Typology of employers offering line manager training for mental health.

BACKGROUND: Mental ill health has a high economic impact on society and employers. National and international policy advocates line manager (LM) training in mental health as a key intervention, but little is known about employer training provisions. AIMS: To explore the prevalence and characteristics of organizations that offer LM training in mental health. METHODS: Secondary analysis of existing longitudinal anonymised organizational-level survey data derived from computer-assisted telephone interview surveys collected in four waves (2020:1900 firms, 2021:1551, 2022:1904, 2023:1902) in England, before, during and after a global pandemic. RESULTS: The proportion of organizations offering LM training in mental health increased pre- to post-pandemic (2020:50%, 2023:59%) but 41% do not currently provide it. Logistic regression confirmed that LM training is more likely to be offered by large-sized enterprises, organizations with a larger proportion of employees who are younger (aged 25-49), female, disabled and from ethnic minority communities. Sector patterns were inconsistent, but in 2023, organizations from the 'Hospitality' and 'Business Services' sectors were more likely to provide LM training than other sectors. CONCLUSIONS: Continued efforts are needed to increase the proportion of employers offering LM training in mental health, particularly small- to medium-sized enterprises, and organizations with predominantly male, White and/or older workforces.

Studies of association between the gene for calpain-10 and type 2 diabetes mellitus in the United Kingdom.

Variation in CAPN10, the gene encoding the ubiquitously expressed cysteine protease calpain-10, has been associated with type 2 diabetes in Mexican Americans and in two northern-European populations, from Finland and Germany. We have studied CAPN10 in white subjects of British/Irish ancestry, using both family-based and case-control studies. In 743 sib pairs, there was no evidence of linkage at the CAPN10 locus, which thereby excluded it as a diabetes-susceptibility gene, with an overall sib recurrence risk, lambda(S), of 1.25. We examined four single-nucleotide polymorphisms (SNP-44, -43, -19, and -63) previously either associated with type 2 diabetes or implicated in transcriptional regulation of calpain-10 expression. We did not find any association between SNP-43, -19, and -63, either individually or as part of the previously described risk haplotypes. We did, however, observe significantly increased (P=.033) transmission of the less common C allele at SNP-44, to affected offspring in parents-offspring trios (odds ratio 1.6). An independent U.K. case-control study and a small discordant-sib study did not show significant association individually. In a combined analysis of all U.K. studies (P=.015) and in combination with a Mexican American study (P=.004), the C allele at SNP-44 is associated with type 2 diabetes. Sequencing of the coding region of CAPN10 in a group of U.K. subjects revealed four coding polymorphisms-L34V, T504A, R555C, and V666I. The T504A polymorphism was in perfect linkage disequilibrium with the diabetes-associated C allele at SNP-44, suggesting that the synthesis of a mutant protein and/or altered transcriptional regulation could contribute to diabetes risk. In conclusion, we were not able to replicate the association of the specific calpain-10 alleles identified by Horikawa et al. but suggest that other alleles at this locus may increase type 2 diabetes risk in the U.K. population.

A genomewide scan for loci predisposing to type 2 diabetes in a U.K. population (the Diabetes UK Warren 2 Repository): analysis of 573 pedigrees provides independent replication of a susceptibility locus on chromosome 1q.

