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A method has been developed for obtaining measurements of the systolic time intervals during uninterrupted graded exercise in the upright position on a bicycle ergometer. The method has been applied to 112 subjects divided into 4 groups: two of normal subjects below and above the age of 40, respectively (mean ages 30 and 48 years), and two of patients (mean ages for each about 50 years), both with coronary insufficiency, but one without and the other with abnormality of left ventricular function as shown by ventriculography. The measurements obtained yield linear relations between total electromechanical systole (QS2) and heart rate, between pre-ejection period (PEP) and RR interval for each subject. The average standard deviation about the regression is less than 6 ms for all three regression lines; the average correlation coefficient is greater than 0-93. The younger group of normal subjects have a significantly shorter PEP compared to the older group. Indices have been derived which separate the patient groups from each other and from the normal subjects. Using these indices 86 per cent of all the subjects were correctly classified according to the group to which they belonged. It is concluded that measurements of STI during uninterrupted exercise offer valuable information in the assessment of cardiac patients. [ABSTRACT FROM PUBLISHER]

Protein phosphatase PP1 has two active-site metals (Zn 2+ /Fe 2+ ) that are essential for catalysis. However, when expressed in bacteria, PP1 has two Mn 2+ -ions in its active site, indicating that the incorporation of Zn 2+ /Fe 2+ depends on additional eukaryotic component(s). Here, we used purified, metal-deficient PP1 to study metal incorporation. Fe 2+ was incorporated spontaneously, but Zn 2+ was not. Mn 2+ -incorporation at physiological pH depended on the co-expression of PP1 with PPP1R2 (Inhibitor-2) or PPP1R11 (Inhibitor-3), or a pre-incubation of PP1 at pH 4. We also demonstrate that PPP1R2 and PPP1R11 are Zn 2+ -binding proteins but are, by themselves, not able to load PP1 with Zn 2+ . Our data suggest that PPP1R2 and PPP1R11 function as metal chaperones for PP1 but depend on co-chaperone(s) and/or specific modification(s) for the transfer of associated Zn 2+ to PP1., (© 2024 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Protein Ser/Thr phosphatase PP1 is always associated with one or two regulatory subunits or RIPPOs. One of the earliest evolved RIPPOs is PPP1R2, also known as Inhibitor-2. Since its discovery nearly 5 decades ago, PPP1R2 has been variously described as an inhibitor, activator or (metal) chaperone of PP1, but it is still unknown how PPP1R2 affects the function of PP1 in intact cells. Here, using specific research tools, we demonstrate that PPP1R2 stabilises a subgroup of PP1 holoenzymes, exemplified by PP1:RepoMan, thereby promoting the dephosphorylation of their substrates. Mechanistically, the recruitment of PPP1R2 disrupts an inhibitory, fuzzy interaction between the C-terminal tail and catalytic domain of PP1, and generates an additional C-terminal RepoMan-interaction site. The resulting holoenzyme is further stabilized by a direct PPP1R2:RepoMan interaction, which renders it refractory to competitive disruption by RIPPOs that do not interact with PPP1R2. Our data demonstrate that PPP1R2 modulates the function of PP1 by altering the balance between holoenzymes through stabilisation of specific subunit interactions., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

NIPP1 is a ubiquitously expressed regulatory subunit of PP1. Its embryonic deletion in keratinocytes causes chronic sterile skin inflammation, epidermal hyperproliferation, and resistance to mutagens in adult mice. To explore the primary effects of NIPP1 deletion, we first examined hair cycle progression of NIPP1 skin knockouts (SKOs). The entry of the first hair cycle in the SKOs was delayed owing to prolonged quiescence of hair follicle stem cells. In contrast, the entry of the second hair cycle in the SKOs was advanced as a result of precocious activation of hair follicle stem cells. The epidermis of SKOs progressively accumulated senescent cells, and this cell-fate switch was accelerated by DNA damage. Primary keratinocytes from SKO neonates and human NIPP1-depleted HaCaT keratinocytes failed to proliferate and showed an increase in the expression of cell cycle inhibitors (p21, p16/Ink4a, and/or p19/Arf) and senescence-associated-secretory-phenotype factors as well as in DNA damage (γH2AX and 53BP1). Our data demonstrate that the primary effect of NIPP1 deletion in keratinocytes is a cell cycle arrest and premature senescence that gradually progresse to chronic senescence and likely contribute to the decreased sensitivity of SKOs to mutagens., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

