Myocardial late gadolinium enhancement: a head-to-head comparison of motion-corrected balanced steady-state free precession with segmented turbo fast low angle shot.

AIM: To evaluate the image quality and diagnostic agreement with a head-to-head comparison of late gadolinium enhancement (LGE) images acquired by the motion-corrected (MOCO) balanced steady-state free precession (bSSFP) phase sensitivity inversion recovery (PSIR) and conventional segmented fast low angle shot (FLASH) PSIR methods15,16 in a patient cohort with a wide spectrum of cardiovascular disease. MATERIALS AND METHODS: In 59 consecutive patients, signal-to-noise ratios (SNRs), contrast-to-noise ratios (CNRs) of the normal myocardium (NM), LGE, and blood pool (BP) were pair-wise compared between the two different sequences. A further semi-qualitative score system (graded 1 -4) was used to compare the overall image quality (OIQ). The diagnostic agreement of the two techniques were evaluated by both transmural severity and absolutely quantitative size of LGE. RESULTS: The SNRs of the NM, LGE, and BP of MOCO bSSFP were 4.8±3.4, 53.6±35.6 and 43.2±29.3, compared with 3.9±3.6 (p=0.126), 27.7±18.5 (p<0.001) and 24.3±13.4 (p<0.001) of FLASH LGE, respectively. The CNRs of LGE to NM, LGE to BP, and BP to NM were 48.3±33.1 versus 23.8±16.7 (p<0.001), 6.5±21.6 versus 3.8±10.8 (p<0.001), and 38.3±27.2 versus 20.3±10.7 (p=0.448), respectively. The OIQ of MOCO bSSFP was higher than that of segmented FLASH (median 4 versus median 3, p<0.001). For quantification of LGE size, there is good agreement and high correlation (r=0.992, p<0.001) between the two methods. CONCLUSIONS: MOCO bSSFP is a feasible, robust sequence for LGE imaging, especially for patients with arrhythmia and those incapable of breath-holding due to severe heart failure.

Myocardial fat overgrowth in Proteus syndrome.

Proteus syndrome (PS) is a rare, mosaic disorder with asymmetric and distorting overgrowth of the skeletal system, skin, and adipose tissues. Cardiac abnormalities are rare in this syndrome and only two prior cases have been reported. Many patients with PS followed at our institution underwent transthoracic echocardiograms for preoperative evaluation or as work-up for associated pulmonary disease. Some were noted to have prominent, focal echodense areas in the myocardium. We further investigated cardiac findings in a cohort of children and adult patients with PS. Patients with abnormal echocardiograms were referred for cardiac magnetic resonance imaging, Holter monitoring, and exercise treadmill testing. Twenty children and adults with PS, age 24 months to 50 years old, underwent transthoracic echocardiograms. Seven patients (35%) had focal bright echodense areas within the myocardium suggesting fatty infiltration. The majority of patients had significant involvement of the interventricular septum. The cardiac characteristics of all patients with fatty infiltration on transthoracic echocardiograms were compared to Proteus patients without these findings. There were no significant differences in chamber sizes, mass, systolic or diastolic function. No increased risk of conduction defects or arrhythmias was found. This study shows that abnormal fat overgrowth is a common finding in the myocardium in patients with Proteus syndrome; however, it is not associated with functional derangements or arrhythmias. Further evaluation of a larger number of Proteus patients is needed in order to determine the frequency and prognosis of cardiac involvement. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Implementation of municipal mobility support services for older people who have stopped driving in Japan.

OBJECTIVES: In a motorized society, increasing numbers of drivers and their family members will have to face the issue of driving cessation late in life due to dementia or age-related conditions. Mobility support for driving retirees should be considered from a public health perspective. Compared with alternative forms of transportation, relying on family members and friends, municipality-provided mobility support services would be more reliable and practical. The present study aimed to explore the provision of mobility support measures at the community level. STUDY DESIGN: A cross-sectional study of all municipal governments in Japan. METHODS: A nationwide survey was conducted using a postal self-administered questionnaire to explore the allocation of municipality-provided mobility support measures for two target groups: (1) healthy older residents and (2) older residents with dementia. The possible sociodemographic characteristics of municipalities affecting the implementation of such measures were examined. RESULTS: Data from 1027 (56.8%) municipal governments were analysed. The present study demonstrated that mobility support measures for older residents, particularly dementia sufferers, were not sufficiently developed in municipalities. Moreover, the analyses showed that the following three characteristics of municipalities were related to the implementation of mobility support measures for healthy older residents: longer roads, low percentage of older residents per unit of road length, and low population density. CONCLUSIONS: These findings provide insight into the possible incentives for implementing mobility support for healthy older residents, and indicate the prospective mobility needs of driving retirees, including dementia sufferers.

