Analysis of differential HLA-DQB expression in autologous B cell lines.

Class II major histocompatibility complex genes are differentially expressed during cellular activation and differentiation, often in a locus-specific manner. We investigated the differential expression of the HLA-DQB gene, using B cell lines LAZ221 and LAZ388: LAZ221, derived from an early B cell leukemia, expresses HLA-DR but not HLA-DQ: LAZ388, the autologous Epstein-Barr virus-transformed B cell line, expresses both DR and DQ. Transfection experiments demonstrate differential function of class II gene upstream regulatory regions in the two lines, which correlates with differential class II gene expression. Using gel retardation and DNase I footprint assays, we demonstrate that absence of DQB gene expression is associated with characteristic nuclear protein-binding interactions in the proximal DQB gene upstream regulatory region. These interactions are visualized as DNA-protein complexes that are seen with nuclear proteins from the DQ-negative cell line, LAZ221, and involve consensus promoter Y box and W box elements, as well as novel upstream sites. Transcriptional regulatory proteins that differ in these autologous B cell lines may be stage-specific factors involved in the developmental regulation of HLA genes.

Locus- and allele-specific DNA-protein interactions in the HLA-DQB1 X box.

Expression of MHC class II genes is regulated by a complex series of protein-DNA interactions which lead to the initiation of transcription. Although the different MHC class II loci are generally coordinately expressed, important differences in expression can be seen among loci and among individual alleles. The major sites of transcriptional control in the human MHC consist of several highly conserved nucleotide sequence elements located upstream of each MHC class II gene. We have analyzed the interlocus and interallelic variation in one of these key regulatory regions of the HLA-DQB1 promoter, the X box, and identified several sites of protein-DNA interaction. Two protein-DNA complexes were found which differ between the DQ and DR loci as well as two distinct complexes which differed between DQ alleles. These nuclear protein-X box interactions are likely to influence the differential expression of the MHC class II loci and alleles in tissue-specific or developmentally regulated pathways.

Allele-specific DNA-protein interactions associated with the X-box regulatory region of the DQB1*0302 gene.

The X box is an essential transcriptional regulatory region for both constitutive and inducible expression of HLA-class II genes, and, while highly conserved among class II genes, both locus- and allele-specific polymorphisms exist. Using gel regardation analysis, we have analyzed the binding of B cell nuclear proteins to the X box regions of the DQB1*0302, *0301, and DRA genes and have identified two distinct X box binding complexes which differ for the diabetes-associated DQB1*0302 allele.

Differential expression of related HLA class II DQ beta genes caused by nucleotide variation in transcriptional regulatory elements.

HLA class II genes comprise a large multigene family with intra- and interlocus variation in structure and expression. Within this family of related genes, the HLA-DX alpha and beta loci (HLA DQA2 and DQB2) are highly homologous to functional HLA-DQ loci (HLA DQA1 and DQB1) but are frequently termed pseudogenes because DX gene transcription has not been observed, even in cells expressing HLA-DQ. Analysis of upstream transcriptional regulatory elements for the DX beta and DQ beta genes identified a high degree of nucleotide homology, consistent with their derivation from a common ancestral class II gene. However, transient expression assays with plasmids utilizing promoter elements from the DX beta gene had no activity in transfected human B cells, in contrast to homologous DQ beta sequences. Reciprocal exchange of specific sequences from the DQ beta gene with those of the DX beta gene restored expression to wild-type DQ beta levels, as did mutagenesis of only three DX-specific nucleotides in the upstream regulatory region. These three nucleotides mark two binding sites for distinct nuclear DNA-binding proteins that differentially recognize DQ beta and DX beta sequences. Transcription of these genes is critically dependent on interactions between these two upstream regulatory region sites which distinguish DX beta from its closely related homologue, DQ beta.

Nonprogression of subclinical beta-cell dysfunction among first-degree relatives of IDDM patients. 5-yr follow-up of the Seattle Family Study.

It is unknown among first-degree relatives of individuals with insulin-dependent diabetes mellitus (IDDM) whether the disease process occurs in relatively few but always progresses to clinical IDDM or whether subclinical disease is more common but remains nonprogressive in many cases. Islet cell antibodies (ICAs) were found in 21 of 724 (2.9%) first-degree relatives during screening in the greater Seattle area between 1983 and 1988. Measures of beta-cell function (glucose disappearance rate [Kg], fasting insulin, acute insulin response to intravenous arginine [AIRarg], acute insulin response to intravenous glucose [AIRgluc], slope of glucose potentiation of AIRarg) and insulin sensitivity were obtained. Twenty individuals, 9 ICA+ relatives and 11 ICA- relatives, were evaluated prospectively. When expressed in relation to the expected AIRgluc based on each subject's sensitivity index, AIRgluc in 18 of 20 relatives fell below 100%, indicating inappropriately low insulin secretion (subclinical beta-cell dysfunction). After a median follow-up of 42 mo, 10 of 11 ICA- relatives remained ICA-. None showed deteriorating beta-cell dysfunction, and none developed diabetes. Five ICA+ relatives showed persistent immunologic positivity. beta-Cell function remained remarkably stable in all except 2 relatives. One was a 15-yr-old boy who developed IDDM shortly after screening and before evaluation of beta-cell function could be carried out. The other was an 18-yr-old monozygotic twin who developed IDDM after 27 mo. Both of these individuals had ICAs of 80 Juvenile Diabetes Foundation U and had been discordant for less than 5 yr.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of vasopressin receptors and phosphoinositide hydrolysis in the septum of heterozygous and homozygous Brattleboro rats.

