very different from other CCK receptors studied. Also, unlike pancreatic and gallbladder CCK receptors, affinity labeling of this receptor was not affected by L364,718. These observations confirm that the gastric smooth muscle tumor CCK receptor represents a receptor subtype that is distinct from other peripheral CCK receptors, biochemically as well as functionally.

Effects of DNA heterologies on bacteriophage lambda recombination.

Previous studies of bacteriophage lambda recombination have provided indirect evidence that substantial sequence nonhomologies, such as insertions and deletions, may be included in regions of heteroduplex DNA. However, the direct products of heterology-containing heteroduplex DNA--heterozygous progeny phage--have not been observed. We have constructed a series of small insertion and deletion mutations in the cI gene to examine the possibility that small heterologies might be accommodated in heterozygous progeny phage. Genetic crosses were carried out between lambda cI- Oam29 and lambda cI+ Pam80 under replication-restricted conditions. Recombinant O+P+ progeny were selected on mutL hosts and tested for cI heterozygosity. Heterozygous recombinants were readily observed with crosses involving insertions of 4 to 19 base pairs (bp) in the cI gene. Thus, nonhomologies of at least 19 bp can be accommodated in regions of heteroduplex DNA during lambda recombination. In contrast, when a cI insertion or deletion mutation of 26 bp was present, few of the selected recombinants were heterozygous for cI. Results using a substitution mutation, involving a 26-bp deletion with a 22-bp insertion, suggest that the low recovery of cI heterozygotes containing heterologies of 26 bp or more is due to a failure to encapsulate DNA containing heterologies of 26 bp or more into viable phage particles.

Effects of DNA heterologies on bacteriophage lambda packaging.

We have examined the impact of DNA heterologies on the packaging of lambda DNA in vitro. Heterology-containing DNA molecules were constructed by denaturing and reannealing a mixture of DNA from cI+ phage and DNA front phage carrying small insertion or deletion mutations in the cI gene. We found that molecules with heterologies of up to 19 base pairs (bp) can be packaged as viable heterozygous phage with approximately the same efficiency as molecules with a base pair mismatch. In contrast, with a heterology of 26-bp heterozygous plaque formers are rare. In principle, the absence of cI heterozygotes among packaged phage may be due either to a failure to encapsulate the DNA or a failure to inject the packaged DNA on infection. Southern blot analysis of DNA isolated from packaged phage indicates that DNA harboring a 26-bp heterology is almost completely absent in packaged phage. Thus, an upper limit has been established for the size of heterology that can be accommodated by the packaging apparatus The size of the connector portal could be the basis for this limit.

Analysis of the carbohydrate composition of the pancreatic plasmalemmal glycoprotein affinity labeled by short probes for the cholecystokinin receptor.

Affinity labeling of the rat pancreatic cholecystokinin (CCK) receptor with decapeptide probes has identified an Mr = 85,000-95,000 protein, distinct from the Mr = 80,000 component previously labeled with 125I-Bolton Hunter-CCK-33. We have characterized the carbohydrate composition of this novel protein labeled with 125I-D-Tyr-Gly-[(Nle28,31)-CCK-26-33] and disuccinimidyl suberate by using chemical and enzymatic deglycosylation and lectin chromatography. The Mr = 85,000-95,000 component was demonstrated to be an N-linked sialoglycoprotein based on neuraminidase digestion to Mr = 75,000-85,000 and endo-beta-N-acetylglucosaminidase F (Endo F) digestion to Mr = 42,000. This was distinct from the Mr = 65,000 product of Endo F digestion of the protein labeled with 125I-Bolton Hunter-CCK-33. Lack of an effect of endo-beta-N-acetylglucosaminidase H demonstrated the absence of N-linked simple oligosaccharides, while products of chemical deglycosylation with hydrogen fluoride and endo-alpha-N-acetylgalactosaminidase supported the absence of O-linked carbohydrate. The presence of at least four oligosaccharide chains on the core protein was suggested by Endo F digestion of the Mr = 85,000-95,000 protein using limiting enzyme conditions. This glycoprotein was retained on wheat germ agglutininagarose and eluted by N,N',N"-triacetylchitotriose. Identification of the Mr = 85,000-95,000 component on the ectodomain of the plasmalemma of intact pancreatic acini confirmed this to be the fully processed form of the CCK-binding protein.

Establishment of a new short, protease-resistant, affinity labeling reagent for the cholecystokinin receptor.

