Care pathways in atopic dermatitis: a retrospective population-based cohort study.

BACKGROUND: Atopic dermatitis (AD) is a complex disease with variations in severity and healthcare utilization. Examining patient pathways through analyses of longitudinal patient data provides an opportunity to describe real-world clinical patient care and evaluate healthcare access and treatment. OBJECTIVE: To describe longitudinal care pathways including health care management, treatment patterns and disease progression (by proxy measures) in patients with AD. MATERIALS AND METHODS: This was a longitudinal observational study, which used linked data from national and regional healthcare registers in Sweden. Patients with AD were identified through diagnosis in primary or secondary care or by dispensed medications. Descriptive statistics for number of healthcare visits, type of dispensed drug class, rate of - and time to - referral to secondary care and treatment escalation were calculated. RESULTS: A total of 341 866 patients with AD distributed as 197 959 paediatric (age < 12), 36 133 adolescent (age ≥ 12- < 18) and 107 774 adult (age ≥ 18) patients were included in this study. Healthcare visits to primary and secondary care and dispensation of AD-indicated treatments were more common during the year in which managed AD care was initiated. Topical corticosteroids (TCSs) and emollients were the most frequently used treatments across all age cohorts while systemic treatment was uncommon in all age cohorts. Among patients who initiated treatment with TCSs, 18.2% escalated to TCSs with higher potency following the start of managed AD care. CONCLUSIONS: We found that healthcare contacts and use of AD-indicated treatments were concentrated in the year during which managed AD care was initiated and decreased significantly thereafter. Since a significant proportion of patients with AD have flares and persistent AD, our results suggest that patients with AD may be monitored infrequently and are undertreated. There is a need to inform practitioners about adequate treatment options to provide individualized care, in particular for patients with persistent severe AD.

A novel economic framework to assess the cost-effectiveness of bone-forming agents in the prevention of fractures in patients with osteoporosis.

A novel cost-effectiveness model framework was developed to incorporate the elevated fracture risk associated with a recent fracture and to allow sequential osteoporosis therapies to be evaluated. Treating patients with severe osteoporosis after a recent fracture with a bone-forming agent followed by antiresorptive therapy can be cost-effective compared with antiresorptive therapy alone. Incorporating these novel technical attributes in economic evaluations can support appropriate policy and reimbursement decision-making. PURPOSE: To develop a cost-effectiveness model accommodating increased fracture risk after a recent fracture and treatment sequencing. METHODS: A micro-simulation cost-utility model was developed to accommodate both treatment sequencing and increased risk with recent fracture. The risk of fracture was estimated and simulated using the FRAX® algorithms combined with Swedish registry data on imminent fracture relative risk. In the base-case cost-effectiveness analysis, a sequential treatment starting with a bone-forming agent for 12 months followed by an antiresorptive agent for 48 months initiated immediately after a major osteoporotic fracture (MOF) in a 70-year-old woman with a T-score of 2.5 or less was compared to an antiresorptive treatment alone for 60 months. The model was populated with data relevant for a UK population reflecting a personal social service perspective. RESULTS: The cost per additional quality-adjusted life year (QALY) gained in the base-case setting was estimated at £34,584. Sensitivity analyses revealed the sequential treatment to be cost-saving compared with administering a bone-forming treatment alone. Without simulating an elevated fracture risk immediately after a recent fracture, the cost per QALY changed from £34,584 to £62,184. CONCLUSION: Incorporating imminent fracture risk in economic evaluations has a significant impact on the cost-effectiveness when evaluating fracture prevention treatments in patients with osteoporosis who sustained a recent fracture. Bone-forming treatment followed by antiresorptive therapy can be cost-effective compared to antiresorptive therapy alone depending on treatment acquisition costs.

Cost-effectiveness of romosozumab for the treatment of postmenopausal women with severe osteoporosis at high risk of fracture in Sweden.

