Binding sites for hepatocyte nuclear factor 3 beta or 3 gamma and pancreas transcription factor 1 are required for efficient expression of the gene encoding pancreatic alpha-amylase.

Efficient expression of genes under the control of alpha-amylase 2 5'-flanking sequences in exocrine pancreatic cells requires, in addition to the pancreas transcription factor 1 binding site (M. Cockell, B.J. Stevenson, M. Strubin, O. Hagenbüchle, and P. K. Wellauer, Mol. Cell. Biol. 9:2464-2476, 1989), another cis-acting element at positions -60 to -86. This DNA element, which contains an AT-rich core, site for nuclear proteins present not only in the pancreas but also in other tissues and cell lines derived from the endoderm. Purification of binding activities from pancreatic cells by DNA affinity chromatography reveals several distinct proteins ranging in size from 45 to 54 kDa (p45, p47/48, and p54). All of these proteins interact with the specific DNA sequence upon renaturation in vitro. Protein sequencing, electrophoretic mobility shift assay, and immunoblot analyses identify p54 and p47/48 as members of the hepatocyte nuclear factor 3 (HNF3 [forkhead]) family of transcription factors. p54 belongs to the subfamily of HNF3 beta proteins, while p47/48 binding activity includes HNF3 gamma. The cDNAs for two HNF3 beta proteins differing only in N-terminal amino acid sequences were isolated from a pancreatic cDNA library. The mRNAs encoding the two protein species accumulate to different steady-state levels in poly(A)+ RNA of pancreatic cells. Our results support a model by which the pancreas-specific expression of the alpha-amylase gene is mediated by a combination of cell-specific and cell lineage-specific transcription factors.

Thiol-disulfide redox buffers maintain a structure of immunoglobulin A that is essential for optimal in vitro binding to secretory component.

We have shown that human secretory component (SC) binds in vitro to different samples of human and murine dimeric immunoglobulin A (IgA). The binding ratio in the IgA/SC complex is 1:1. IgA which is stably bound to SC is separated from unreacted IgA by anion exchange chromatography. A part of IgA/SC complexes formed in vitro is unstable to this elution; the proportion varies between different samples of IgA; it increases following prolonged incubation of IgA at 37 degrees C. Incubation of IgA with glutathione/glutathione disulfide (GSH/GSSG) redox buffers increases the proportion able to form a stable complex with SC to approximately 90%. The presence of bound SC is not essential for this process but does allow it to occur at a lower GSH/GSSG concentration. The stable IgA/SC complex consists of a structure with a disulfide bond between IgA and SC apparently in equilibrium with a structure in which this bond is absent. The proportion bound covalently is similar for different samples of IgA and is insensitive to incubation with GSH/GSSG. It is significantly greater for secretory IgA (sIgA) and for IgA and SC incubated together with a starting mixture of cysteine/cystine. Monoclonal, antigen-specific IgA, all of which is optimally bound to SC in essentially the same way as in native sIgA, can be isolated in high yield. Our results support a mechanism for optimal binding of IgA to SC, that can occur both in vitro and in vivo, in which a thiol disulfide interchange occurs between a free IgA thiol and a sensitive SC disulfide following the initial non-covalent interaction.

Elevation of apolipoprotein E in the CSF of cattle affected by BSE.

The cerebrospinal fluid (CSF) of patients suffering from Creutzfeldt-Jakob disease (CJD) display two unique polypeptide chains by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In the absence of a well-defined ante-mortem diagnostic test for bovine spongiform encephalopathy (BSE), spinal fluid samples of eight normal cows and eight cows known to carry BSE by post-mortem histological analysis were investigated to verify if equivalent polypeptides were present. Proteins with similar migration to human CJD polypeptides were not detected. But surprisingly, a cluster of polypeptide spots that was faint or not detected in normal bovine CSF samples was found to be elevated or massively increased in BSE CSF samples (more than 10-fold increase). These elevated polypeptide chains were identified as apolipoprotein E.

cDNA cloning of a human 100 kDa de-ubiquitinating enzyme: the 100 kDa human de-ubiquitinase belongs to the ubiquitin C-terminal hydrolase family 2 (UCH2).