Improved molecular understanding of the pathogenesis of type 2 diabetes is essential if current therapeutic and preventative options are to be extended. To identify diabetes-susceptibility genes, we have completed a primary (418-marker, 9-cM) autosomal-genome scan of 743 sib pairs (573 pedigrees) with type 2 diabetes who are from the Diabetes UK Warren 2 repository. Nonparametric linkage analysis of the entire data set identified seven regions showing evidence for linkage, with allele-sharing LOD scores > or =1.18 (P< or =.01). The strongest evidence was seen on chromosomes 8p21-22 (near D8S258 [LOD score 2.55]) and 10q23.3 (near D10S1765 [LOD score 1.99]), both coinciding with regions identified in previous scans in European subjects. This was also true of two lesser regions identified, on chromosomes 5q13 (D5S647 [LOD score 1.22] and 5q32 (D5S436 [LOD score 1.22]). Loci on 7p15.3 (LOD score 1.31) and 8q24.2 (LOD score 1.41) are novel. The final region showing evidence for linkage, on chromosome 1q24-25 (near D1S218 [LOD score 1.50]), colocalizes with evidence for linkage to diabetes found in Utah, French, and Pima families and in the GK rat. After dense-map genotyping (mean marker spacing 4.4 cM), evidence for linkage to this region increased to a LOD score of 1.98. Conditional analyses revealed nominally significant interactions between this locus and the regions on chromosomes 10q23.3 (P=.01) and 5q32 (P=.02). These data, derived from one of the largest genome scans undertaken in this condition, confirm that individual susceptibility-gene effects for type 2 diabetes are likely to be modest in size. Taken with genome scans in other populations, they provide both replication of previous evidence indicating the presence of a diabetes-susceptibility locus on chromosome 1q24-25 and support for the existence of additional loci on chromosomes 5, 8, and 10. These data should accelerate positional cloning efforts in these regions of interest.

PTEN and myotubularin phosphoinositide phosphatases: bringing bioinformatics to the lab bench.

Phosphoinositides play an integral role in a diverse array of cellular signaling processes. Although considerable effort has been directed toward characterizing the kinases that produce inositol lipid second messengers, the study of phosphatases that oppose these kinases remains limited. Current research is focused on the identification of novel lipid phosphatases such as PTEN and myotubularin, their physiologic substrates, signaling pathways and links to human diseases. The use of bioinformatics in conjunction with genetic analyses in model organisms will be essential in elucidating the roles of these enzymes in regulating phosphoinositide-mediated cellular signaling.

Ligneous conjunctivitis: a local manifestation of a systemic disorder?

Ligneous conjunctivitis is a descriptive term. It refers to the "woody" consistency of the pseudomembrane that usually forms on the palpebral conjunctiva of those affected. It is rare and probably only one manifestation of a multiorgan, pseudomembranous disease. (1-4) We report a case of ligneous conjunctivitis in which investigation revealed a plasminogen deficiency in the heterozygous range (previously reported only in association with a homozygous plasminogen deficiency). We suggest a strategy for investigating known and new cases of ligneous conjunctivitis and/or pseudomembranous disease.

Gathering STYX: phosphatase-like form predicts functions for unique protein-interaction domains.

The effects of tyrosine phosphorylation are manifested and regulated through protein domains that bind to specific phosphotyrosine motifs. STYX is a unique modular domain found within proteins implicated in mediating the effects of tyrosine phosphorylation in vivo. Individual STYX domains are not catalytically active; however, they resemble protein tyrosine phosphatase (PTP) domains and, like PTPs, contain core sequences that recognize phosphorylated substrates. Thus, the STYX domain adds to the repertoire of modular domains that can mediate intracellular signaling in response to protein phosphorylation.

A family of putative tumor suppressors is structurally and functionally conserved in humans and yeast.

In Saccharomyces cerevisiae the CDC14 gene is essential for cell cycle progression. Strains carrying the cdc14-1(ts) allele enter the cell cycle and arrest at restrictive temperatures. We have identified two human cDNAs encoding proteins which share sequence identity to the yeast CDC14p. The cell cycle arrest in cdc14-1(ts) can be specifically complemented by the human cDNAs suggesting that they are functionally equivalent to the yeast CDC14 gene. Another clone identified in the search for human CDC14-like proteins corresponded to the putative tumor suppressor gene PTEN/MMAC1 (phosphatase and tensin homologue deleted on chromosome 10 or mutated in multiple advanced cancers 1). Analysis of the PTEN/MMAC1 showed that it did not complement the cdc14-1(ts) allele and that it is more closely related to the yeast open reading frame YNL128W. Human CDC14p and PTEN/MMAC1 were expressed as recombinant proteins, and both were shown to have kinetic properties characteristic of dual specific phosphatases. The human CDC14p was localized in the nucleus while PTEN/MMAC1 has been reported to be localized in the cytoplasm. Our results suggest that CDC14 and YNL128W/PTEN/MMAC1 represent two related, but distinct, families of human and yeast phosphatases.