Romosozumab is a novel bone-building drug that reduces fracture risk. This health economic analysis indicates that sequential romosozumab-to-alendronate can be a cost-effective treatment option for postmenopausal women with severe osteoporosis at high risk of fracture. PURPOSE: To estimate the cost-effectiveness of sequential treatment with romosozumab followed by alendronate ("romosozumab-to-alendronate") compared with alendronate alone in patients with severe osteoporosis at high risk of fracture in Sweden. METHODS: A microsimulation model with a Markov structure was used to simulate fractures, costs, and quality-adjusted life years (QALYs), for women treated with romosozumab-to-alendronate or alendronate alone. Patients aged 74 years with a recent major osteoporotic fracture (MOF) were followed from the start of treatment until the age of 100 years or death. Treatment with romosozumab for 12 months was followed by alendronate for up to 48 months or alendronate alone with a maximum treatment duration of 60 months. The analysis had a societal perspective. Efficacy of romosozumab and alendronate were derived from phase III randomized controlled trials. Resource use and unit costs were collected from the literature. Cost-effectiveness was estimated using incremental cost-effectiveness ratio (ICER) with QALYs as effectiveness measures. RESULTS: The base case analysis showed that sequential romosozumab-to-alendronate treatment was associated with 0.089 additional QALYs at an additional cost of €3002 compared to alendronate alone, resulting in an ICER of €33,732. At a Swedish reference willingness-to-pay per QALY of €60,000, romosozumab-to-alendronate had a 97.9% probability of being cost-effective against alendronate alone. The results were most sensitive to time horizon, persistence assumptions, patient age, and treatment efficacy. CONCLUSION: The results of this study indicate that sequential romosozumab-to-alendronate can be a cost-effective treatment option for postmenopausal women with severe osteoporosis at high risk of fracture.

Enabling sense-making for patients receiving outpatient palliative treatment: A participatory action research driven model for person-centered communication.

OBJECTIVES: In clinical palliative cancer care, the diversity of patient concerns over time makes information provision a critical issue, the demands of information-seeking patients presenting a challenge to both the communicative and organizational skills of the health provider. This study puts forward a practice model for communication between patients, their family members, and professional health providers during ongoing palliative chemotherapy; a model which supports the providers in enabling person-centered communication. METHOD: A constant comparative analysis adapted to participatory action research was applied. The model was developed step-wise in three interrelated cycles, with results from previous studies from palliative cancer care processed in relation to professional health providers' experience-based clinical knowledge. In doing this, focus group discussions were carried out with providers and patients to develop and revise the model. RESULTS: The Enabling Sense Making model for person-centered communication gave rise to three domains (which are also the major communicative actors in palliative care): the patient, the family, and the provider. These actors were placed in the context of a communicative arena. The three respective domains were built up in different layers discriminating between significant aspects of person-centered communication, from the manifest that is most usually explicated in dialogues, to the latent that tends to be implicitly mediated. SIGNIFICANCE OF RESULTS: The model intends to facilitate timely reorientation of care from curative treatment or rehabilitation to palliation, as well as the introduction of appropriate palliative interventions over time during palliative phases. In this way the model is to be regarded a frame for directing the awareness of the professionals, which focuses on how to communicate and how to consider the patient's way of reasoning. The model could be used as a complement to other strategic initiatives for the advancement of palliative care communication. It needs to be further evaluated in regard to practice evidence.

7B2 facilitates the maturation of proPC2 in neuroendocrine cells and is required for the expression of enzymatic activity.