The full length cDNA encoding a 100 kDa human de-ubiquitinating enzyme, referred to as de-ubiquitinase was obtained using one clone selected from a randomly sequenced human brain cDNA library and specific primers. The sequence of 18 peptides generated from the de-ubiquitinase isolated from out-dated human erythrocytes matched perfectly with the predicted amino acid sequence, which would encode a protein containing 858 amino acids (calculated M(r) = 95,743 Da). Homology search disclosed that the protein is a member of a large family of ubiquitin C-terminal hydrolases (UCH2), that was defined on the basis of the presence of two specific patterns, 'the Cys- and His-domains', which are likely to be involved in the de-ubiquitinating activity [7]. An additional conserved region, 'the aspartic acid domain', was also identified, the functional role of which is unknown.

A human de-ubiquitinating enzyme with both isopeptidase and peptidase activities in vitro.

Some enzymatic and physicochemical properties of a human ubiquitin-specific isopeptidase are reported. The enzyme was purified to homogeneity from red blood cells and its specificity towards polymeric ubiquitin substrates suggests a de-ubiquitinating activity capable of cleaving 'head-to-tail' polyUb chains as well as isoamide 'branched' Ub dimers. KM values show a 10 fold preference for the cleavage of branched Ub dimers over head-to-tail Ub dimers. The enzymatic activity can be strongly inhibited by various peptides containing either of the cleavage site sequences found in Ub polymers, but not by unrelated peptides. The enzyme is monomeric under reducing conditions and exhibits a globular shape with an average diameter of 9 nm, an S20,w value of 5.2 S and a molar mass of 110 kDa +/- 10%. Because the enzyme cleaves both peptide-linked and isopeptide-linked Ub moieties from substrates, we propose to name it de-ubiquitinase rather than isopeptidase.

Human free secretory component is composed of the first 585 amino acid residues of the polymeric immunoglobulin receptor.

The main objective of this work was to unequivocally determine the C-terminal sequence of human milk free secretory component (SC). It was found to end at arginine-585, i.e. 33 amino acids downstream from the major heterogeneous C-terminal residue previously identified for colostrum SC. In contrast, our data showed that the C-terminal end of SC was found to be homogeneous. Conflicting assignments, Asp/Gln, a missing Asn-211, Asp/Asn, Glu/Gln were corrected and found to agree with the cDNA sequence. An Ala/Val substitution at position 562 (domain VI) was identified. Its genetic significance is uncertain at present.

Two-dimensional polyacrylamide gel electrophoresis analysis of cryoglobulins and identification of an IgM-associated peptide.

The clonality of immunoglobulins (Igs) in cryoprecipitates (n = 41) was studied by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Our series included 24 cryoglobulins characterized by immunofixation electrophoresis (IF), 12 'trace amount' cryoglobulins, defined by a protein content in the precipitate of less than 0.05 mg/ml of serum, and five cryoglobulins of undetermined protein composition by IF. 2-D PAGE analysis showed polyclonal IgG associated either with monoclonal Igs (type II cryoglobulins; n = 14) or with polyclonal IgM (type III cryoglobulins; n = 14). In ten cryoprecipitates (two 'trace amount' cryoglobulins as well as seven of 19 type II and as one of five type III cryoglobulins by IF) polyclonal IgG were associated with a mixture of polyclonal and monoclonal IgM. These cryoglobulins were tentatively named type II-III cryoglobulins. A monoclonal IgM was observed in one cryoprecipitate (type I cryoglobulins). Two cryoglobulins presented unexpected 2-D patterns, characterized by the presence of oligoclonal IgM, with trace amounts of Igs of different isotypes (tentatively named type II-III(variant) cryoglobulins). A peptide of 44 kDa with a pI of 5.45 was observed in all cryoglobulins containing IgM (n = 40). This peptide was also present in purified monoclonal or polyclonal IgM fractions. N-terminal microsequencing (12 amino acid residues) revealed that this IgM-associated peptide was an unknown protein. Our results highlight the role of 2-D PAGE as an aid in the analysis of cryoglobulins.

