Electronic reporting of rare endocrine conditions within a clinical network: results from the EuRRECa project.

Objective: The European Registries for Rare Endocrine Conditions (EuRRECa, eurreb.eu) includes an e-reporting registry (e-REC) used to perform surveillance of conditions within the European Reference Network (ERN) for rare endocrine conditions (Endo-ERN). The aim of this study was to report the experience of e-REC over the 3.5 years since its launch in 2018. Methods: Electronic reporting capturing new encounters of Endo-ERN conditions was performed monthly through a bespoke platform by clinicians registered to participate in e-REC from July 2018 to December 2021. Results: The number of centres reporting on e-REC increased to a total of 61 centres from 22 countries. A median of 29 (range 11, 45) paediatric and 32 (14, 51) adult centres had reported cases monthly. A total of 9715 and 4243 new cases were reported in adults (age ≥18 years) and children, respectively. In children, sex development conditions comprised 40% of all reported conditions and transgender cases were most frequently reported, comprising 58% of sex development conditions. The median number of sex development cases reported per centre per month was 0.6 (0, 38). Amongst adults, pituitary conditions comprised 44% of reported conditions and pituitary adenomas (69% of cases) were most commonly reported. The median number of pituitary cases reported per centre per month was 4 (0.4, 33). Conclusions: e-REC has gained increasing acceptability over the last 3.5 years for capturing brief information on new encounters of rare conditions and shows wide variations in the rate of presentation of these conditions to centres within a reference network. Significance statement Endocrinology includes a very wide range of rare conditions and their occurrence is often difficult to measure. By using an electronic platform that allowed monthly reporting of new clinical encounters of several rare endocrine conditions within a defined network that consisted of several reference centres in Europe, the EuRRECa project shows that a programme of e-surveillance is feasible and acceptable. The data that have been collected by the e-reporting of rare endocrine conditions (e-REC) can allow the continuous monitoring of rare conditions and may be used for clinical benchmarking, designing new studies or recruiting to clinical trials.

Supporting international networks through platforms for standardised data collection-the European Registries for Rare Endocrine Conditions (EuRRECa) model.

Rare endocrine pathology is manifested by either a deficiency or excess of one or more hormones. These conditions can be life-threatening and are almost universally associated with long-term morbidity. Understanding the aetiology of these conditions requires multicentre collaboration and expertise, most often across national boundaries, with the capacity for long-term follow-up. The EuRRECa (European Registries for Rare Endocrine Conditions) project ( www.eurreca.net ), funded by the EU Health Programme, aims to support the needs of the wider endocrine community by maximising the opportunity for collaboration between patients, health care professionals and researchers across Europe and beyond. At the heart of the EuRRECa collaboration is a Core Endocrine Registry that collects a core dataset for all rare endocrine conditions that are covered within Endo-ERN. The registry incorporates patient reported markers of clinical outcome and will signpost participants to high-quality, disease-specific registries. Furthermore, an electronic surveillance programme (e-REC) captures clinical activity and epidemiology for these rare conditions. EuRRECa receives guidance compliant with the highest ethical standards from Expert Working Groups that align with the Main Thematic Groups of Endo-ERN. Security, data quality and data governance are cornerstones of this platform. Clear policies that are acceptable to patients, researchers and industry for data governance coupled with widespread dissemination and knowledge exchange through closely affiliated stakeholders will ensure sustainability beyond the current lifetime of the project. This paper describes the infrastructure that has been developed, stakeholder involvement, the data fields that are captured within the registry and details on the process for using the platform.

Are multiple checkpoint mediators involved in a checkpoint linking histone gene expression with DNA replication?

In metazoans, accurate replication of chromosomes is ensured by the coupling of DNA synthesis to the synthesis of histone proteins. Expression of replication-dependent histone genes is restricted to S-phase by a combination of cell cycle-regulated transcriptional and post-transcriptional control mechanisms and is linked to DNA replication by a poorly understood mechanism involving checkpoint kinases [Su, Gao, Schneider, Helt, Weiss, O'Reilly, Bohmann and Zhao (2004) EMBO J. 23, 1133-1143; Kaygun and Marzluff (2005) Nat. Struct. Mol. Biol. 12, 794-800]. Here we propose a model for the molecular mechanisms that link these two important processes within S-phase, and propose roles for multiple checkpoints in this mechanism.

Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing.

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block-induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.

INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow.

Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins [1], which include the inner centromere protein (INCENP [2]), the Aurora-related serine-threonine protein kinase AIRK2 [3,4] and the unidentified human autoantigen TD-60 [5], have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of INCENP function affects both chromosome segregation and completion of cytokinesis [6,7], whereas interference with AIRK2 function primarily affects cytokinesis [3,8]. Here, we report that INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and that INCENP and AIRK2 kinase bind one another in vitro. This association was found to be evolutionarily conserved. Sli15p, the binding partner of yeast Aurora kinase Ipl1p, can be recognized as an INCENP family member because of the presence of a conserved carboxy-terminal sequence region, which we term the IN box. This interaction between INCENP and Aurora kinase was found to be biologically relevant. INCENP and AIRK2 colocalized exactly in human cells, and INCENP was required to target AIRK2 correctly to centromeres and the central spindle.

Incorporation of the nuclear pore basket protein nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs.

In cell-free extracts of Xenopus eggs that support the assembly of replication-competent nuclei, we found that lamin B(3) specifically associates with four polypeptides (termed SLAPs, soluble lamin associated proteins). Here, one SLAP is identified as the nuclear pore complex protein Nup153, one member of the F/GXFG motif-containing nucleoporins. In vitro translated Nup153 and lamin B(3) co-immunoprecipitate, and lamin B(3) interacts specifically with the C-terminal domain of Nup153. During nuclear envelope assembly, other F/GXFG-containing nucleoporins are incorporated into the nuclear envelope preceding lamina assembly. Incorporation of Nup153 occurs at the same time as lamina assembly. When lamina assembly is prevented using the dominant-negative mutant XlaminB delta 2+, Nup153 does not appear at the nuclear envelope, while other F/GXFG-containing nucleoporins and Nup93 are recruited normally. When the lamina of pre-assembled nuclei is disrupted using the same dominant-negative mutant, the distribution of other nucleoporins is unaffected. However, Nup153 recruitment at the nuclear envelope is lost. Our results indicate that both the recruitment and maintenance of Nup153 at the pore are dependent upon the integrity of the lamina.

Noninflammatory phagocytosis of monosodium urate monohydrate crystals by mouse macrophages. Implications for the control of joint inflammation in gout.

OBJECTIVE: We have hypothesized that the process of monocyte to macrophage differentiation may alter the inflammatory response of mononuclear phagocytes to the uptake of monosodium urate monohydrate (MSU) crystals. METHODS: Eight mouse monocyte/macrophage cell lines were arranged in increasing order of differentiation, as judged by expression of the macrophage markers F4/80 and BM 8 and by phagocytic capacity. Secretion of tumor necrosis factor alpha (TNFalpha) in response to MSU was measured by enzyme-linked immunosorbent assay. RESULTS: The panel of monocyte/macrophage cell lines revealed a close linkage between the state of differentiation and the capacity of the cells to ingest MSU crystals. TNFalpha production, however, was not linked to phagocytic ability. Peak TNFalpha levels were synthesized by cells at an intermediate state of differentiation (3.2-14.1 ng/ml), whereas mature macrophages, which efficiently phagocytosed crystals, did not secrete TNFalpha. Mature cell lines produced TNFalpha when stimulated with zymosan (5.9-6.2 ng/ml), but this was abolished by coincubation with MSU crystals. Suppression of the zymosan response was not due to apoptosis or steric hindrance by MSU crystals. Culture supernatants from mature macrophages did not stimulate endothelial cell activation, in contrast to MSU-treated cells at an earlier stage of differentiation, which stimulated intercellular adhesion molecule 1 expression on sEND endothelioma cells through the release of TNFalpha (inhibited 80.6% by anti-TNFa). CONCLUSION: We demonstrated that phagocytosis and TNFalpha production are distinct events in the response of mononuclear phagocytes to urate crystals, and these events can be distinguished at the level of macrophage differentiation. The noninflammatory removal of urate crystals by mature macrophages defines a new pathway that may be important in controlling the development of acute gout in patients with hyperuricemia.