The prohormone convertase PC2, which is thought to mediate the proteolytic conversion of many peptide hormones, has recently been shown to interact with the neuroendocrine-specific polypeptide 7B2 in Xenopus intermediate lobe (Braks, J. A. M., and G. J. M. Martens. Cell. 78:263. 1994). In the present work we have stably transfected neuroendocrine cell lines with rat 7B2 constructs and found that overexpression of 27 kD 7B2 greatly facilitates the kinetics of maturation of proPC2, both in AtT-20/PC2 cells and in Rin5f cells. The half-life of conversion of proPC2 was reduced from 2.7 to 1.7 h in AtT-20/PC2 cells stably transfected with 27 kD 7B2 cDNA. The previously proposed "chaperone" domain was not sufficient for this facilitation event; however, a construct corresponding to the 21-kD 7B2 protein (which represents the naturally occurring maturation product) functioned well. A 7B2 construct in which maturation of 27 kD 7B2 to its 21-kD form was blocked was unable to facilitate maturation of proPC2. To correlate effects on PC2 maturation with the actual generation of PC2 enzymatic activity, a similar transfection of 21 kD 7B2 was performed using CHO cells previously amplified for the expression of proPC2. Enzymatic activity cleaving the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC was highly correlated with the expression of immunoreactive 21 kD 7B2 in the conditioned medium; medium obtained from the parent cell line was completely inactive. Enzymatic activity was identified as PC2 on the basis of inhibition by the carboxy-terminal peptide of 7B2, which has previously been shown to represent a potent and specific PC2 inhibitor. Taken together, our in vivo results indicate that the interesting secretory protein 7B2 is a bifunctional molecule with an amino-terminal domain involved in proPC2 transport as well as activation.

Mechanism of the facilitation of PC2 maturation by 7B2: involvement in ProPC2 transport and activation but not folding.

Among the members of the prohormone convertase (PC) family, PC2 has a unique maturation pattern: it is retained in the ER for a comparatively long time and its propeptide is cleaved in the TGN/ secretory granules rather than in the ER. It is also unique by its association with the neuroendocrine protein 7B2. This interaction results in the facilitation of proPC2 maturation and in the production of activatable proPC2 from CHO cells. In the present study, we have investigated the mechanism of this interaction. ProPC2 binds 7B2 in the ER, but exits this compartment much more slowly than 7B2. We found that proPC2 was also slow to acquire the capacity to bind 7B2, whereas 7B2 could bind proPC2 rapidly after synthesis. This indicated that proPC2 folding was the limiting step in the formation of the complex. Indeed, sensitivity of native proPC2 to N-glycanase F digestion and inhibition of proPC2 folding supported the notion that 7B2 is not involved in the early steps of proPC2 folding, and that proPC2 must fold before binding 7B2. Under experimental conditions that prevent propeptide cleavage, 7B2 expression increased proPC2 transport to the Golgi. This increase exhibited the same kinetics as the facilitation of the removal of the propeptide. Finally, proPC2 activation could be reconstituted in Golgi- enriched subcellular fractions. In vitro, 7B2 was required for proPC2 activation at an acidic pH. Taken together, our results demonstrate that rather than promoting proPC2 folding, 7B2 acts as a helper protein involved in proPC2 transport and is required in the proPC2 activation process. We propose, therefore, that 7B2 stabilizes proPC2 in a conformation already competent for these two events.

Detection, distribution, and genetic variability of European mountain ash ringspot-associated virus.

European mountain ash ringspot-associated virus (EMARAV) was recently characterized from mountain ash (rowan) (Sorbus aucuparia) in Germany. The virus belongs tentatively to family Bunyaviridae but is not closely related to any classified virus. How commonly EMARAV occurs in ringspot disease (EMARSD) affected mountain ash trees was not reported and was investigated here. Virus-specific detection tools such as reverse transcription-polymerase chain reaction and dot blot hybridization using digoxigenin-labeled RNA probes were developed to test 73 mountain ash trees including 16 trees with no virus-like symptoms from 16 districts in Finland and Viipuri, Russia. All trees were infected with EMARAV. Hence, EMARAV is associated with EMARSD and can also cause latent infections in mountain ash. Symptom expression and the variable relative concentrations of viral RNA detected in leaves showed no correlation. Infectious EMARAV was detected also in dormant branches of trees in winter. Subsequently, genetic variability, geographical differentiation, and evolutionary selection pressures were investigated by analyzing RNA3 sequences from 17 isolates. The putative nucleocapsid (NP) gene sequence (944 nucleotides) showed little variability (identities 97 to 99%) and was under strong purifying selection. Amino acid substitutions were detected in two positions at the N terminus and one position at the C terminus of NP in four isolates. The 3' untranslated region (442 nucleotides) was more variable (identities 94 to 99%). Six isolates from a single sampling site exhibited as wide a genetic variability as isolates from sites that were hundreds of kilometers apart and no spatial differentiation of populations of EMARAV was observed.