Changes in self-stimulation at stimulation-bound eating and drinking sites in the lateral hypothalamus during food or water deprivation, glucoprivation, and intracellular or extracellular dehydration.

These studies were designed to examine the effects of "hunger" induced by food deprivation, 2-deoxy-D-glucose (200 mg/kg), or insulin (2 U/kg) and "thirst" induced by water deprivation, sodium chloride (4 M), or polyethylene glycol (5 ml of 30% w/w) on lateral hypothalamic self-stimulation in 40 male Long-Evans rats. Changes in self-stimulation were evaluated at electrodes that produced stimulation-bound eating and/or drinking or neither behavior. Daily 30-min test sessions consisted of three 5-min periods of self-stimulation alternated with three 5-min periods when bar presses resulted in a 5-s time-out from experimenter-delivered stimulation (stimulation escape). Food deprivation significantly increased self-stimulation; insulin, 2-deoxy-D-glucose, and sodium chloride significantly suppressed self-stimulation; water deprivation mildly inhibited self-stimulation; and polyethylene glycol had no effect. This pattern of findings was noted at electrodes that did and those that did not elicit eating and/or drinking. These findings argue against the hypothesis that the magnitude of lateral hypothalamic self-stimulation is differentially and predictably controlled by specific drive mechanisms indexed by the consummatory behaviors also elicited by the stimulation.

Effect of glucoprivation on self-stimulation rate-frequency functions.

In a series of two experiments using rats, the effect of glucoprivation, induced by 2-deoxy-D-glucose (2-DG) or insulin injections, on self-stimulation rate-frequency functions, was evaluated at two levels of current intensity. At the higher current intensity, neither insulin nor 2-DG produced a significant change in rate-frequency function parameters. At the lower current intensity, insulin suppressed asymptotic responding while 2-DG produced a lateral curve shift. Results of this study would argue that glucoprivation produces changes in self-stimulation at lateral hypothalamic electrodes that are: a) unrelated to the involvement of the neurons in stimulation-induced eating, b) are most notable when a smaller number of reward relevant neurons is stimulated, and c) can be differentially attributed to changes in motoric performance capacity during insulin tests and to changes in the reward value of stimulating current during 2-DG tests.

A symbolic environment for visualizing activated foci in functional neuroimaging datasets.

This paper presents a symbolic visualization environment known as the Corner Cube environment, which was developed to facilitate rapid examination and comparison of activated foci defined by analyses of functional neuroimaging datasets. We have performed a comparative evaluation of this environment against maximum-intensity projection and 'gallery of slices' displays, and the results suggest that the Corner Cube environment has definite advantages over both conventional display techniques. We conclude that the Corner Cube is an effective tool for summarizing the spatial characteristics of activated foci within an easily understood visual context and is especially useful for displaying the similarities and differences in functional neuroimaging datasets.

Neuropsychological correlates of methylphenidate treatment in adult ADHD with and without depression.

The purpose of this study was to characterize the neuropsychological profiles of adult patients with attention deficit hyperactivity disorder (ADHD) alone and ADHD with active comorbid depression, and to evaluate changes in the neuropsychological profile in these two groups following a trial of methylphenidate. Forty patients with ADHD were classified into two groups based on their affective status resulting in a group of 21 patients with ADHD alone and 19 patients with ADHD and active comorbid symptoms of depression (ADHD-D). All subjects received a comprehensive neuropsychological evaluation including measures of cognitive, motor and affective functioning before and after treatment. Fifteen normal controls were also assessed at a yoked time interval. At baseline, both patient groups showed impairment in verbal memory, motor and processing speed, visual scanning, and auditory and visual distractibility. Following treatment, both patient groups showed improvement across all neuropsychological measures while controls remained relatively stable over time. Improvement in neuropsychological test performance was not related to gender, affective status or referral source. Patients with active comorbid symptoms of depression show a similar neuropsychological profile and appear equally likely to benefit from methylphenidate intervention as patients with ADHD alone.

Recent studies of the interaction of rabbit dimeric IgA with its polymeric immunoglobulin receptor.