Assembly of U7 small nuclear ribonucleoprotein particle and histone RNA 3' processing in Xenopus egg extracts.

In animals, replication-dependent histone genes are expressed in dividing somatic cells during S phase to maintain chromatin condensation. Histone mRNA 3'-end formation is an essential regulatory step producing an mRNA with a hairpin structure at the 3'-end. This requires the interaction of the U7 small nuclear ribonucleoprotein particle (snRNP) with a purine-rich spacer element and of the hairpin-binding protein with the hairpin element, respectively, in the 3'-untranslated region of histone RNA. Here, we demonstrate that bona fide histone RNA 3' processing takes place in Xenopus egg extracts in a reaction dependent on the addition of synthetic U7 RNA that is assembled into a ribonucleoprotein particle by protein components available in the extract. In addition to reconstituted U7 snRNP, Xenopus hairpin-binding protein SLBP1 is necessary for efficient processing. Histone RNA 3' processing is not affected by addition of non-destructible cyclin B, which drives the egg extract into M phase, but SLBP1 is phosphorylated in this extract. SPH-1, the Xenopus homologue of human p80-coilin found in coiled bodies, is associated with U7 snRNPs. However, this does not depend on the U7 RNA being able to process histone RNA and also occurs with U1 snRNPs; therefore, association of SPH1 cannot be considered as a hallmark of a functional U7 snRNP.

ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK.

ATR is a large, > 300 kDa protein containing a carboxy-terminus kinase domain related to PI-3 kinase, and is homologous to the ATM gene product in human cells and the rad3/MEC1 proteins in yeast. These proteins, together with the DNA-PK, are part of a new family of PI-3 kinase related proteins. All members of this family play important roles in checkpoints which operate to permit cell survival following many forms of DNA damage. We have expressed ATR protein in HEK293 cells and purified the protein to near-homogeneity. We show that pure ATR is a protein kinase which is activated by circular single-stranded, double-stranded or linear DNA. Thus ATR is a new member of a sub-family of PIK related kinases, founded by the DNA-PK, which are activated in the presence of DNA. Unlike DNA-PK, ATR does not appear to require Ku proteins for its activation by DNA. We show directly that, like ATM and DNA-PK, ATR phosphorylates the genome surveillance protein p53 on serine 15, a site which is up-regulated in response to DNA damage. In addition, we find that ATR has a substrate specificity similar to, but unique from, the DNA-PK in vitro, suggesting that these proteins have overlapping but distinct functions in vivo. Finally, we find that the kinase activity of ATR in the presence and absence of DNA is suppressed by caffeine, a compound which is known to induce loss of checkpoint control. Our results are consistent with the notion that ATR plays a role in monitoring DNA structure and phosphorylation of proteins involved in the DNA damage response pathways.

Temporal differences in the appearance of NEP-B78 and an LBR-like protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types.

In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.

Information and communication systems for the assistance of carers based on ACTION.

Recent advances in telecommunication technologies allow the design of information and communication systems for people who are caring for others in the home as family members or as professionals in the health or community centres. The present paper analyses and classifies the information flow and maps it to an information life cycle, which governs the design of the deployed hardware, software and the data-structure. This is based on the initial findings of ACTION (assisting carers using telematics interventions to meet older persons' needs) a European Union funded project. The proposed information architecture discusses different designs such as centralized or decentralized Web and Client server solutions. A user interface is developed reflecting the special requirements of the targeted user group, which influences the functionality and design of the software, data architecture and the integrated communication system using video-conferencing. ACTION has engineered a system using plain Web technology based on HTML, extended with JavaScript and ActiveX and a software switch enabling the integration of different types of videoconferencing and other applications providing manufacturer independence.