SGNE1/7B2 is epigenetically altered and transcriptionally downregulated in human medulloblastomas.

In a genome-wide screen using differential methylation hybridization (DMH), we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in medulloblastomas compared to fetal cerebellum. Bisulfite sequencing and combined bisulfite restriction assay were performed to confirm the methylation status of this CpG island in primary medulloblastomas and medulloblastoma cell lines. Hypermethylation was detected in 16/23 (70%) biopsies and 7/8 (87%) medulloblastoma cell lines, but not in non-neoplastic fetal (n=8) cerebellum. Expression of SGNE1 was investigated by semi-quantitative competitive reverse transcription-polymerase chain reaction and found to be significantly downregulated or absent in all, but one primary medulloblastomas and all cell lines compared to fetal cerebellum. After treatment of medulloblastoma cell lines with 5-aza-2'-deoxycytidine, transcription of SGNE1 was restored. No mutation was found in the coding region of SGNE1 by single-strand conformation polymorphism analysis. Reintroduction of SGNE1 into the medulloblastoma cell line D283Med led to a significant growth suppression and reduced colony formation. In summary, we have identified SGNE1 as a novel epigenetically silenced gene in medulloblastomas. Its frequent inactivation, as well as its inhibitory effect on tumor cell proliferation and focus formation strongly argues for a significant role in medulloblastoma development.

The cell biology of the prohormone convertases PC1 and PC2.

Mature peptide hormones and neuropeptides are typically synthesized from much larger precursors and require several posttranslational processing steps--including proteolytic cleavage--for the formation of the bioactive species. The subtilisin-related proteolytic enzymes that accomplish neuroendocrine-specific cleavages are known as prohormone convertases 1 and 2 (PC1 and PC2). The cell biology of these proteases within the regulated secretory pathway of neuroendocrine cells is complex, and they are themselves initially synthesized as inactive precursor molecules. ProPC1 propeptide cleavage occurs rapidly in the endoplasmic reticulum, yet its major site of action on prohormones takes place later in the secretory pathway. PC1 undergoes an interesting carboxyl terminal processing event whose function appears to be to activate the enzyme. ProPC2, on the other hand, exhibits comparatively long initial folding times and exits the endoplasmic reticulum without propeptide cleavage, in association with the neuroendocrine-specific protein 7B2. Once the proPC2/7B2 complex arrives at the trans-Golgi network, 7B2 is internally cleaved into two domains, the 21-kDa fragment and a carboxy-terminal 31 residue peptide. PC2 propeptide removal occurs in the maturing secretory granule, most likely through autocatalysis, and 7B2 association does not appear to be directly required for this cleavage event. However, if proPC2 has not encountered 7B2 intracellularly, it cannot generate a catalytically active mature species. The molecular mechanism behind the intriguing intracellular association of 7B2 and proPC2 is still unknown, but may involve conformational rearrangement or stabilization of a proPC2 conformer mediated by a 36-residue internal segment of 21-kDa 7B2.

PCSK1 Variants and Human Obesity.