SDS22 forms an inactive complex with nascent protein phosphatase PP1 and Inhibitor-3. SDS22:PP1:Inhibitor-3 is a substrate for the ATPase p97/VCP, which liberates PP1 for binding to canonical regulatory subunits. The exact role of SDS22 in PP1-holoenzyme assembly remains elusive. Here, we show that SDS22 stabilizes nascent PP1. In the absence of SDS22, PP1 is gradually lost, resulting in substrate hyperphosphorylation and a proliferation arrest. Similarly, we identify a female individual with a severe neurodevelopmental disorder bearing an unstable SDS22 mutant, associated with decreased PP1 levels. We furthermore find that SDS22 directly binds to Inhibitor-3 and that this is essential for the stable assembly of SDS22:PP1: Inhibitor-3, the recruitment of p97/VCP, and the extraction of SDS22 during holoenzyme assembly. SDS22 with a disabled Inhibitor-3 binding site co-transfers with PP1 to canonical regulatory subunits, thereby forming non-functional holoenzymes. Our data show that SDS22, through simultaneous interaction with PP1 and Inhibitor-3, integrates the major steps of PP1 holoenzyme assembly., (© 2024. The Author(s).)

Protein phosphatase-1 (PP1) complexed to nuclear inhibitor of PP1 (NIPP1) limits DNA repair through dephosphorylation of NIPP1-recruited substrates. However, the PP1:NIPP1 holoenzyme is completely inactive under basal conditions, hinting at a DNA damage-regulated activation mechanism. Here, we report that DNA damage caused the activation of PP1:NIPP1 after a time delay of several hours through phosphorylation of NIPP1 at the C-terminal tyrosine 335 (Y335) by a Src-family kinase. PP1:NIPP1 activation partially resulted from the dissociation of the C terminus of NIPP1 from the active site of PP1. In addition, the released Y335-phosphorylated C terminus interacted with the N terminus of NIPP1 to enhance substrate recruitment by the flanking forkhead-associated (FHA) domain. Constitutive activation of PP1:NIPP1 by knock-in of a phospho-mimicking (Y335E) NIPP1 mutant led to the hypo-phosphorylation of FHA ligands and an accumulation of DNA double-strand breaks. Our data indicate that PP1:NIPP1 activation through circularization of NIPP1 is a late response to DNA damage that contributes to the timely recovery from damage repair., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

RepoMan is a chromosome-associated scaffold protein that integrates signaling of multiple kinases and phosphatases to coordinate spindle-kinetochore interactions, chromosome (de)condensation and nuclear envelope (dis)assembly during mitosis. Another key mitotic event is the assembly of a microtubule-based spindle, which involves redundant pathways emanating from the centrosomes, microtubules and chromosomes. Here we describe a novel mitotic function of RepoMan in regulating chromosome-dependent microtubule assembly. At limiting concentrations of microtubule-destabilizing agents, RepoMan-depleted cells showed enhanced chromosome clustering. This clustering was completely dependent on the partial inhibition of microtubule growth originating from the chromosome-dependent pathway. We also demonstrated that RepoMan interacts with prime regulators of the chromosome-dependent spindle assembly such as NuSAP1, NuMA, and TPX2. In addition, RepoMan was required to enable or maintain phosphorylation of NuSAP1 at CDK sites, thereby enabling activation of NuSAP1 through dissociation of inhibitory importin β/7. Our data identify RepoMan as an enhancer of microtubule assembly at chromosomes.