Quality of life after esophagectomy and endoscopic therapy for Barrett's esophagus with dysplasia.

Esophagectomy (EG) and endoscopic therapy (ET) can eradicate Barrett's esophagus with early neoplasia. Their relative effect on quality of life is unknown. The 36-item Short Form Health Survey (SF-36) and Gastrointestinal Quality of Life Index (GIQLI) questionnaires were sent to all patients who underwent either EG or ET at our institution over the last 9 years. Groups were stratified by age and American Society of Anesthesia (ASA) class. Surveys were sent to 77 patients and completed by 14 EG (50%) and by 28 ET patients (57%). The average time between treatment and survey was 4 years in the ET group and 5 years in the EG group. There were no significant differences in SF-36 scores between EG and ET patients except for superior physical functioning among EG patients 65 and older QOL scores among EG and ET groups were not significantly different than sex age-matched controls. GIQLI scores were similar between ET and EG patients of all ages (P= 0.60). GIQLI scores were higher among younger ET patients than young EG patients (P= 0.049). GIQLI scores also tended to be higher among ASA 1 and 2 ET patients than ASA 1 and 2 EG patients, but this did not reach statistical significance (P= 0.09). EG and ET for early Barrett's neoplasia appear to have similar impact on QOL 1 year or more after treatment compared with age-matched controls. Negative QOL impact appears to be greater for younger patients undergoing EG than for ET.

Safety and efficacy of PD-1/PD-L1 blockade in patients with preexisting antinuclear antibodies.

PURPOSE: Immune checkpoint inhibitors (ICIs) show promising clinical activity in advanced cancers. However, the safety and efficacy of PD-1/PD-L1 blockade in patients with preexisting antinuclear antibodies (ANA) are unclear. METHODS: 191 patients treated with nivolumab, pembrolizumab, atezolizumab, or durvalumab for unresectable advanced cancers between September 2014 and December 2018 were identified retrospectively. Patients were divided into positive (ANA titers ≥ 1:160) and negative ANA groups (ANA titers < 1:160). Development of immune-related adverse events (irAEs), the overall response rate (ORR), and disease control rate (DCR) were monitored. RESULTS: Positive ANA titers were seen in 9 out of 191 patients. Four patients in the positive ANA group and 69 patients in the negative group developed irAEs of any grade without a significant difference between the groups. The development of endocrine, pulmonary, and cutaneous irAEs was not significant, whereas positive ANA was significantly higher in patients who developed colitis (2/9) than in patients who did not (3/182, P = 0.0002). DCR in the positive and negative ANA group was 37.5% and 67.5%, respectively, and was not statistically significant, but had better efficacy in patients without ANA (P = 0.08). ANA-related autoimmune diseases such as SLE, Sjögren's syndrome, MCTD, scleroderma, dermatomyositis, and polymyositis was not induced in either group. However, one patient with preexisting dermatomyositis had a flare up after initiation of atezolizumab. CONCLUSION: Further studies to identify predictive factors for the development of irAEs are required to provide relevant patient care and maximize the therapeutic benefits of ICIs.

BCR/ABL and IL-3 activate Rap1 to stimulate the B-Raf/MEK/Erk and Akt signaling pathways and to regulate proliferation, apoptosis, and adhesion.

The Ras family small GTPase Rap1 is activated by hematopoietic cytokines, such as interleukin (IL)-3, to induce beta1 integrin-mediated cell adhesion or by the BCR/ABL fusion tyrosine kinase to stimulate the MEK/Erk signaling pathway. Here, we demonstrate that the abrogation of Rap1 activation by SPA-1, a Rap1-specific GAP, inhibits activation of B-Raf, MEK, Erk, and Akt in a murine hematopoietic cell line, Ton.B210, stimulated with IL-3 or inducibly expressing BCR/ABL. Furthermore, Rap1 inactivation had an inhibitory effects on proliferation and survival of Ton.B210 cells, which were more remarkable when cells were stimulated by BCR/ABL than by IL-3. Induction of BCR/ABL expression increased adhesion of Ton.B210 cells to fibronectin in a manner at least partly dependent on its kinase activity, and Rap1 inhibition by SPA-1 partially inhibited BCR/ABL-induced adhesion of cells. Thus, IL-3- or BCR/ABL-induced activation of Rap1 may play important roles in regulation of cell proliferation and survival through activation of the B-Raf/MEK/Erk and Akt signaling pathways and in induction of integrin-mediated cell adhesion. Furthermore, as compared with IL-3, BCR/ABL is more dependent on Rap1-mediated signaling to induce cell proliferation and survival and, thus, Rap1 may represent an attractive target for novel therapies for leukemias caused by BCR/ABL.