Purification and identification of a 28-kDa calcium-regulated heat-stable protein. A novel secretagogue-regulated phosphoprotein in exocrine pancreas.

This study reports the purification and identification of a novel 28 kDa phosphoprotein from rat pancreatic acini, previously described as being highly regulated by calcium mobilizing secretagogues, which we have designated calcium-regulated heat-stable protein 28 (CRHSP-28). Internal amino acid sequences of purified CRHSP-28 were obtained following trypsin digestion and found to match with >95% identity the predicted amino acid sequence of a novel cDNA recently identified as being highly expressed in human breast carcinomas. Verification that this cDNA codes for human CRHSP-28 was demonstrated by the ability of antiserum raised against purified rat CRHSP-28 to recognize the recombinant human protein when expressed in bacteria. Furthermore, this antibody was found to specifically react with CRHSP-28 in rat acini following one- and two-dimensional electrophoresis and underwent a marked acidic shift in mobility after cholecystokinin stimulation, a phenomenon indicative of an increase in its phosphorylation. CRHSP-28 is predicted to be extremely hydrophilic, is phosphorylated entirely on serine residues, and bears little homology to any known proteins. Finally, the distribution of the CRHSP-28 protein in various rat tissues revealed that although it was present at low levels in almost all tissues, it was most highly expressed in pancreas, followed by the gastric, intestinal, and colonic mucosa. In view of its relative abundance throughout the digestive system and its apparent regulation by calcium-mobilizing agents, this protein may provide valuable insight into the mechanism(s) of calcium signaling in these tissues.

Relationships between gastric emptying, intragastric meal distribution and blood glucose concentrations in diabetes mellitus.

UNLABELLED: The aim of this study was to evaluate the prevalence of disordered intragastric meal distribution and the relationships between gastric emptying, intragastric distribution, glycemic control and gastrointestinal symptoms in diabetes mellitus. METHODS: Eighty-six patients with diabetes mellitus had measurements of gastric emptying and intragastric distribution of a radioisotopically labeled solid/liquid meal (100 g beef and 150 ml 10% dextrose), glycemic control (plasma glucose concentrations), upper gastrointestinal symptoms (questionnaire) and autonomic nerve function (cardiovascular reflexes). Results were compared to those obtained in 20 normal volunteers. RESULTS: Solid and liquid gastric emptying were delayed in the diabetic patients and correlated weakly. Intragastric meal distribution was also often abnormal, with increased retention of both solid and liquid in the proximal stomach and increased retention of solid but not liquid in the distal stomach. In all patients with increased retention of solid in the proximal stomach, emptying from the total stomach was delayed. Gastric emptying of liquid was slower in those subjects who had a mean plasma glucose > 15 mmol/liter during the gastric emptying measurement, when compared to the remainder of the group. CONCLUSION: In patients with diabetes mellitus, there is poor relationship between solid and liquid gastric emptying and intragastric meal distribution is frequently abnormal. Interpretation of the results of gastric emptying measurements should consider meal composition and plasma glucose concentrations.

A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase.

Dual-specificity protein-tyrosine phosphatases (dsPTPases) have been implicated in the inactivation of mitogen-activated protein kinases (MAPKs). We have identified a novel phosphoserine/threonine/tyrosine-binding protein (STYX) that is related in amino acid sequence to dsPTPases, except for the substitution of Gly for Cys in the conserved dsPTPase catalytic loop (HCXXGXXR(S/T)). cDNA subcloning and Northern blot analysis in mouse shows poly(A+) hybridization bands of 4.6, 2.4, 1.5, and 1.2 kilobases, with highest abundance in skeletal muscle, testis, and heart. Polymerase chain reaction amplification of reverse-transcribed poly(A+) RNA revealed an alternatively spliced form of STYX containing a unique carboxyl terminus. Bacterially expressed STYX is incapable of hydrolyzing Tyr(P)-containing substrates; however, mutation of Gly120 to Cys (G120C), which structurally mimics the active site of dsPTPases, confers phosphatase activity to this molecule. STYX-G120C mutant hydrolyzes p-nitrophenyl phosphate and dephosphorylates both Tyr(P) and Thr(P) residues of peptide sequences of MAPK homologues. The kinetic parameters of dephosphorylation are similar to human dsPTPase, Vaccinia H1-related, including inhibition by vanadate. We believe this is the first example of a naturally occurring "dominant negative" phosphotyrosine/serine/threonine-binding protein which is structurally related to dsPTPases.