Proteolytic degradation of radioligands is an important source of artifact in affinity labeling of receptor proteins. To complement our previous characterization of the pancreatic acinar cell cholecystokinin (CCK) receptor, we synthesized D-Tyr-Gly[(Nle28,31)CCK-26-33]. The amino terminal D-enantiomer of tyrosine provided a site for oxidative iodination, a free amino group for cross-linking, and rendered the peptide resistant to aminopeptidases. The decapeptide was oxidatively iodinated and purified by reverse-phase HPLC to 2,000 Ci/mmol, to yield a probe which was equal in potency and efficacy to CCK-8, and which bound to rat pancreatic membranes in a rapid, reversible, temperature-dependent, specific, saturable and high affinity manner. This probe was resistant to aminopeptidase degradation, and maintained its ability to bind to receptor after incubation with pancreatic membranes or dispersed cells. Affinity labeling of pancreatic membranes with this analogue identified an Mr = 85,000-95,000 molecule. This analogue offers several advantages over existing probes and should be useful for future studies of this and other CCK receptors.

Fatal soft-tissue infection complicating pulmonary arteriovenous fistulae.

A patient with pulmonary arteriovenous (AV) fistulae presented with an ultimately fatal anaerobic soft tissue infection. This represents the first report of such a complication, but the association of intracerebral anaerobic abscess with pulmonary AV fistulae suggests the possibility of a common pathophysiologic mechanism.

Affinity labeling of a novel cholecystokinin-binding protein in rat pancreatic plasmalemma using new short probes for the receptor.

Previous biochemical characterizations of the cholecystokinin (CCK) receptor have used the "long" probe 125I-Bolton-Hunter-CCK-33 since it was the only CCK analogue with high affinity and high specific radioactivity which possessed an amino group available for chemical cross-linking. These studies have consistently identified a major binding protein of approximately 81 kilodaltons and have identified several minor proteins which were obtained under different cross-linking conditions and in different laboratories. Because the receptor-binding region of CCK-33 (carboxyl-terminal heptapeptide) is so far removed from the radiolabel and from available amino groups (positions 1 and 11), this probe carries potential for proteolytic cleavage of label from receptor and for labeling "near neighbors" instead of the binding site. We therefore designed two "short" probes for the CCK receptor. 125I-Bolton-Hunter-Lys-Gly-CCK-8 has an epsilon-amino group available for cross-linking. 125I-Tyr-[Thr28,Nle31]CCK-25-33 has an alpha-amino group for cross-linking and has the major advantage of being labeled by oxidative means, unique for CCK derivatives. Both radioiodinated decapeptides were purified by reverse-phase high pressure liquid chromatography to yield specific radioactivity of 2,000 Ci/mmol; demonstrated saturable, specific, and high affinity binding to rat pancreatic plasma membranes; and retained full biological activity to stimulate amylase secretion. Using a variety of cross-linking methods, these probes each identified the same Mr = 85,000-95,000 protein in rat pancreatic plasmalemma, and CCK-8 competed for this labeling in a concentration-dependent manner (IC50 = 1 nM). No change in apparent mobility of this band was observed under reducing or nonreducing conditions, suggesting lack of covalent attachment to other subunits. The Mr = 85,000-95,000 species migrated differently on sodium dodecyl sulfate gels than any of the components previously identified using 125I-Bolton-Hunter-CCK-33, confirming the novel nature of this binding protein. These short probes should be very useful for further characterization of CCK receptors on this and other tissues.

Biochemical characterization of the pancreatic cholecystokinin receptor using monofunctional photoactivatable probes.

Receptor characterization by affinity labeling can be enhanced by taking multiple complementary approaches. To extend our observations on the subunit structure of the rat pancreatic cholecystokinin (CCK) receptor (made using bifunctional cross-linking reagents), we synthesized two monofunctional photoactivatable receptor probes. CCK-8 was acylated with the iodinated aryl azide derivatives, methyl-3-azido-4-hydroxy-5-[125I]iodobenzimidate and N-[4-(4'-azido-3'-[125I]iodophenylazo)benzoyl]-3-aminopropionyl-N- oxy- succinimide. The products were purified by reverse-phase HPLC to a specific radioactivity of 2,000 Ci/mmol. Both analogs demonstrated saturable and specific binding to rat pancreatic plasma membranes. Photoaffinity labeling of pancreatic membranes with these monofunctional probes identified an Mr 85,000-95,000 protein that was not part of a larger disulfide-linked complex. High affinity for CCK was demonstrated by the concentration-dependent inhibition of labeling observed with competing CCK-8 (IC50 = 1 nM). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) this protein co-migrates with the major component we identified using a series of cross-linkable, iodinated decapeptide analogs of CCK, and is different from the major protein labeled using 125I-Bolton Hunter-CCK-33. Thus, these results support the presence of an Mr 85,000-95,000 subunit in the pancreatic CCK receptor, while the small size of these photoaffinity probes and their monovalency suggest that this subunit may contain or be spatially apposed to the active binding site. These probes should be very useful in the further characterization of this and other receptors for this hormone.