Intra- and inter-tester reliability and validity of normal finger size measurement using the Japanese ring gauge system.

This study was designed to investigate the reliability and validity of measurements of finger diameters with a ring gauge. A reliability study enrolled two independent samples (50 participants and seven examiners in Study I; 26 participants and 26 examiners in Study II). The sizes of each participant's little fingers were measured twice with a ring gauge by each examiner. To investigate the validity of the measurements, five hand therapists compared the finger size and hand volume of 30 participants with the ring gauge and with a figure-of-eight technique (Study III). The intra-class correlation coefficient for intra-observer reliability ranged from 0.97 to 0.99 in Study I, and 0.90 to 0.97 in Study II. The intra-class correlation coefficient for inter-observer reliability was 0.95 in Study I and 0.94 in Study II. The validity study showed a Pearson product moment correlation coefficient of 0.75. The ring gauge showed high reliability and validity for measurement of finger size. LEVEL OF EVIDENCE: III, diagnostic.

Positive modulation of AMPA receptors increases neurotrophin expression by hippocampal and cortical neurons.

This study investigated whether positive modulators of AMPA-type glutamate receptors influence neurotrophin expression by forebrain neurons. Treatments with the ampakine CX614 markedly and reversibly increased brain-derived neurotrophic factor (BDNF) mRNA and protein levels in cultured rat entorhinal/hippocampal slices. Acute effects of CX614 were dose dependent over the range in which the drug increased synchronous neuronal discharges; threshold concentrations for acute responses had large effects on mRNA content when applied for 3 d. Comparable results were obtained with a second, structurally distinct ampakine CX546. Ampakine-induced upregulation was broadly suppressed by AMPA, but not NMDA, receptor antagonists and by reducing transmitter release. Antagonism of L-type voltage-sensitive calcium channels blocked induction in entorhinal cortex but not hippocampus. Prolonged infusions of suprathreshold ampakine concentrations produced peak BDNF mRNA levels at 12 hr and a return to baseline levels by 48 hr. In contrast, BDNF protein remained elevated throughout a 48 hr incubation with the drug. Nerve growth factor mRNA levels also were increased by ampakines but with a much more rapid return to control levels during chronic administration. Finally, intraperitoneal injections of CX546 increased hippocampal BDNF mRNA levels in aged rats and middle-aged mice. The present results provide evidence of regional differences in mechanisms via which activity regulates neurotrophin expression. Moreover, these data establish that changes in synaptic potency produce sufficient network level physiological effects for inducing neurotrophin genes, indicate that the response becomes refractory during prolonged ampakine exposure, and raise the possibility of using positive AMPA modulators to regulate neurotrophin levels in aged brain.

A new enzyme, maltobionate alpha-D-glucohydrolase, from alkalophilic Bacillus sp. N-1053.

A new enzyme, maltobionate alpha-D-glucohydrolase, was purified to apparent homogeneity from a cell-free extract of alkalophilic Bacillus sp. N-1053 about 930-fold with a yield of 18% and some of its properties were investigated. The enzyme showed optimum activity at about pH 7.0, and was stable over the range of pH 6.0-9.5. The molecular weight was estimated to be 152,000 and 71,000 by HPLC gel filtration on TSKgel G3000SWXL and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme hydrolyzed maltobionate more effectively than disaccharides such as maltose and maltitol or trisaccharides such as maltotrionate, maltotriose and maltotriitol, but showed no activity toward polysaccharides such as amylose, amylopectin and soluble starch. The reaction products from 1 mol of maltobionate were found to be 1 mol of beta-D-glucose and 1 mol of D-gluconate. The Km value for maltobionate was 1.63 mM and the Vmax/Km value for maltobionate was the largest among the substrates tested. The enzyme activity was almost completely inhibited by Hg2+, Ag+, iodine and N-bromosuccinimide, and also inhibited by p-nitrophenyl alpha-D-glucoside, maltose and maltitol.

