[Comparison of two therapeutic regimes for diabetes-stricken children. Social and mental resources of the family are often crucial for the prognosis]. / Två behandlingsregimer vid diabetesdebut hos barn jämförda. Familjens sociala och mentala resurser avgör ofta prognosen.

In a prospective, randomized intervention study, 36 children with diabetes mellitus type I were followed, the aim being to study if a family psychosocial intervention at diagnosis could improve glycemic control and minimise hospital admissions. The control group was treated initially in a hospital ward, while the whole family of the children in the study group received therapeutic and social support in an out-hospital training apartment. In the study group only, both parents reported a significant improvement of the family climate. An increased risk for poor glycemic control was recorded in children living in one-parent families and in families where the father had a low level of education. Younger age, a single-parent family and poor glycemic control were factors associated with more days of rehospitalization. The divorce rate in the whole group was at least as high as in the normal population but, surprisingly, maternal dysfunction was associated with lower levels of HbA1c. The conclusion is that the social family background is a most important factor for the glycemic control and need for hospital readmission of the diabetic child.

Extrapancreatic trypsin-2 cleaves proteinase-activated receptor-2.

Proteinase-activated receptors (PARs) are activated by proteolytic removal of a short amino terminal peptide, thus exposing a new amino terminus that functions as a tethered ligand that activates the receptor. With the aim to identify and study potential activators of PAR-2 we have developed a new method to measure proteolytic cleavage of PARs. PAR-2 was tagged with the insulin C-peptide that upon receptor cleavage is released and quantified using an ELISA. The modified receptor, shown to be functional in mouse 3T3 cells, was expressed in an insect cell line and the ability of different proteinases to cleave PAR-2 was studied. Two different mast cell tryptases cleaved PAR-2 in a concentration dependent manner, but were much less potent than pancreatic trypsin and trypsin-2 isolated from a carcinoma cell line. Pancreatic trypsin and trypsin-2 were almost equally effective at cleaving PAR-2 suggesting that extrapancreatic trypsins are potential in vivo activators of PAR-2.

Influence of the initial management regimen and family social situation on glycemic control and medical care in children with type I diabetes mellitus.

It is well known that social family factors are of importance in diabetes care, but it is not clear whether the initial management regimen can buffer these factors. In a prospective, randomized intervention study, 36 children with diabetes mellitus (type I) were followed, the aim being to study if a family psychosocial intervention at diagnosis could improve glycemic control and minimize hospital admissions. The control group was treated initially in a hospital ward, while the study group received problem-based learning and family-therapeutic and social support in an out-hospital training apartment. A number of family social variables were evaluated at the time of diagnosis and 6, 12 and 24 mo later. Family function was assessed using the self-estimated Family Climate Test at these same time-points. HbAlc values and information concerning in- and out-hospital visits to the pediatric clinic were collected for the 5-y period following diagnosis. We found no association between the offered management regimen and glycemic control or rate of readmission. In the study group only, both parents reported a significant improvement of the family climate. An increased risk for poor glycemic control was recorded in children living in one-parent families (p = 0.03) or in families where the father had a low level of education (p = 0.04). Younger age (p = 0.05), a single-parent family (p = 0.05) and poor glycemic control (p = 0.02) were associated with more days of rehospitalization. The rate of divorce in the whole group was at least as high as in the normal population but, surprisingly, maternal dysfunction was associated with lower HbAlc value. The conclusion is that even with an initial management regimen designed to offer a family-individual care regimen based on accurate estimation of the psychological and pedagogical needs, the social family background is a most important factor for the glycemic control and need for readmission.

Stimulation of proteinase activated receptor-2 causes endothelial cells to promote blood coagulation in vitro.