Secretagogue regulation of pancreatic acinar cell protein phosphorylation shown by large-scale 2D-PAGE.

High-resolution large-scale two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with computer-assisted image analysis was used to construct a database of secretagogue/second messenger-induced phosphoprotein modifications in intact rat pancreatic acinar cells. Isolated acini were labeled with 32Pi, exposed to hormones and other test agents, and subjected to large-scale 2D-PAGE and autoradiography. This procedure resolved 500 phosphoproteins in pancreatic acinar whole cell lysates, approximately 90% of which were localized in the soluble fraction of centrifuged samples. Soluble proteins were further characterized as to heat and acid stability. Cholecystokinin (CCK), carbachol, and bombesin altered the phosphorylation state of about 27 proteins with both increases and decreases observed. Subsets of proteins were phosphorylated in response to phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), calcium ionophore A-23187, and adenosine 3',5'-cyclic monophosphate (cAMP) analogue 8-bromo-cAMP. One of these proteins was identified as the myristoylated, alanine-rich, C-kinase substrate (MARCKS) protein by immunoprecipitation. The time course and dose response of phosphorylation changes due to CCK showed considerable variation between proteins, although a temporal hierarchy of phosphorylation events was clearly exhibited. Particularly striking was the rapid dephosphorylation within 30 s of a 19-kDa soluble protein to a minimum of 20 +/- 1% of control. Increased phosphorylation of the MARCKS and other TPA-regulated proteins suggests that CCK, carbachol, bombesin, and the CCK partial agonist, JMV-180, all activate protein kinase C in intact acini.

GP-3, a newly characterized glycoprotein on the inner surface of the zymogen granule membrane, undergoes regulated secretion.

We have recently reported the cloning of the rat zymogen granule membrane glycoprotein GP-3 and the related pancreatic secretory lipase (Wishart, M. J., Andrews, P. C., Nichols, R., Blevins, G. T., Logsdon, C.D., and Williams, J. A. (1993) J. Biol. Chem. 268, 10303-10311). Specific antipeptide antibodies were generated against both GP-3 and secretory lipase and used for the biochemical and physiological characterization of GP-3. Western blotting confirmed that GP-3 was found exclusively in zymogen granule membranes and was absent from zymogen granule content which contains the majority of secretory lipase. Extraction of zymogen granule membranes with Triton X-114 showed GP-3 to be significantly more hydrophobic than lipase. The GP-3 amino acid sequence contains one potential N-linked glycosylation site at Asn-336. The loss of concanavalin A labeling after both chemical deglycosylation with trifluoromethanesulfonic acid and enzymatic deglycosylation with N-glycanase showed GP-3 to possess a small N-linked oligosaccharide side chain. Digestion of intact and permeabilized zymogen granules with the nonspecific protease Pronase localized GP-3 to the inner surface of zymogen granule membranes. Since GP-3 is resident on the inner surface of the zymogen granule membrane, it should appear on the outer cellular surface after exocytosis. Although membrane attachment of GP-3 was resistant to treatment with phosphatidylinositol-specific phospholipase C, we observed that GP-3 is released into the pancreatic juice and that secretion of GP-3 was greatly enhanced by cholecystokinin.

Identification and cloning of GP-3 from rat pancreatic acinar zymogen granules as a glycosylated membrane-associated lipase.