PCSK1, encoding prohormone convertase 1/3 (PC1/3), was one of the first genes linked to monogenic early-onset obesity. PC1/3 is a protease involved in the biosynthetic processing of a variety of neuropeptides and prohormones in endocrine tissues. PC1/3 activity is essential for the activating cleavage of many peptide hormone precursors implicated in the regulation of food ingestion, glucose homeostasis, and energy homeostasis, for example, proopiomelanocortin, proinsulin, proglucagon, and proghrelin. A large number of genome-wide association studies in a variety of different populations have now firmly established a link between three PCSK1 polymorphisms frequent in the population and increased risk of obesity. Human subjects with PC1/3 deficiency, a rare autosomal-recessive disorder caused by the presence of loss-of-function mutations in both alleles, are obese and display a complex set of endocrinopathies. Increasing numbers of genetic diagnoses of infants with persistent diarrhea has recently led to the finding of many novel PCSK1 mutations. PCSK1-deficient infants experience severe intestinal malabsorption during the first years of life, requiring controlled nutrition; these children then become hyperphagic, with associated obesity. The biochemical characterization of novel loss-of-function PCSK1 mutations has resulted in the discovery of new pathological mechanisms affecting the cell biology of the endocrine cell beyond simple loss of enzyme activity, for example, dominant-negative effects of certain mutants on wild-type PC1/3 protein, and activation of the cellular unfolded protein response by endoplasmic reticulum-retained mutants. A better understanding of these molecular and cellular pathologies may illuminate possible treatments for the complex endocrinopathy of PCSK1 deficiency, including obesity.

Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells.

A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Enkephalins in the rat pituitary gland: immunohistochemical and biochemical observations.

The immunohistochemical distribution of opioid peptides derived from proenkephalin A in the rat pituitary was studied by indirect immunofluorescence; immunoreactive peptides were also characterized by column chromatography followed by specific RIAs. Nerve terminals in the neural lobe were immunoreactive (ir) for Tyr-Gly-Gly-Phe-Met-Arg-Phe (YGGFMRF), Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu (YGGFMRGL), and met-enkephalin [Tyr-Gly-Gly-Phe-Met (YGGFM)]. All cells in the intermediate lobe were ir for YGGFMRF, while only occasional cells exhibited YGGFMRGL-like immunoreactivity, and YGGFM-ir cells were not detected in this lobe. In the anterior lobe, some large ovoid cells, identified as gonadotrophs, were immunoreactive for enkephalins. The number of YGGFMRF-ir cells was larger than the number of YGGFMRGL- and YGGFM-ir cells, and these opioid peptides were present in cells that did not contain beta-endorphin immunoreactivity. Twenty times more YGGFMRF than YGGFMRGL-immunoreactivity was present in the anterior lobe, whereas the neurointermediate lobe obtained 4 times more ir YGGFMRF than YGGFMRGL. Pituitary lobe extracts contained substantial amounts of high mol wt forms of ir YGGFMRF and YGGFMRGL, but not of YGGFM or Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu). Low mol wt ir peptides present in both lobes consisted largely of the authentic peptides when analyzed by HPLC; however, an unidentified YGGFMRF-ir peptide was also detected. The results indicate that the proenkephalin A molecule may be processed differentially in the various compartments of the pituitary gland and that opioid peptides derived from this precursor may have functional roles in all three lobes. The relatively large amount of YGGFMRF immunoreactivity, which was detected both biochemically and immunohistochemically, indicates that YGGFMRF-ir peptides may be important proenkephalin A-derived products in the pituitary gland.

Cleavage of proenkephalin by a chromaffin granule processing enzyme.

Human proenkephalin generated by means of a recombinant vaccinia virus expression vector was used as the substrate for a putative processing enzyme obtained from bovine adrenal chromaffin granules. The adrenal enzyme successfully cleaved proenkephalin to generate low mol wt enkephalins as well as other enkephalin-containing intermediates. Radioactively labeled proenkephalin prepared with this system was also cleaved; however, under identical conditions bovine proinsulin was not cleaved. These results provide support for the notion that the adrenal trypsin-like enzyme is involved in the processing of proenkephalin in vivo and demonstrate the usefulness of protein substrates prepared by expression vector systems in testing the reactivity and specificity of proposed prohormone processing enzymes in vitro.

Partial purification and characterization of a putative prohormone-processing enzyme complex from bovine pituitary.