Proteolytically activated receptors define a new subclass among the G-protein coupled receptors. Proteinase activated receptor-2 (PAR-2), the second member to be identified of this growing receptor subclass, can be activated by trypsin and trypsin-like serine proteases such as mast cell tryptase. PAR-2 is expressed in endothelial cells. Here we have studied if activation of PAR-2 changes the coagulation properties of cultured human umbilical vein endothelial cells. We show that activation of PAR-2 induces rapid and transient formation of tissue factor mRNA with a maximum level 1 hour after receptor stimulation. The increased mRNA level was accompanied by an increased tissue factor activity at the endothelial cell surface, shortening coagulation time in a standard clotting assay. The level of tissue factor activity after PAR-2 activation was comparable with the effects of thrombin receptor (PAR-1) activation although neither of the two protease receptors were as strong inducers of tissue factor as tumor necrosis factor-alpha.

Structure and expression of the murine thromboxane A2 receptor gene.

The gene encoding the murine thromboxane A2 receptor (TP) is split into three exons and spans 6.2 kilobase pairs. Primer extension analysis revealed two transcription initiation sites located approximately 210 nucleotides 5' of the first intron. The 1.2-kb 5'-flanking region lacks typical TATA and CAAT boxes but contains several potential regulatory elements including binding sites for Sp-1, NF-kappaB, AP-2, and a glucocorticoid response element. A 192-bp murine repetitive B2 element is located in the 5'-flanking region, but did not exert a negative effect on the basal promoter activity in transient transfection experiments with reporter constructs. Ribonuclease protection assays showed expression of TP RNA in several organs including thymus, spleen, kidney, and lung. In the thymus, in situ hybridization revealed transcripts in the cortex, but not in the medulla, suggesting that thromboxane A2 may play a role in the development of T-lymphocytes.

Metabolic control in children with insulin-dependent diabetes mellitus 5 y after diagnosis. Early detection of patients at risk for poor metabolic control.

Children (n = 38) aged 3-15 y were randomly chosen, at the time of diabetes diagnosis, for conventional management at a hospital ward, or for treatment partly in a training apartment where the family was offered problem-based education and special therapeutic support. HbA1c, blood glucose stability, urinary C-peptide excretions and incidence of hypoglycaemic attacks and diabetes ketoacidosis (DKA) were monitored and some standardized, self-estimated psychological tests were performed during the first 2 y after diagnosis. During the 3 y thereafter, HbA1c, presence of DKA, microalbuminuria, retinopathy and hypertension were monitored. None of the patients demonstrated signs of diabetes microangiopathy or DKA. The overall mean HbA1c level was 7.2% 5 y after diagnosis and 30% of the children had HbA1c values <6.3%. There were no differences in the HbA1c values for the patients treated by the different management regimens. Blood glucose variability (SD) was also similar, with 75% of the values in the range of 3-10 mmol/l. Patients with poor glycaemic control (mean HbA1c >8.3%) year 5 after diagnosis had already the second year after diagnosis significantly higher HbA1c values and blood glucose variability. The fathers of these patients demonstrated a higher degree of maladjustment. On the basis of increasing HbA1c values, high blood glucose variability and psychosocial risk factors such as their fathers' emotional responses, patients at risk for poor metabolic control in the future can be identified within 2 y after diagnosis. Efforts and resources can thus be focused at an early stage on this group.

Vascular effects of proteinase-activated receptor 2 agonist peptide.

Proteinase-activated receptor 2 (PAR-2) is a G protein-coupled receptor related to the thrombin receptor. PAR-2 can be activated by trypsin and by synthetic peptides corresponding to the new amino terminus generated by activating proteolytic cleavage. We show in this report that intravenous injection of PAR-2 agonist peptides has dramatic effects on arterial blood pressure in anesthetized rats. The peptide SLIGRLETQPPI, at 150 nmol/kg, transiently decreased the mean arterial pressure from 104 to 60 mm Hg. The hypotensive response was dose-dependent, and was not secondary to effects on central vasoregulatory systems, heart rate, or the kidneys. A nitric oxide synthase inhibitor attenuated the hypotensive response induced by the PAR-2 agonist peptide. Further experiments in vitro, on preparations of rat femoral artery and vein, showed that PAR-2 agonist peptide elicited a dose-dependent relaxation of both types of vessel. Removal of the endothelium abolished the agonist peptide-induced relaxation. Our results demonstrate that activation of PAR-2 can modulate vascular tone, and that this response was an effect mediated at least partly by nitric oxide. The effect on blood vessels further suggests that the physiological activator of this proteolytically activated receptor is an enzyme present and active in the blood, possibly after a vascular injury.