The activity of HMG-CoA reductase and acetyl-CoA carboxylase in human apocrine sweat glands, sebaceous glands, and hair follicles is regulated by phosphorylation and by exogenous cholesterol.

Human apocrine and sebaceous glands function to secrete lipids, predominantly triglycerides, fatty acids, cholesterol and its esters, and, in the sebaceous gland, squalene. The enzymes that catalyze the important regulatory steps in cholesterol and fatty acid biosyntheses, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl-CoA carboxylase, respectively, were therefore studied in isolated human skin appendages, and their relevant kinetic parameters determined. The enzyme activities that were observed can account for previously described rates of incorporation of radiolabeled substrates into the appropriate lipids by glands in vitro. Reduced enzyme activities following homogenization in the presence of fluoride indicated that both of these enzymes in skin appendages are inactivated by phosphorylation. The activity of the enzyme known to catalyze this phosphorylation, the AMP-activated protein kinase, was also measured. Compactin was shown to inhibit HMG-CoA reductase in homogenates of these appendages. Conversely, incubation of whole sebaceous glands with compactin resulted in the stimulation of enzyme activity, which suggests that these appendages can respond to diminishing cholesterol levels. The effect of exogenous low density lipoprotein and 25-hydroxycholesterol on HMG-CoA reductase activity from skin appendages was investigated. HMG-CoA reductase activity in both apocrine and sebaceous glands was reduced following incubation with either low density lipoprotein or 25-hydroxycholesterol. Low density lipoprotein receptor and lipoprotein lipase mRNA expression was also detected in skin appendages. These results indicate that apocrine and sebaceous glands have the capacity to sequester dietary cholesterol and fatty acids that may have important implications for the understanding of both acne and axillary odor.

PD 98059 prevents establishment of the spindle assembly checkpoint and inhibits the G2-M transition in meiotic but not mitotic cell cycles in Xenopus.

Most chemotherapeutic agents block DNA replication, damage DNA, or interfere with chromosome segregation. The existence of checkpoints, which monitor these events, indicates that mechanisms exist to avoid death when essential cellular events are inhibited. A molecular understanding of cellular checkpoints should therefore provide opportunities for the development of inhibitors of checkpoint controls which may increase the potency of chemotherapeutic drugs by inducing catastrophic cell cycle progression. The molecular dissection of cell cycle arrest points is facilitated in the Xenopus egg/oocyte system, in which cell-free systems retain both S/M and spindle assembly checkpoints. Members of the MAP kinase family have been shown to play a role in the induction of G2 to M transition during oocyte maturation and have been implicated in the maintenance of either cytostatic factor- or spindle assembly checkpoint-induced M-phase arrest. Here, we have examined the effects of the inhibitor of MAP kinase kinase activation, PD 98059, on cell cycle progression in Xenopus oocytes and in cell-free extracts. This inhibitor is highly specific for the kinase which activates the classical p42/p44 MAP kinase, having no effect on upstream activators of stress-activated protein kinases. We have found that PD 98059 inhibits oocyte maturation, consistent with a role for p42 MAP kinase as a rate-limiting component in the induction of meiosis, but had no effect on the timing of G2-M transition in cell-free extracts indicating that, unlike meiosis, p42 MAP kinase activation is not limiting for normal mitotic M phase entry. However, we found that cytostatic factor-induced metaphase arrest, as well as the spindle assembly checkpoint, were both abolished in the presence of the drug. These results demonstrate that p42 MAP kinase, and not some other member of the MAP kinase family, is responsible for both CSF- and checkpoint-induced metaphase arrest and suggest that PD 98059 and similar agents may have considerable therapeutic potential for the potentiation of chemotherapeutic regimes.

Some lessons learned in the care of the elderly.

This article is designed to acquaint physicians not trained in geriatrics with some precepts concerning the care of the elderly. It presents a summary of the experience in the care of over 2,000 outpatients over the age of 65 who were seen at a community hospital geriatric evaluation unit and resource center. The findings reaffirmed the importance of concentrating on the preservation and restoration of function in the care of the frail elderly. Although there are no clinical skills unique to geriatrics, but the approach to the gathering of clinical data in people with multiple and overlapping problems and disabilities must be structured differently. There is less emphasis on precise diagnosis, much less emphasis on the concept of cure, and more emphasis on structuring a medical/social response to the dominant problem or problems.