SDS22 is an ancient regulator of protein phosphatase-1 (PP1). Our crystal structure of SDS22 shows that its twelve leucine-rich repeats adopt a banana-shaped fold that is shielded from solvent by capping domains at its extremities. Subsequent modeling and biochemical studies revealed that the concave side of SDS22 likely interacts with PP1 helices α5 and α6, which are distal from the binding sites of many previously described PP1 interactors. Accordingly, we found that SDS22 acts as a "third" subunit of multiple PP1 holoenzymes. The crystal structure of SDS22 also revealed a large basic surface patch that enables binding of a phosphorylated form of splicing factor BCLAF1. Taken together, our data provide insights into the formation of PP1:SDS22 and the recruitment of additional interaction proteins, such as BCLAF1., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The spindle assembly checkpoint (SAC) generates a diffusible protein complex that prevents anaphase until all chromosomes are properly attached to spindle microtubules. A key step in SAC initiation is the recruitment of MAD1 to kinetochores, which is generally thought to be governed by the microtubule-kinetochore (MT-KT) attachment status. However, we demonstrate that the recruitment of MAD1 via BUB1, a conserved kinetochore receptor, is not affected by MT-KT interactions in human cells. Instead, BUB1:MAD1 interaction depends on BUB1 phosphorylation, which is controlled by a biochemical timer that integrates counteracting kinase and phosphatase effects on BUB1 into a pulse-generating incoherent feedforward loop. We propose that this attachment-independent timer serves to rapidly activate the SAC at mitotic entry, before the attachment-sensing MAD1 receptors have become fully operational. The BUB1-centered timer is largely impervious to conventional anti-mitotic drugs, and it is, therefore, a promising therapeutic target to induce cell death through permanent SAC activation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aims: Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/-)) deficiency on leukocyte subsets relevant to atherosclerosis., Methods and Results: LDL receptor deficient mice that were transplanted with Sgpl1(-/-) bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1(-/-) chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response., Conclusions: Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution.

Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [γ-(32)P]-ATP, followed by extraction, thin-layer chromatography, and detection of the formed labeled ceramide-1-phosphate. To eliminate the use of radioactivity, we developed similarly but independently from the approach by Don and Rosen (2008), a fluorescence-based ceramide kinase assay, using N-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl-sphingenine (NBD-C(6)-ceramide) as substrate. Its K(m) value (4 μM) was comparable to that of N-hexanoyl-sphingenine (C(6)-ceramide). The produced fluorescent NBD-C(6)-ceramide-1-phosphate was captured by means of solid-phase extraction on an aminopropyl phase, resulting in a fast and sensitive CERK measurement. By performing this assay in a 96-well format, it is also suitable for high-throughput screening (HTS) to search for CERK modulators. A limited screen revealed that some protein kinase inhibitors (e.g., U-0126; IC(50) 4 μM) and ceramide analogues (e.g., fenretinide, AMG-9810; IC(50) 1.1 μM) affect CERK in vitro.

Upon exposure of truncated ceramides, such as N-acetyl-sphingenine, and long-chain ceramides to moderate acidic conditions, three derivatives are formed. Two of them turned out to be O-acylated sphingenine, 1-O- and 3-O-acyl-sphingenine, and the other was identified as sphingenine. Truncated dihydroceramides (e.g., N-acetyl- and N-hexanoyl-sphinganine) also show this type of rearrangement, which is therefore not related to the presence of the allylic hydroxy group or to the length of the N-acyl chain. Of particular concern is the fact that the O-acylated compounds, which can be considered sphingoid base analogs, can be formed in chloroform or chloroform-methanol mixtures upon storage. For long-term storage, methanol or dichloromethane is the preferred solvent. A procedure to document the presence/formation of such O-acylated sphingoid bases in ceramide solutions in the picomole range, based on reversed-phase chromatography after derivatization of their amino group with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, is presented.

The enzymatic phosphorylation of sphingoid bases was analysed in rat tissues, using D-erythro-[4,5-(3)H]sphinganine as substrate. After optimisation of the assay, taking care to block sphingosine-phosphate lyase and sphingosine phosphatase, highest ATP-dependent kinase activities were present in testis, followed by kidney, and intestinal mucosa. Approximately two thirds of the kidney activity were membrane bound, the remaining being cytosolic. Classical cell fractionation studies of kidney and liver did not allow to identify unequivocally the subcellular site of the membrane bound kinase. Separation of a particulate fraction from kidney homogenates by Percoll gradient and sucrose density gradient centrifugation revealed that kinase activities are associated with vesicles derived from the endoplasmic reticulum and the plasma membrane. Based on indirect data, such as the effect of detergents and divalent ions, the cytosolic and both membrane bound activities appear to reside in different proteins. N,N-Dimethylsphingenine was inhibitory to all three different kinases, which were mainly active towards the D-erythro isomers of sphingenine and sphinganine.