Rabbit secretory components (SC) constitute a highly heterogeneous population of glycoprotein molecules that are present in secretions as free or bound forms to polymeric immunoglobulins (Ig). Two SC families are known, one of high molecular weight (approximately equal to 80 Kd) composed of five (perhaps six) domains related to Ig variable domains, and one of low molecular weight (approximately equal to 55 Kd). An account of our most recent experimental data is reviewed in this article. We have shown: 1) that both the high and low Mr SC families possess the same relative avidity for binding to dimeric IgA of the g-subclass; 2) that the first NH2-terminal domain of SC derived from the high and low Mr polypeptides is necessary and sufficient for efficient non-covalent binding to dimeric IgA of the g-subclass; 3) that the low Mr SC polypeptide derives from the high Mr SC by the internal deletion of the entire second and third domains, suggesting that these domains are not involved in the binding reaction with polymeric Ig; 4) that the heterogeneity of rabbit secretory components is, in large part, due to the expression of several polymorphic forms (allotypes) susceptible to be recognized by specific alloantisera; the biochemical characterization of the three known SC allotypes (t61, t62 and t63) reveals that t62 and t63 are structurally very similar to each other and markedly divergent from the t61 homologue; 5) that by using non-cross-reactive alloantisera, the major immunodominant allotopes are confined within the COOH-terminal domains 3, 4 and 5 of SC; 6) that the location of the residues involved in the attachment of the carbohydrate unit within domain 1 varies according to the allotype: t61 is N-linked glycosylated at position 70, whereas about 75% of t62 molecules are devoid of sugars; the remaining 25% of t62 molecules are glycosylated at residue position 90; these oligosaccharide chain units are linked to asparagine residues in the acceptor site consensus sequence, Asn-X-Thr/Ser; 7) that the presence of the carbohydrate unit in domain 1 is not required for efficient binding of this domain to polymeric Ig: indeed, after enzymatic deglycosylation, domain 1 exhibits a relative binding avidity which is indistinguishable from that of the native glycosylated domain 1.

Rabbit secretory components of different allotypes vary in their carbohydrate content and their sites of N-linked glycosylation.

The asparagine-linked glycosylation sites in rabbit high and low Mr secretory components (SC) have been determined for the three known allotypes, t61, t62, and t63. Purified SC polypeptides were subjected to mild periodate oxidation of terminal nonreducing sugars followed by selective reduction with [3H]sodium borohydride, SC polypeptides were further proteolytically cleaved, and the 3H-labeled peptides were isolated and characterized. Both high and low Mr SCs of the three allotypes possess a common glycosylation site at the asparagine residue position 400, whereas the second site, in the amino-terminal domain of SC, was found to be variable: the t61 and t63 allotypes contained a glycosylation site at positions 70 and 90, respectively. Moreover, although the t62 allotype was found to contain a triplet acceptor site (N-X-S) at positions 90-92, analyses showed that less than 30% of the t62 allotype peptides encompassing this region were glycosylated at residue 90. Furthermore, the amino acid sequence of the t61 SC in the region of residues 69-90 varies by 8 and 10 amino acid substitutions when compared with the t62 and t63 allotype sequences, respectively. However, neither the variation in amino acid sequence nor the variation in degree or site of glycosylation measurably affected the non-covalent binding of domain 1 to dimeric IgA.

Structural variability of rabbit secretory components. Allotype-associated differences in the third, fourth, and fifth domains.