A putative prohormone-processing enzyme complex with specificity toward basic residues was partially purified from whole bovine pituitary glands. The complex is basic, binding to S-Sepharose at pH 8.2. The pH optimum of the enzyme is around 8.0. The enzyme is capable of cleaving proenkephalin and is present in at least three forms with relative molecular masses of about 36,000, 58,000, and 90,000 Da. The proteinase complex is inhibited by soybean trypsin inhibitor, limabean trypsin inhibitor, and aprotinin, but not by inhibitors of thiol proteinases or metal chelators. Our results indicate that this proteinase is a trypsin-like serine esterase with properties appropriate to that of a prohormone-processing enzyme.

Characterization of enkephalin-immunoreactive peptides generated by peptic digestion of rat plasma proteins.

Met-Enkephalin-immunoreactive peptides (MEIP) and Leu-enkephalin-immunoreactive peptides (LEIP), generated from plasma proteins after digestion with pepsin, were characterized with gel filtration, reverse phase HPLC, and isoelectric focusing. Pooled rat plasma was found to generate 170 pmol LEIP and 76 pmol MEIP/ml plasma. Two peaks of enkephalin (enk)-immunoreactive peptides (EIP) were observed after gel filtration of plasma digests; the early eluting peak (which contained 90% of the total LEIP and 75% of the total MEIP) eluted close to, but not in, the void volume of the column, whereas the elution volume of a later eluting peak (which contained 25% of the total MEIP and about 5% of the total LEIP) was close to that of authentic enk. Five peaks of EIP with pI values of 3.7, 5.8, 7.3, 8.0, and 9.4, were observed after isoelectric focusing of plasma digests; with the exception of the pI 3.7 peak, all of these immunoreactive species could be detected with greater efficiency using the Leu-enk RIA. Peptic digestion of rat serum albumin generated a MEIP with the same pI and HPLC retention time as the plasma pI 3.7 MEIP. No other EIP could be generated from rat serum albumin. No immunoreactive peptides were found which coeluted with synthetic Met-enk or its sulfoxide on HPLC; however, a portion of low mol wt LEIP was eluted with the retention time of authentic Leu-enk. With certain exceptions, these results support and extend the original findings of Singer and Carraway concerning the ability of pepsin to generate extremely high concentrations of EIPs from plasma protein(s).

Posttranslational processing of proenkephalin in AtT-20 cells: evidence for cleavage at a Lys-Lys site.

Proteolytic processing of proenkephalin was examined in several subclones of AtT-20 cells stably transfected with rat proenkephalin cDNA (AT/PE cells). Proenkephalin is synthesized in both N-glycosylated and unglycosylated forms, as demonstrated by treatment with tunicamycin. RIAs and Western blot studies showed that AT/PE clones process proenkephalin at some, but not all, Lys-Arg sequences in a limited processing profile reminiscent of bovine adrenal chromaffin cells. Pulse-chase studies using Met5-enkephalin-Arg-Gly-Leu antiserum demonstrated that 50% of the precursor is processed within 1 h, and processing is complete after 2.5 h with the production of the 5.3-kilodalton (kDa) peptide. Further cleavage to the octapeptide Met5-enkephalin-Arg-Gly-Leu is minimal. Radiosequencing results verified the efficient cleavage of a Lys-Lys site within proenkephalin that resulted in the production of the 5.3-kDa peptide. Proenkephalin cleavage products stored within cells, which included the 5.3-kDa peptide, could be released upon stimulation of cells with BaCl2 (2-fold above basal levels), 8-bromo-cAMP or CRF (7- and 8-fold above basal levels, respectively), and a mixture of BaCl2 and 8-bromo-cAMP (20-fold above basal levels). An important difference between the processing of proenkephalin and the ACTH/endorphin precursor (POMC) in AtT-20 cells is efficient cleavage of a Lys-Lys site in proenkephalin and not in POMC. The ability of AT/PE to process proenkephalin in a natural manner makes it a suitable model system to investigate elements involved in the processing of proenkephalin at Lys-Lys sites.

Proenkephalin gene expression in the PC12 pheochromocytoma cell line: stimulation by sodium butyrate.