The introduction of the double bond in the sphingoid backbone of sphingolipids occurs at the level of dihydroceramide via an NADPH-dependent desaturase, as discovered in permeabilized rat hepatocytes. In the rat, the enzyme activity, which has now been further characterized, appeared to be mostly enriched in liver and Harderian gland. By means of subcellular fractionation of rat liver homogenates and density gradient separation of microsomal fractions, the desaturase was localized to the endoplasmic reticulum. Various detergents were inhibitory to the enzyme, and maximal activities were obtained in the presence of NADPH and when the substrate was complexed to albumin. In the presence of albumin, the chain length of the fatty acid of the truncated dihydroceramides hardly affected the activity. Finally, in view of a likely evolutionary relationship between desaturases and hydroxylases, the formation of hydroxylated intermediates was analyzed. No evidence for their presence was found under our assay conditions.

Pulse wave transmission times and pulse wave velocities in central aorta and peripheral arteries were measured in a group of 85 healthy children and adolescents between the ages of 1 and 21 years. A new external plethysmograph is described which gives reliable tracings of external pulsations. A method is given to calculate real pulse wave transmission time from the aortic valves to the groin.

Because of the inherent risks of intra-arterial blood pressure monitoring a new non-invasive device, Finapres, which measures blood pressure continuously in the finger, was evaluated in 14 hypertensive and one normotensive subject. Brachial intra-arterial and finger pressures were compared during a control period and a subsequent Valsalva manoeuvre. Visually, blood pressures measured by Finapres faithfully reproduced the intra-arterial recordings in all subjects. From each pressure signal beat to beat systolic, diastolic, and mean blood pressure values and their differences were obtained and the time course of the response and its characteristic features were analysed. During the control period the Finapres measurements were lower than intra-arterial systolic, mean, and diastolic pressures (mean(SD) 1(9.6), 9(6.8), and 4(6.1) mmHg respectively). During the response to the Valsalva manoeuvre the brachial-finger pressure differences showed limited deviation from those during the control period; median differences were at most 6 mmHg occurring late during the intrathoracic strain period and 7 mmHg during the post-release blood pressure overshoot. In general, the Finapres device reproduced intra-arterial patterns faithfully. This device appears to offer a reliable alternative to intra-arterial blood pressure monitoring.

Using noninvasive techniques only, the fall in mean pressure and the pulse amplification between brachial and finger arterial pressure were measured in six anaesthetised female subjects during surgery. Brachial pressure was measured every 2 min with an oscillometric technique. Finger pressure was measured continuously using an arterial volume clamp method. In addition changes in the degree of peripheral vasoconstriction were established on an adjacent finger with a photo reflection plethysmograph. On the average finger mean pressure is 10 mmHg below brachial pressure. The difference tends to decrease with increasing constriction. The change in the difference between full constriction and maximal dilatation is 8 mmHg. The average finger to brachial pulse amplitude ratio changes from 110% at maximal dilatation to 170% at full constriction. Finger systolic pressure overshoot is responsible for the pulse wave amplification. On the average it is + 7 mmHg and ranges between maximal dilatation and full constriction over 26 mmHg. The standard error deviation on the volume clamp method could be established at 5% for mean pressure, about equal to that of the oscillometric technique in the literature.

Systolic time intervals (STI), measured non-invasively and during uninterrupted exercise, may contain information about cardiac function. A processing system was developed, which determines STI automatically from 8-sec recordings at each load level. A preprocessing unit provides automatic recording and optimal signal-to-noise ratios. One of the main tasks of the computer program, which is modular in structure and written in FORTRAN IV, is to recognize disturbances and artefacts in the signals. After selection of the complexes, usually with some kind of correlation method, a representative beat is found and the inclination points are localized. Besides these points, indicateions of possible errors are given. Preliminary results indicate that the system may be able to replace the human observer in estimating STI.