The mouse genes for the EP1 prostanoid receptor and the PKN protein kinase overlap.

PKN is a newly discovered protein kinase that has been shown to mediate GTPase Rho dependent intracellular signalling. We show in this report that the mouse PKN gene is situated at the mouse EP1 prostanoid receptor gene locus and that the two genes are overlapping in a tail-to-tail manner. An "exon trap" strategy was used to identify the overlap phenomenon. By using RT-PCR and 3' RACE we have identified two major PKN transcripts that are produced by alternative polyadenylation. The 3' end of the short PKN transcript overlaps the 3' untranslated region of the EP1 gene with approximately 280 bp, while the long PKN transcript overlaps the whole EP1 gene. Remarkably, none of the three transcripts originating from this locus display the consensus AAUAAA polyadenylation signal. The last seven exons of the PKN gene, corresponding to the last third of the PKN cDNA, have been recognised in 7.2 kb of continuous genomic sequence that we have collected from the EP1/PKN genetic locus. The 3' part of the PKN gene is highly fragmented and its intron/exon organisation is reminiscent of that of the Drosophila protein kinase C gene. The possibility of a natural antisense regulation of these genes is discussed.

Ligand cross-reactivity within the protease-activated receptor family.

Recently, a second member of the protease-activated receptor (PAR) family, named PAR-2, has been identified. Similar to the thrombin receptor, PAR-2 appears to be activated by proteolytic-mediated exposure of a "tethered ligand" sequence and can also be activated by the corresponding synthetic peptides. Similarities in the amino acid sequence of the receptors' tethered ligand sequences suggest that their respective agonist peptides might not be absolutely specific for their particular receptors. To test this, the receptor specificity of each agonist has been determined by measuring the responses of Xenopus oocytes expressing the thrombin receptor or PAR-2 to agonist peptides or enzymes. Thrombin receptors responded to thrombin, the human thrombin receptor-activating peptide SFLLRNP-NH2 (TRAP) (EC50 = 0.1 microM), and Xenopus TRAP, TFRIFD-NH2 (EC50 = 1 microM), but did not show any increase in calcium efflux over control levels with trypsin (50 nM) or PAR-2 agonist peptides (100 microM). Human and murine PAR-2 receptors responded comparably to human and murine PAR-2 agonist peptides (SLIGKVD and SLIGRL, respectively) (EC50 = 0.5-2.0 microM) and trypsin, but not to thrombin. PAR-2 was also found to be responsive to TRAP (EC50 = 1 microM) but was unresponsive to Xenopus TRAP (50 microM). Responses to additional peptide agonist analogs suggest that an amino-terminal serine is critical for PAR-2 agonist activity.

The proteinase-activated receptor 2 is induced by inflammatory mediators in human endothelial cells. Comparison with the thrombin receptor.