The differentiation promoter sodium butyrate increases the content of Met5-enkephalin-Arg6-Gly7-Leu8 (Met5-enk-RGL)-immunoreactive peptides in PC12 pheochromocytoma cells, which, unlike mature adrenomedullary chromaffin cells, contain exceedingly low levels of opioid peptides. These butyrate-induced enkephalin-immunoreactive peptides, which are specific products of the proenkephalin gene, consist principally of two high mol wt forms of amino-terminally extended Met5-enk-RGL. These high mol wt peptides, with apparent mol wt of 20,000 and 10,000, are approximately the same size as the two major immunoreactive peptides found in adult New England Deaconess Hospital rat adrenal. The low mol wt Met5-enk-RGL-immunoreactive peptide found in butyrate-treated cells is similar in size to authentic Met5-enk-RGL, which is not found in the adrenal medulla of the adult rat. When PC12 cells are grown as a tumor in vivo, the amount of Met5-enk-RGL-immunoreactive peptide increased only slightly above the level found in control cells grown in vitro and consisted exclusively of the highest mol wt immunoreactive species. In PC12 cells, the butyrate-stimulated elevation in the content of Met5-enk-RGL-immunoreactive peptides may involve changes in transcription, since the peptide increase is preceded by a 2- to 3-fold increase in the level of proenkephalin mRNA. These results suggest that the PC12 cell line may be useful for investigating those factors that control the initial expression and processing of proenkephalin-derived peptides during embryogenesis.

Posttranslational modifications of rat proenkephalin overexpressed in Chinese hamster ovary cells.

Rat proenkephalin was overexpressed in Chinese hamster ovary cells using the dihydrofolate reductase-coupled genetic amplification method. About 2 mg purified protein could be obtained from 250 ml conditioned medium; multiple successive harvests could be obtained from the same roller bottle. Degradation of proenkephalin released into the conditioned medium was reduced significantly in the presence of 2% fetal bovine serum. Forty-eight percent of recombinant proenkephalin was glycosylated; glycosylation could be entirely prevented by the addition of tunicamycin. Two-dimensional isoelectric focusing experiments showed that recombinant proenkephalin exhibited considerable charge heterogeneity, with two major unglycosylated isoelectric forms and six or seven glycosylated isoelectric forms. The estimated isoelectric points of the major unglycosylated proenkephalins were 6.0 and 6.1, while glycosylated proenkephalins ranged in pI from 5.7-6.1. Some of this isoelectric heterogeneity is due to phosphorylation; [32P] orthophosphate was readily incorporated into serine residues within newly synthesized proenkephalin.

The SAAS granin exhibits structural and functional homology to 7B2 and contains a highly potent hexapeptide inhibitor of PC1.

Prohormone convertases (PCs) 1 and 2 are thought to mediate the proteolytic cleavage of many peptide precursors. Endogenous inhibitors of both PC1 and PC2 have now been identified; the 7B2 protein is a nanomolar inhibitor of PC2, while the novel protein proSAAS was recently reported to be a micromolar inhibitor of PC1 [Fricker et al. (2000) J. Neurosci. 20, 639-648]. We here report evidence that 7B2 and proSAAS exhibit several elements of structural and functional homology. Firstly, 26 kDa human, mouse and rat proSAAS, like all vertebrate 7B2s, contain a proline-rich sequence within the first half of the molecule and also contain a C-terminal 40 residue peptide (SAAS CT peptide) separated from the remainder of the protein by a furin consensus sequence. The SAAS CT peptide contains the precise sequence of a hexapeptide previously identified by combinatorial peptide library screening as a potent inhibitor of PC1, and the vast majority of the inhibitory potency of proSAAS can be attributed to this hexapeptide. Further, like the 7B2 CT peptide, SAAS CT-derived peptides represent tight-binding competitive convertase inhibitors with nanomolar potencies. Lastly, recombinant PC1 is able to cleave the proSAAS CT peptide to a product with a mass consistent with cleavage following the inhibitory hexapeptide. Taken together, our results indicate that proSAAS and 7B2 may comprise two members of a functionally homologous family of convertase inhibitor proteins.