Accurel polypropylene mini-devices, loaded with arginine vasopressin (AVP) and implanted in the lateral cerebral ventricle were used to centrally treat heterozygous (HE) and homozygous (HO) Brattleboro (BB) rats. After 1 week of treatment, the concentration of AVP receptors in the HO-BB rat septum decreased from 19.4 +/- 2.6 to 12.4 +/- 1.1 fmol/mg protein, but remained unchanged in the HE-BB rat (10.7 +/- 0.8 and 7.0 +/- 1.1 fmol/mg protein). In the HO-BB rat the [3H]-AVP equilibrium dissociation constant (KD) of the septal AVP receptor decreased following AVP treatment (from 4.17 +/- 0.7 to 1.97 +/- 0.3 nM) compared to that of control animals. This decrease in receptor number following AVP treatment was accompanied by a decrease in the postreceptor response to AVP as measured by the AVP-stimulation of [3H]-inositol-1-phosphate (IP1) accumulation (22.0 +/- 6.1%) when compared to untreated animals (54.3 +/- 8.3%). This apparent AVP-induced down-regulation was not due to occupancy of the binding sites by AVP since preincubation of the tissue at 37 degrees C for 60 min (which was able to cause near-complete dissociation of the hormone-receptor complex) did not result in an increased number of binding sites upon reexposure to [3H]-AVP. This study thus provides evidence for the homologous down-regulation and desensitization in terms of [3H]-IP1 accumulation (phosphoinositide hydrolysis) of AVP receptors in the septum of the BB rat.

Enhanced phosphoinositol hydrolysis in response to vasopressin in the septum of the homozygous Brattleboro rat.

Arginine8-vasopressin (AVP) receptors in the septum of the Long-Evans rat have been shown to be both pharmacologically (displacement profiles) and functionally (ability to stimulate phosphoinositide hydrolysis) similar to the peripheral V1-type receptor for AVP. Previous binding studies of AVP receptors in the septum of heterozygous (HE) and homozygous (vasopressin-deficient, HO) Brattleboro (BB) rats revealed an increased number of receptors with a lower affinity for AVP in the HO-BB rat when compared to the HE-BB rat. To determine the effect of these receptor changes in the HO-BB rat septum on the postreceptor response of the tissue to AVP, concentration-response relationships for AVP-stimulated phosphoinositide hydrolysis were examined in septal slices from age-matched, adult male HE- and HO-BB rats. AVP-stimulated accumulation of [3H]inositol-1-phosphate (IP1) was significantly greater in the HO-BB (43.7%) than in the HE-BB (13.7%) at AVP concentrations of 10(-08) to 10(-05) M. The two groups did not, however, differ in their ability to stimulate [3H]IP1 accumulation in response to 2.0 mM carbachol. When the AVP-stimulated phosphoinositide response in both genotypes was compared to that obtained for the Long-Evans (LE) rat (the parent strain of the Brattleboro rat) septum under the same assay condition, it was found that the response in the HE-BB was much lower than in the LE. AVP receptor binding capacity (Bmax) correlated (r = 0.975) with release of IP1 ([3H]IP1 accumulation) for all 3 groups studied (LE, HE, HO).(ABSTRACT TRUNCATED AT 250 WORDS)

V1-type vasopressin receptors in rat brain septum: binding characteristics and effects on inositol phospholipid metabolism.

Specific binding sites for 3H-arginine8-vasopressin (AVP) have been characterized in rat septal membranes. Scatchard analyses revealed a single class of high-affinity binding sites having an equilibrium dissociation constant of 1.7 +/- 0.3 nM and total binding capacity of 22.6 +/- 4.2 fmol/mg protein. Binding displacement studies with peptide analogs of AVP indicate that this binding site is similar to the V1 (pressor)-type receptor for AVP. When added to rat brain septal slices that had been prelabeled with 3H-myo-inositol, vasopressin stimulated the accumulation of 3H-inositol-1-phosphate (IP1) in the presence of 7 mM lithium. This effect was dose dependent with maximal stimulation (65% over basal) occurring at a concentration of 0.5 microM AVP. Higher concentrations, however, tended to inhibit phosphoinositide hydrolysis. The vasopressin-stimulated accumulation of 3H-IP1 was completely inhibited by the vasopressin V1 antagonist, d(CH2)5[Tyr(Me)2]AVP, in a concentration-dependent manner. Oxytocin, at concentrations of 10(-8) and 10(-5) M, only slightly increased 3H-IP1 accumulation (17-20% over basal). In contrast, the V2 agonist deamino-D-arginine vasopressin (dDAVP), failed to produce significant stimulation of 3H-IP1 accumulation, even at high concentrations. The effects of these analogs on phosphoinositide hydrolysis is consistent with their potencies in displacing 3H-AVP from septal binding sites. These results indicate that vasopressin stimulates hydrolysis of inositol phospholipids in rat brain septum through an interaction with V1-type vasopressin receptors.