The proteinase-activated receptor 2 (PAR-2) belongs to the family of seven transmembrane region receptors, and, like the related thrombin receptor, it is activated by specific proteolytic cleavage of its extracellular amino terminus. It is not known which proteinase is the physiological activator of the PAR-2, but candidates can be found among the enzymes involved in the inflammatory cascade systems. Here, we have studied the effects of various mediators on the expression of the PAR-2 and the thrombin receptor in cultured human umbilical vein endothelial cells. Stimulation with the cytokines tumor necrosis factor alpha or interleukin-1 alpha as well as bacterial lipopolysaccharide elevated the expression of PAR-2 in a dose-dependent manner. The time course of induction after cytokine stimulation was similar to those published for the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. After 20 h of stimulation, PAR-2 mRNA and protein levels were increased to 5-10-fold basal values, and, in the continued presence of tumor necrosis factor alpha, PAR-2 mRNA expression was found to remain elevated for up to 4 days. In contrast, the thrombin receptor gene was not induced by any of these inflammatory mediators. The responses to phorbol ester treatment also differed between the two genes. Thrombin receptor mRNA levels decreased steadily up to 20 h, whereas PAR-2 mRNA levels first rose to about 3-fold basal values at 4 h before decreasing again. Cell surface protein levels of both receptors were decreased after 20 h of phorbol ester stimulation. Elevating intracellular cAMP levels by treatment with forskolin resulted in decreased expression of both receptors, and inhibition of cAMP degradation appeared to blunt the cytokine-induced increase in PAR-2 expression. The induction of the PAR-2 by cytokine treatment supports the concept of PAR-2 involvement in the acute inflammatory response.

Family-oriented support at the onset of diabetes mellitus: a comparison of two group conditions during 2 years following diagnosis.

As part of a prospective, randomized study, the psychological effects of two different treatment regimens on the diagnosis of children and adolescents aged 3-15 years with insulin-dependent diabetes insipidus were evaluated. Conventional treatment was compared to a new regimen with a crisis programme which included a milieu therapeutic setting. A total of 38 families were randomly assigned to the 2 groups and followed over a period of 2 years after initial treatment. Parents' experiences of family climate and function over this period were registered and a test battery for the children was administered on five separate occasions. No decisive difference between the two groups was found. Few significant differences were found. Further investigation of the effects of the new treatment regimen on selected groups of families with defined extra needs is suggested.

Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2.

We previously reported the molecular cloning of a mouse guanosine-nucleotide-binding-protein-coupled receptor similar to the thrombin receptor. Since the physiological agonist was unknown, the receptor was named proteinase-activated receptor 2. We describe here the cloning and functional expression of the gene encoding the corresponding human receptor. The gene is divided into two exons separated by about 14 kb intronic DNA. The deduced protein sequence is 397 amino acids long and 83% identical to the mouse receptor sequence. Within the extracellular amino terminus, the residues predicted to form the tethered agonist ligand differ between the two receptors; of the first six residues only four are conserved. At positions five and six, a lysine residue and a valine residue, respectively, have replaced arginine and leucine residues found in the mouse sequence. When the human receptor is expressed in Chinese hamster ovary cells, it can be activated by low nanomolar concentrations of the serine proteinase trypsin and by peptides made from the receptor sequence. Northern-blot analysis of receptor expression showed that the receptor transcript is widely expressed in human tissues with especially high levels in pancreas, liver, kidney, small intestine and colon. Moderate expression was detected in many organs but none in brain or skeletal muscle. By fluorescence in situ hybridization, the human proteinase-activated receptor 2 gene was mapped to chromosomal region 5q13, where, previously, the related thrombin receptor gene has been located.

Molecular characterization of the mouse prostanoid EP1 receptor gene.

A partial cDNA, corresponding to the mouse prostaglandin E2 receptor subtype EP1, was isolated from mouse brain cDNA using a degenerate primer PCR strategy. Using the cDNA fragment as a probe, the EP1 receptor gene was isolated and characterized. The gene consists of three exons, of which the first is non-coding, and is contained within a 3.5-kb region. The coding nucleotide sequence determined is identical to that of the published mouse EP1 cDNA. The positions of the introns correspond to those of the thromboxane A2 and prostaglandin D receptor genes. No alternative splicing of the EP1 receptor gene could be detected. PCR and specific primers designed from the genomic sequence were used to amplify the coding part of the isolated gene from kidney cDNA. The cDNA obtained was cloned into a eukaryotic expression vector, and stably transfected Chinese hamster ovary cell lines were established. The cells respond to prostaglandin E2 with intracellular Ca2+ mobilization, as expected for this prostanoid receptor subtype. In situ hybridization was used to localize the EP1 receptor transcript in different mouse tissues. Significant hybridization was detected only in the collecting ducts of the kidney, and in the paraventricular and supraoptic nuclei of the hypothalamus. The expression of the EP1 receptor in the hypothalamus suggests that this prostanoid receptor is involved in mediating the fever response evoked by prostaglandin E2.