Cell-cycle arrest and inhibition of Cdk4 activity by small peptides based on the carboxy-terminal domain of p21WAF1.

BACKGROUND: A common event in the development of human neoplasia is the inactivation of a damage-inducible cell-cycle checkpoint pathway regulated by p53. One approach to the restoration of this pathway is to mimic the activity of key downstream effectors. The cyclin-dependent kinase (Cdk) inhibitor p21(WAF1) is one such molecule, as it is a major mediator of the p53-dependent growth-arrest pathway, and can, by itself, mediate growth suppression. The primary function of the p21(WAF1) protein appears to be the inhibition of G1 cyclin-Cdk complexes. Thus, if we can identify the region(s) of p21(WAF1) that contain its inhibitor activity they may provide a template from which to develop novel anti-proliferative drugs for use in tumours with a defective p53 pathway. RESULTS: We report on the discovery of small synthetic peptides based on the sequence of p21(WAF1) that bind to and inhibit cyclin D1-Cdk4. The peptides and the full-length protein are inhibitory at similar concentrations. A 20 amino-acid peptide based on the carboxy-terminal domain of p21(WAF1) inhibits Cdk4 activity with a concentration for half-maximal inhibition (l0.5) of 46 nM, and it is only four-fold less active than the full-length protein. The length of the peptide has been minimized and key hydrophobic residues forming the inhibitory domain have been defined. When introduced into cells, both a 20 amino-acid and truncated eight amino-acid peptide blocked phosphorylation of the retinoblastoma protein (pRb) and induced a potent G1/S growth arrest. These data support a physiological role for the carboxyl terminus of p21(WAF1) in the inhibition of Cdk4 activity. CONCLUSIONS: We have discovered that a small peptide is sufficient to mimic p21(WAF1) function and inhibit the activity of a critical G1 cyclin-Cdk complex, preventing pRb phosphorylation and producing a G1 cell-cycle arrest in tissue culture cell systems. This makes cyclin D1-Cdk4 a realistic and exciting target for the design of novel synthetic compounds that can act as anti-proliferative agents in human cells.

Vacuum erection device in spinal cord injured men: patient and partner satisfaction.

OBJECTIVE: To assess the efficacy of and patient and partner satisfaction with a vacuum erection device (VED) to treat erectile dysfunction of spinal cord injury. DESIGN: Case series. SETTING: University hospital outpatient clinic. PATIENTS: Twenty spinal cord injured men with erectile dysfunction and their heterosexual partners, recruited from outpatient population and by advertisement. INTERVENTION: Use of a VED to obtain erections for sexual activity. MAIN OUTCOME MEASURES: Efficacy in obtaining adequate penile erection, and patient and partner satisfaction with the device (survey). RESULTS: At 3 months, 93% of the men and 83% of the women reported rigidity sufficient for vaginal penetration, with an average duration of 18 minutes. These numbers decreased somewhat at the 6-month control. At 6 months, 41% of the men and 45% of the women were satisfied with the device, with premature loss of rigidity during intercourse the most commonly reported complaint. Sixty percent of men and 42% of women indicated an improvement of the sexual relationship. Minor side effects, such as petechiae and penile skin edema, occurred frequently, but there were no complications that required treatment. CONCLUSION: The VED is effective in many couples in the treatment of erectile dysfunction associated with spinal cord injury. The devices were not universally accepted, but had a significant impact on sexual activity and sexual satisfaction for nearly half the couples. Vacuum erection devices should be presented to SCI men along with other options for treatment of erectile dysfunction.

Addictive behavior of older adults.