We have previously shown (Frutiger, S., Hughes, G. J., Hanly, W. C., Kingzette, M., and Jaton, J.-C. (1986) J. Biol. Chem. 261, 16673-16681) that limited tryptic digestion of the high Mr form of rabbit secretory component of allotypes t61, t62, and t63 generates two major fragments, the NH2-terminal domain and a 40-kDa fragment encompassing domains 3, 4, and 5. Similarly, from the low Mr form of secretory component, (SC) the NH2-terminal domain, together with a 30-kDa fragment containing domains 4 and 5, were released. These fragments were used as inhibitors in a sensitive competitive binding radioimmunoassay with noncross-reactive rabbit alloantisera to study the distribution and localization of the major allotype-specific allotopes within the SC polypeptide. The 40-kDa fragments were shown to inhibit the 125I-labeled intact SC/anti-SC allotype reaction to the extent of 90%, i.e. nearly as well as the intact homologous high Mr SC form. In contrast, the NH2-terminal fragments (domain 1) were not inhibitory. The low Mr SC of each allotype was less inhibitory on a molar basis than the homologous high Mr SC polypeptide, an observation compatible with the deletion of domains 2 and 3 in the smaller polypeptide (Deitcher, D. L., and Mostov, K. E. (1986) Mol. Cell. Biol. 6, 2712-2715; Frutiger, S., Hughes, G. J., Fonck, Ch., and Jaton, J.-C. (1987) J. Biol. Chem. 262, 1712-1715). The structural correlates of the allotypic specificities were evaluated by comparative peptide mapping of the 40-kDa fragments (allotypes t61, t62, and t63). The data suggest that the t61 allotype structure differs significantly from the t62 and t63 structures, the latter two being much more related to each other than to t61. These findings are in full agreement with the serological data. The inhibition results suggest that the major allotype-specific, noncross-reactive allotopes of SC are distributed throughout domains 3, 4, and 5, even though domain 4 appears to be more conserved than domains 3 and 5 between the allotypes t61 and t63. Seven amino acid substitutions between t61 and t63 have been detected within domains 3, 4, and 5.

Structural and electron-microscopic studies of jacalin from jackfruit (Artocarpus integrifolia) show that this lectin is a 65 kDa tetramer.

The 133-amino-acid sequences of the alpha-subunit of jacalin (a lectin from Artocarpus integrifolia) and of the slightly larger alpha'-subunit were determined. The alpha'- and alpha-subunits, in the approximate ratio of 1:3, were found to be virtually identical in their primary structures, except for one valine for isoleucine substitution at position 113. Although both alpha'- and alpha-chains were glycosylated, the extent of glycosylation in the alpha'-chain was much greater than that in the alpha-subunit. In the alpha'-polypeptide, all molecules contained an N-linked oligosaccharide at position 74 and some contained sugar at position 43. The alpha- and alpha'-subunits were found to be strongly non-covalently associated with three distinct beta-subunits containing 20 amino acids each. Electron-microscopic visualization of native jacalin disclosed a structure composed of four alpha-type subunits with a clear-cut 4-fold symmetry. Analytical-ultracentrifugation studies of jacalin revealed an average molecular mass of 65 kDa, a value compatible with a tetrameric structure of the alpha(alpha')-subunits. The recalculated number of sugar-binding sites per jacalin molecule, given a molecular mass of 65 kDa, would yield 0.8 sites per alpha(alpha')-promoter, i.e. about twice the value previously determined [Appukutan & Basu (1985) FEBS Lett. 180, 331-334; Ahmed & Chatterjee (1989) J. Biol. Chem. 264, 9365-9372].

High and low molecular weight rabbit secretory components. Evidence for the deletion of the second and third domains in the smaller polypeptide.

Rabbit secretory components exist in two forms which differ in apparent mass by about 25 kDa. Each of these two forms were reduced, carboxymethylated, and extensively digested with trypsin. The resulting peptides were purified by reverse-phase high performance liquid chromatography and characterized by NH2- and COOH-terminal sequence determination and/or amino acid analysis. They were aligned with the protein sequence predicted from the cDNA nucleotide sequence encoding the rabbit poly(Ig) receptor (Mostov, K. E., Friedlander, M., and Blobel, G. (1984) Nature 308, 37-43). All peptides belonging to the fourth and fifth domains except one (positions 488-496) were accounted for in both forms. In addition, limited tryptic proteolysis of the native low Mr secretory components produced the intact 18-kDa NH2-terminal domain (positions 1-117) and the 30-kDa fragment encompassing the fourth and fifth domains. These results suggest that the smaller polypeptide derives from the larger secretory component form by the deletion of the second and third domains.

The p48 DNA-binding subunit of transcription factor PTF1 is a new exocrine pancreas-specific basic helix-loop-helix protein.