The mouse proteinase-activated receptor-2 cDNA and gene. Molecular cloning and functional expression.

We have reported the cloning from mouse genomic DNA of a fragment encoding a G-protein-coupled receptor related to the receptor for the blood clotting enzyme thrombin. Like the thrombin receptor this receptor is activated by proteolytic cleavage of its extracellular amino terminus. Because the physiological agonist at the receptor was unknown, we provisionally named it proteinase-activated receptor 2 (PAR-2). Here we present a PAR-2 cDNA of 2729 nucleotides that differs from the published genomic sequence at the 5' end, including a part of the protein coding region. The differences do not affect the peptide sequence of the activating proteinase cleavage site proper, but may include amino acid residues important for enzyme-substrate recognition. Analysis of the PAR-2 gene structure showed that the cDNA 5' end is derived from a separate exon located about 10 kilobases away from the 3' exon. Results from a primer extension experiment indicate that transcription starts at a unique site around nucleotide -203 respective to the translation initiation ATG. Chinese hamster ovary cells transfected with either the PAR-2 cDNA or a construct made from the published PAR-2 genomic sequence responded with intracellular calcium mobilization to stimulation with 1 nM trypsin, 10 microM PAR-2-activating peptide (SLIGRL), or 1 microM thrombin receptor-activating peptide (SFLLRN). Untransfected cells responded only to stimulation with thrombin receptor activating peptide. Only transcripts corresponding to the PAR-2 cDNA could be detected in three mouse tissues examined.

Molecular cloning of a potential proteinase activated receptor.

A DNA sequence encoding a G-protein-coupled receptor was isolated from a mouse genomic library. The predicted protein is similar in structure to the thrombin receptor and has a similar activation mechanism. When expressed in Xenopus laevis oocytes, the receptor was activated by low concentrations of trypsin (EC 3.4.21.4) and by a peptide (SLIGRL) derived from the receptor sequence, but was not activated by thrombin (EC 3.4.21.5). Trypsin failed to activate a mutant receptor in which the presumed cleavage site Arg-34-Ser-35 was changed to an Arg-Pro sequence. The agonist peptide (SLIGRL) activated equally well mutant and wild-type receptors. Northern blot analysis demonstrated receptor transcripts in highly vascularized tissues such as kidney, small intestine, and stomach. Because this, to our knowledge, is the second example, besides the thrombin receptor, of a proteolytically activated seven-transmembrane G-protein-coupled receptor, we have provisionally named it proteinase activated receptor 2.

Expression of cellular retinoid-binding proteins during normal and abnormal epidermal differentiation.

Retinoids have important roles in growth and differentiation of epidermal cells. We have analyzed the expression of two intracellular retinoid-binding proteins, the cellular retinol-binding protein type I and the cellular retinoic acid-binding protein type I, during normal and abnormal epidermal differentiation. Both proteins were found to be expressed in normal epidermis with increasing expression from basal layer towards superficial layers. In psoriatic lesions, a hyperproliferative condition of the skin, the epidermal expression of cellular retinol-binding protein I was induced, whereas expression of cellular retinoic acid-binding protein I was sharply down-regulated. This and other features of psoriatic lesions indicate that down-regulation of cellular retinoic acid-binding protein I expression might cause aberrant retinoid-regulated gene expression in skin. In basal and squamous cell carcinomas, cellular retinoic acid-binding protein I expression was down-regulated, whereas cellular retinol-binding protein I was expressed. Apart from epidermal cells, a mesenchymal, dendritic cell-type, strongly expressing cellular retinoic acid-binding protein I, was identified in the dermis. In several hyperproliferative conditions of the skin, including psoriasis, and squamous and basal cell carcinomas, this cell type was abundant. These results have implications for the role of retinoids in normal and abnormal epidermal differentiation and suggest that part of the phenotype of psoriasis is due to inappropriate metabolism of retinoic acid in skin.