Very little is known about addictive alcohol use by older people. In the present paper personal effects reasons for drinking (i.e. drinking for the effects of alcohol) and concerns about drinking were used as indicators of addictive drinking behavior among a sample of 826 people aged 65 and older who participated in survey interviews in their homes. The relationship of addictive drinking behavior to frequency of drinking, quantity of drinks per occasion, and depressant drug use was examined. Alcohol use was higher among males and young-old (aged 65-74), while depressant medication use was higher among females and old-old (aged 75+). However, with the exception of use of over-the-counter medications containing codeine (which was significantly higher among current drinkers), no relationship existed between alcohol use and use of depressant medications. Personal effects reasons for drinking and concerns about drinking were related both to alcohol and depressant medication use. Frequency of drinking was associated with higher endorsement of both personal effects and social reasons, whereas volume of alcohol consumption (drinks per drinking day) was associated only with personal effects drinking. In addition, use of depressant medications by drinkers was significantly related to consuming alcohol for personal effects reasons (but unrelated to consuming for social reasons) and with having concerns about one's own drinking. These results suggest that even within the generally low levels of alcohol consumption of older people, addictive-use patterns emerge. In addition, the results confirm the importance of including depressant medication use in evaluating the drinking behavior of older people.

Activation of the Xenopus cyclin degradation machinery by full-length cyclin A.

The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase. In many cell types, cyclin A-associated kinase activity peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear. Previous work has suggested that while cyclin B is capable of triggering cyclin destruction in Xenopus cell-free systems, cyclin A-associated kinase is not able to support this function. Here we have expressed a full-length human cyclin A in Escherichia coli and purified the protein to homogeneity by virtue of an N-terminal histidine tag. We have found that when added to Xenopus cell-free extracts free of cyclin B and incapable of protein synthesis, the temporal pattern of cyclin A-associated cdc2 kinase activity showed distinct differences that were dependent on the concentration of cyclin A added. When cyclin A was added to a concentration that generated levels of cdc2 kinase activity capable of inducing nuclear envelope breakdown, the histone H1 kinase activity profile was bi-phasic, consisting of an activation phase followed by an inactivation phase. Inactivation was found to be due to cyclin destruction, which was prevented by mos protein. Cyclin destruction was followed by nuclear reassembly and an additional round of DNA replication, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system. It has been suggested that the evolutionary recruitment of cyclin A into an S phase function may have necessitated the loss of an original mitotic ability to activate the cyclin destruction pathway. The results presented here indicate that cyclin A has not lost the ability to activate its own destruction and that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating that there are not distinct cyclin A and cyclin B destruction pathways. Thus the ordered progression of the cell cycle requires the careful titration of cyclin. A concentration in order to avoid activation of the cyclin destruction pathway before sufficient active cyclin B/cdc2 kinase has accumulated.

Isolation and characterisation of a uniquely regulated threonine, tyrosine phosphatase (TYP 1) which inactivates ERK2 and p54jnk.

The recent discovery of the vaccinia virus protein phosphatase VH1, and its mammalian counterparts has highlighted a novel subfamily of protein tyrosine phosphatases that exhibit dual specificity toward phosphotyrosine- and phosphoserine/threonine-residues. We have identified further members of this subfamily. The characterisation of one clone in particular, which we have named threonine-tyrosine phosphatase 1 (TYP 1), encodes a protein homologous to CL100, but differs dramatically in its regulation. TYP 1 is not expressed in human fibroblasts unlike other CL100-like genes. Furthermore, northern analysis has demonstrated that following mitogenic stimulation of squamous cells, induction of TYP 1 mRNA reaches its maximal levels after four hours, in contrast to the immediate early CL100-like genes. Both TYP 1 and CL100 mRNAs are induced upon TGF-beta treatment of squamous cell lines sensitive to the growth factors antiproliferative effects. When TYP 1 is transfected into COS-1 cells, the gene product inhibits both ERK2 and p54 MAP kinase subfamilies. In addition, we show that purified TYP 1 protein efficiently inactivates recombinant ERK2 in vitro by the concomitant dephosphorylation of both its phosphothreonine and -tyrosine residues. TYP 1 encodes a nuclear protein, which when expressed in COS cells is stabilised by EGF treatment.