Preparation and characterization of a new cholecystokinin receptor probe that can be oxidatively radioiodinated.

Oxidative iodination is the simplest, most efficient, and least expensive method for radiolabeling peptide hormones. It has not been applied to cholecystokinin (CCK), however, because this peptide lacks an unsubstituted tyrosine residue and oxidation abolishes its biologic activity. We report the synthesis, purification, and characterization of the first derivative of CCK that can be radioiodinated with an oxidative method while maintaining full biologic activity and receptor binding affinity. Boc-Tyr-[(Thr28, Nle31)CCK-25-33] was synthesized, iodinated using a solid-phase oxidant, and purified on reverse-phase high-pressure liquid chromatography to a specific activity of 2125 Ci/mmol. Both native and iodinated Boc-Tyr-[(Thr28, Nle31)CCK-25-33] exhibited efficacy and potency for stimulation of in vitro pancreatic enzyme secretion that were identical to CCK-8. Binding of Boc-125I-Tyr-[(Thr28, Nle31)CCK-25-33] to rat pancreatic plasma membranes was rapid, reversible, temperature-dependent, saturable, and specific. The abilities of various molecular forms of CCK to compete for this binding paralleled their potencies for stimulation of pancreatic secretion (Kd: CCK-8, 0.8 nM; CCK-33, 3 nM; CCK-8DS, 1 microM; CCK-4, 50 microM), whereas structurally unrelated pancreatic ligands (1 microM) demonstrated no significant competition. These findings suggest that Boc-125I-Tyr-[(Thr28, Nle31)CCK-25-33] interacts with the same high-affinity pancreatic receptor as the CCK receptor probes previously reported, i.e., the Bolton-Hunter-labeled CCK-33 and CCK-8. This probe also possesses the major advantages of simplicity, efficiency, and cost of the labeling procedure, and the stability and relative oxidation-resistance of the product. Boc-125I-Tyr-[(Thr28, Nle31)CCK-25-33] will be useful for the characterization of binding interactions between CCK and its target tissues.

Rat phenol sulfotransferase. Assay procedure, developmental changes, and glucocorticoid regulation.

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic monoamines and phenolic drugs. It has been difficult to measure PST activity in tissue homogenates accurately because of the presence of potent endogenous PST inhibitors. Optimal conditions were determined for the assay of rat PST in very dilute tissue homogenates. These conditions negated the effects of endogenous enzyme inhibitors. Apparent Km values for 3-methoxy-4-hydroxyphenylglycol, the sulfate acceptor substrate used, were 0.15, 0.14, and 0.02 mM for liver, kidney, and brain homogenates respectively. Apparent Km values in the same tissues for 3'phosphoadenosine-5'phosphosulfate, the sulfate donor, were 0.11, 0.07, and 0.07 microM respectively. Rat PST activity expressed per mg protein increased 6.3-fold in the liver, 6.6-fold in the brain, and did not change in the kidney between birth and 10 weeks of age. There was a 5-fold increase in kidney PST activity in both adrenalectomized and sham-operated Sprague-Dawley rats after treatment with dexamethasone (7 mumoles/kg daily for 3 days). Brain enzyme activity was unchanged and liver PST activity increased only 41% during 72 hr of daily treatment with dexamethasone. Basal enzyme activities in all three tissues were no different in adrenalectomized and sham-operated animals. The increase in rat kidney PST activity in response to dexamethasone was dose dependent, and treatment of animals with cycloheximide, a protein synthesis inhibitor, blocked the elevation of kidney PST activity after dexamethasone. Treatment of eight inbred and two outbred rats strains with dexamethasone resulted in striking increases in renal PST, smaller increases in liver PST, and no changes in brain enzyme activity in all ten strains.