Molecular cloning of the murine substance K and substance P receptor genes.

The peptides substance K and substance P evoke a variety of biological responses via distinct, guanosine-nucleotide-binding-regulatory-protein-coupled receptors. We have screened a murine genomic cosmid library using oligonucleotide probes and have isolated, cloned and characterized the substance K receptor and the substance P receptor genes. The coding portion of the substance K receptor gene consists of five exons distributed over 13 kbp. The substance P receptor gene is considerably larger than that of substance K (more than 30 kbp), however, the boundaries of the four exons that have been characterized in the substance P receptor gene correspond exactly to the homologous exons in the substance K receptor gene. To verify the identity of the isolated genes, we have cloned the corresponding cDNA by means of the polymerase chain reaction and we have expressed these cDNA species in Xenopus laevis oocytes. The ligand binding characteristics determined in this system pharmacologically confirm the identity of the two receptors. The deduced amino acid sequence of the mouse substance K receptor is 94% identical to the rat sequence and 85% identical to the bovine and human sequences. The mouse substance P receptor amino acid sequence is 99% identical to the rat sequence. The cloning of the murine substance K and substance P receptor genes should contribute substantially to the generation of in vivo models for the detailed analysis of the functional significance of these receptors.

Isolation and characterization of a cDNA clone corresponding to bovine cellular retinoic-acid-binding protein and chromosomal localization of the corresponding human gene.

A bovine adrenal cDNA library was constructed and a clone corresponding to cellular retinoic-acid-binding protein (CRABP) mRNA was isolated and sequenced. The insert of the clone corresponds to 75 bp of the 5' untranslated portion, the whole translated and the complete 3' untranslated portion of the bovine CRABP mRNA. A genomic Southern blot, probed with CRABP cDNA, indicated that only one copy of the gene is present in the human genome. Hybridizing bands in restricted chicken and fish DNA were also observed. Using the CRABP cDNA as probe we have located the human CRABP gene to chromosome 3 in hybridizations to mouse-human, hamster-human and rat-human cell hybrids. In situ hybridizations on rat testis cells probed with CRABP and cellular retinol-binding protein antisense mRNA indicate that both proteins are expressed in tubuli cells.

The complete amino acid sequence of rat beta 2-microglobulin.

The primary structure of rat beta 2-microglobulin (beta 2m) was determined. It is a polypeptide of 99 amino acids with the following sequence: IQKTPQIQVY SRHPPENGKP NFLNCYVSQF HPPQIEIELL KNGKKIPNIE MSDLSFSKDW SFYILAHTEF TPTETDVYAC RVKHVTLKEP KTVTWDRDM. The primary structure was determined by NH2-terminal sequence analysis together with sequence determination of one cyanogen bromide fragment and one tryptic peptide. Of other known beta 2m sequences, rat beta 2m is most homologous to mouse beta 2m (83% identity). The rabbit, human, and guinea pig sequences are more distant, with 24, 27, and 31% differences, respectively.

Quantitation and tissue localization of the cellular retinoic acid-binding protein.

The distribution of the cellular retinoic acid-binding protein (CRABP) in some rat tissues has been determined, and the protein has been localized by immunocytochemical techniques in sections from rat testis. In the testis CRABP was found in the seminiferous tubuli with Sertoli cells and the spermatogonia most intensely stained. All other cells of the germinal epithelium appeared largely devoid of CRABP. By use of an enzyme-linked immunosorbent assay CRABP was quantitatively estimated in several tissues and the highest levels were found in testis and eye. Comparisons of the tissue levels of CRABP and of the cellular retinol-binding protein (CRBP) did not reveal any apparent correlation.