Interaction of a vasopressin antagonist with vasopressin receptors in the septum of the rat brain.

The ability of d(CH2)5-Tyr(Me)-arginine-8-vasopressin, an antagonist of peripheral pressoric (V1-type) vasopressin receptors, to label vasopressin binding sites in the septum of the rat brain was evaluated. Using crude membrane preparations from the septum, 3H-arginine-8-vasopressin (AVP) specifically labels a single class of binding sites with a Kd of 2.9 nM and maximum binding site concentration of 19.8 fmole/mg protein. 3H-Antag also labels a single class of membrane sites but with higher affinity (Kd = 0.47 nM) and lower capacity (10.1 fmole/mg protein) than 3H-AVP. The rank order of potency of various competitor peptides for 3H-AVP and 3H-Antag binding was similar. Oxytocin was 100-1,000 fold less potent than AVP in competing for binding with both ligands. 3H-AVP and 3H-Antag showed similar labeling patterns when incubated with septal tissue slices. Unlabeled Antag also effectively antagonized vasopressin-stimulated phosphatidylinositol hydrolysis in septal tissue slices.

Enhanced binding of 3H-arginine8-vasopressin in the Brattleboro rat.

Specific binding sites for 3[H]-arginine8-vasopressin (AVP) were characterized using membrane preparations of liver, renal medulla and brain (septal) tissue of heterozygous (HE) and homozygous (HO) Brattleboro (BB) rats. Measurement of binding sites indicated that significantly greater numbers of AVP receptors are present in the liver and septum of HO-BB rats. Similar numbers of AVP receptors were present in renal medullary tissue from HO-BB and HE-BB rats. Higher equilibrium dissociation constants were measured in the HO-BB septal tissue indicating a lower affinity of the brain receptor for 3[H]-AVP than in heterozygotes. No significant differences in AVP receptor affinity were noted in liver or kidney tissue. It is concluded that "up-regulation" of AVP receptor number and, in the brain, alterations in AVP receptor affinity may occur in the absence of endogenous AVP.

Generation of reactive dysmorphogenic intermediates by rat embryos in culture: effects of cytochrome P-450 inducers.

We have investigated the capacity of cultured whole rat embryos to convert 2-acetylaminofluorene (AAF) to reactive metabolites capable of eliciting dysmorphogenic effects in the same embryos. Cultured embryos (Sprague-Dawley) were exposed to AAF for periods of 2 or 24 hr, after which metabolites were isolated from the culture medium and identified with HPLC. Embryotoxic effects were evaluated in the same embryos. Day 10 embryos preexposed in utero to pregnenolone-16 alpha-carbonitrile (PCN) exhibited marked increases in capacity to convert AAF to a variety of hydroxylated metabolites. 3-Methylcholanthrene (3MC) was also a very effective inducer in utero but Aroclor 1254 (PCB), and isosafrole (ISF) evoked only minimal induction while phenobarbital (PB) was not demonstrably effective. Exogenously added hepatic postmitochondrial supernatant (S9) fractions from adult male rats pretreated with PCB, 3MC, or ISF exhibited induced monooxygenase activities as well as increased capacity to convert AAF to dysmorphogenic intermediates in the culture system. PB and PCN displayed much lesser effects. PCN was a very effective inducer of hepatic monooxygenases of pregnant rats but, when this tissue was utilized as an enzyme source, no significant increase in malformations was observed. Embryos with relatively high monooxygenase activities also displayed a high incidence of embryonic abnormalities when cocultured with AAF. Malformation incidence was strongly correlated with hydroxy metabolite generation, suggesting that induction in utero of P-450-dependent, embryonic monooxygenases resulted in the production of embryotoxic metabolites by the embryos own enzymes. The data also indicated that endogenous bioactivation (within the conceptus) was considerably more effective than bioactivation effected by an exogenous (hepatic) enzyme source.

Viral coronary arteritis and myocardial infarction.

Two young male patients are described in whom a viral type of upper respiratory tract infection was followed by myocardial infarction. Based upon previously reported experimental studies in mice and monkeys in which Coxsackie B4 virus produced extensive coronary arterial and capillary injury, and upon the clinical data in these two patients, it is suggested that the myocardial infarcts in our two patients were the result of viral coronary arteritis.