The protein components of highly purified secretory granule membranes and the granule contents from rat exocrine pancreas were characterized by two-dimensional polyacrylamide gel electrophoresis, protein staining, lectin absorption, and Western blotting with anti-secretory protein antibodies. NH2-terminal amino acid sequence was obtained for a approximately 53-kDa glycoprotein denoted GP-3, present only in granule membrane preparations where it was resistant to washing with Na2CO3 and KBr. The sequence of this protein showed homology to pancreatic lipase but was distinct from the NH2-terminal sequence of a 50-kDa content protein presumed to be secretory lipase. Polymerase chain reaction amplification with degenerate oligonucleotide primers to GP-3 and secretory lipase gave partial length subclones that were used to isolate clones from a rat pancreas cDNA library. Dideoxy sequencing of full-length subclones of GP-3 revealed the predicted amino acid sequence for a mature protein of 452 amino acids with a potential N-linked glycosylation site and a deglycosylated molecular weight of 50,860. The GP-3 sequence possesses the serine esterase consensus sequence G-X-S-X-G centered around Ser154 and the catalytic state triad Asp178-His265-Ser154 characteristic of pancreatic lipases. Northern blot analysis of various rat tissues showed GP-3 expression solely in pancreas. Comparison of GP-3 nucleotide and amino acid sequence, along with pancreatic lipases of various species including rat, shows extensive homologies to both proteins and reveals an underlying diversity in the pancreatic lipase family. Close homology is observed between GP-3 and a lipase molecule previously isolated from mouse cytotoxic T cells.

Effects of okadaic acid indicate a role for dephosphorylation in pancreatic stimulus-secretion coupling.

Okadaic acid completely inhibits phosphatase 2A at nanomolar concentrations, while complete inhibition of type 1 phosphatases occurs at 1 microM. Phosphatase 2B is significantly inhibited only at concentrations > 1 microM. In rat pancreatic acini, 1 microM okadaic acid shifted the cholecystokinin (CCK) dose-response curve for stimulating amylase release to the right without reducing maximal secretion. At 3 microM, okadaic acid inhibited maximal CCK-induced amylase release to 78 +/- 7% of control, whereas the inactive analogue 1-Nor-okadaone had no effect. Three lines of evidence indicate that this inhibition by okadaic acid occurs at a late step in stimulus-secretion coupling: 1) intracellular Ca2+ signaling in response to agonist stimulation was not appreciably altered by okadaic acid; 2) stimulation with phorbol ester plus thapsigargin (thus by-passing receptor activation), which gave 85 +/- 4% of maximal CCK-induced amylase release, was inhibited 66 +/- 4% by 3 microM okadaic acid; and 3) Ca(2+)-induced amylase secretion in streptolysin O-permeabilized cells was also reduced by 85 +/- 7%. Two-dimensional polyacrylamide gel electrophoresis of 32P-labeled acini and autoradiography demonstrated that okadaic acid dose dependently increased overall protein phosphorylation. Correspondingly, okadaic acid also led to an inhibition of CCK-induced dephosphorylation. These results show that okadaic acid inhibits pancreatic acinar secretion at a step after generation of intracellular messengers and indicate a role for protein dephosphorylation in stimulus-secretion coupling.

Low molecular mass GTP-binding proteins in subcellular fractions of the pancreas: regulated phosphoryl G proteins.

Low molecular mass guanine nucleotide-binding proteins [small guanosine 5'-triphosphate (GTP)-binding proteins] and phosphoproteins of the pancreatic acinar cell were compared by two-dimensional gel electrophoresis. [35S]GTP alpha S blotting analysis of the total cell protein revealed 20 GTP-binding proteins ranging in molecular mass from 20 to 28 kDa and pI of 4.8-6.4. Analysis of 32P-labeled total cell protein revealed over 300 phosphoproteins. The subcellular distribution of the small GTP-binding proteins was examined: 17 were located in the rough endoplasmic reticulum (RER) fraction, 19 in the smooth microsome fraction, 14 in the zymogen granule membrane fraction, and 11 in the cytosolic fraction, with overlap between fractions. Of the GTP-binding proteins, two were also found to be phosphoproteins, one located on the RER and one on the zymogen granule membrane. The phosphorylation of both small GTP-binding proteins was increased by secretagogue stimulation of the cells but with different time courses. The RER small GTP-binding protein demonstrated a rapid and transient increase in 32P labeling, whereas the granule membrane small GTP-binding protein showed an increase at longer times (30 min). Two of the cytosolic small GTP-binding proteins were also seen in particulate fractions, especially in the zymogen granule membrane fraction, suggesting the possibility of cycling between cytosolic and membrane-associated forms.