We report the isolation of cDNA for the p48 DNA-binding subunit of the heterooligomeric transcription factor PTF1. A sequence analysis of the cDNA demonstrates that p48 is a new member of the family of basic helix-loop-helix (bHLH) transcription factors. The p48 bHLH domain shows striking amino acid sequence similarity with the bHLH domain of proteins that act as developmental regulators, including the twist gene product, myogenic factors and proteins involved in hematopoietic differentiation. We show that reduced p48 synthesis correlates with a diminished expression of genes encoding exocrine pancreas-specific functions. The synthesis of p48 mRNAs, and therefore also the protein, is restricted to cells of the exocrine pancreas in the adult and to the pancreatic primordium in the embryo. Thus the pancreas-specific DNA-binding activity of PTF1 originates from the synthesis of at least one cell-specific component rather than from a cell-specific assembly of more widely distributed proteins.

Disulfide bond assignment in human J chain and its covalent pairing with immunoglobulin M.

The assignment of disulfide bonds in human J chain and its covalent pairing with immunoglobulin M was determined under conditions which minimize disulfide bond interchange. We show that in J chain the three intradisulfide bridges are formed between Cys 12 and 100, Cys 71 and 91, and Cys 108 and 133. Previous reports [reviewed by Koshland, M. E. (1985) Annu. Rev. Immunol. 3, 425-453] have proposed that cysteines 12, 14, or 68 were linked to the penultimate cysteine 575 of two mu chain tails. In this work, we demonstrate that cysteines 14 and 68 are disulfide-bridged to mu chains. A revised, albeit putative, model of J chain folding is presented which takes into account the correct disulfide pairing and the predictive secondary structure assignment.

The amino-terminal domain of rabbit secretory component is responsible for noncovalent binding to immunoglobulin A dimers.

Rabbit secretory components (SC) constitute a family of markedly heterogeneous glycoproteins which are released in the secretions as free SC or as SC bound to polymeric immunoglobulins. The aim of this work was to determine the region of the SC polypeptides which is involved in IgA binding. The high and the low Mr forms of free SC (or IgA-dissociated bound SC) and the native secretory IgA complex were subjected to limited tryptic digestion. Chemically characterized peptides ranging in apparent size from 15 to 20 kDa, depending upon the allotype, were shown to be necessary and sufficient for efficient noncovalent binding to IgA dimers (subclass g). These fragments encompass the amino-terminal first domain of SC, i.e. residues 1-126, when aligned with the predicted amino acid sequence from a cDNA clone encoding the rabbit polymeric Ig receptor (Mostov, K.E., Friedlander, M., and Blobel, G. (1984) Nature 308, 37-43). The high and the low Mr forms of SC exhibited the same relative affinity for IgA dimers, suggesting that the postulated internal deletion in the smaller polypeptide (Kühn, L. C., Kocher, H.-P., Hanly, W.C., Cook, L., Jaton, J.-C., and Kraehenbuhl, J.-P. (1983) J. Biol. Chem. 258, 6653-6659) does not impair the IgA dimer recognition function.

The yeast protein encoded by PUB1 binds T-rich single stranded DNA.

We have characterized binding activities in yeast which recognise the T-rich strand of the yeast ARS consensus element and have purified two of these to homogeneity. One (ACBP-60) is detectable in both nuclear and whole cell extracts, while the other (ACBP-67) is apparent only after fractionation of extracts by heparin-sepharose chromatography. The major binding activity detected in nuclear extracts was purified on a sequence-specific DNA affinity column as a single polypeptide with apparent mobility of 60kDa (ACBP-60). This protein co-fractionates with nuclei, is present at several thousand copies per cell and has a Kd for the T-rich single strand of the ARS consensus between 10(-9) and 10(-10) M. Competition studies with simple nucleic acid polymers show that ACBP-60 has marginally higher affinity for poly dT30 than for a 30 nt oligomer containing the T-rich strand of ARS 307, and approximately 10 fold higher affinity for poly rU. Internal sequence information of purified p60 reveals identity with the open reading frames of genes PUB1 and RNP1 which encode polyuridylate binding protein(s). The second binding activity, ACBP-67, also binds specifically to the T-rich single strand of the ARS consensus, but with considerably lower affinity than ACBP-60. Peptide sequence reveals that the 67kDa protein is identical to the major polyA binding protein in yeast, PAB1.