Hypertrophic cardiomyopathy caused by a novel alpha-tropomyosin mutation (V95A) is associated with mild cardiac phenotype, abnormal calcium binding to troponin, abnormal myosin cycling, and poor prognosis.

BACKGROUND: We report hypertrophic cardiomyopathy (HCM) in a Spanish-American family caused by a novel alpha-tropomyosin (TPM1) mutation and examine the pathogenesis of the clinical disease by characterizing functional defects in the purified mutant protein. METHODS AND RESULTS: HCM was linked to the TPM1 gene (logarithm of the odds [LOD] score 3.17). Sequencing and restriction digestion analysis demonstrated a TPM1 mutation V95A that cosegregated with HCM. The mutation has been associated with 13 deaths in 26 affected members (11 sudden deaths and 2 related to heart failure), with a cumulative survival rate of 73+/-10% at the age of 40 years. Left ventricular wall thickness (mean 16+/-6 mm) and disease penetrance (53%) were similar to those for the ss-myosin mutations L908V and G256E previously associated with a benign prognosis. Left ventricular hypertrophy was milder than with the ss-myosin mutation R403Q, but the prognosis was similarly poor. With the use of recombinant tropomyosins, we identified several functional alterations at the protein level. The mutation caused a 40% to 50% increase in calcium affinity in regulated thin filament-myosin subfragment-1 (S1) MgATPase assays, a 20% decrease in MgATPase rates in the presence of saturating calcium, a 5% decrease in unloaded shortening velocity in in vitro motility assays, and no change in cooperative myosin S1 binding to regulated thin filaments. CONCLUSIONS: In contrast to other reported TPM1 mutations, V95A-associated HCM exhibits unusual features of mild phenotype but poor prognosis. Both myosin cycling and calcium binding to troponin are abnormal in the presence of the mutant tropomyosin. The genetic diagnosis afforded by this mutation will be valuable in the management of HCM.

AMPA receptor modulators have different impact on hippocampal pyramidal cells and interneurons.

Positive modulators of AMPA receptors enhance synaptic plasticity and memory encoding. Facilitation of AMPA receptor currents not only results in enhanced activation of excitatory neurons but also increases the activity of inhibitory interneurons by up-modulating their excitatory input. However, little is known about the effects of these modulators on cells other than pyramidal neurons and about their impact on local microcircuits. This study examined the effects of members from three subfamilies of modulators (mainly CX516, CX546 and cyclothiazide) on excitatory synaptic responses in four classes of hippocampal CA1 neurons and on excitatory and disynaptically induced inhibitory field potentials in hippocampal slices. Effects on excitatory postsynaptic currents (EPSCs) were examined in pyramidal cells, in two types of inhibitory interneurons located in stratum radiatum and oriens, and in stratum radiatum giant cells, a novel type of excitatory neuron. With CX516, increases in EPSC amplitude in pyramidal cells were two to three times larger than in interneurons and six times larger than in radiatum giant cells. The effects of CX546 on response duration similarly were largest in pyramidal cells. However, this drug also strongly differentiated between stratum oriens and radiatum interneurons with increases being four times larger in the latter. In contrast, cyclothiazide had similar effects on response duration in all cell types. In field recordings, CX516 was several times more potent in enhancing excitatory postsynaptic potentials (EPSPs) than feedback or feedforward circuits, as expected from its larger influence on pyramidal cells. In contrast, BDP-20, a CX546 analog, was more potent in enhancing feedforward inhibition than either EPSPs or feedback inhibition. This preference for feedforward over feedback circuits is probably related to its higher potency in stratum radiatum versus oriens interneurons. Taken together, AMPA receptor modulators differ substantially in their potency and/or efficacy across major classes of neurons which is likely to have consequences with regard to their impact on circuits and behavior.

Ionic basis of the chronotropic effect of acetylcholine on the rabbit sinoatrial node.

OBJECTIVE: The aim was to study the ionic basis of the chronotropic effects of bath applied acetylcholine and vagal stimulation on the rabbit sinoatrial node. METHODS: The chronotropic effect of bath applied acetylcholine was measured in single cells and small multicellular preparations from the rabbit sinoatrial node and the chronotropic effect of postganglionic vagal stimulation was measured in the intact sinoatrial node. The roles of the hyperpolarisation activated current, i(f), the acetylcholine activated potassium current, iK,ACh, and the L-type calcium current, iCa, were investigated by blocking the currents with 1-2 mM Cs+ or 10(-6) M UL-FS49, 0.2-1.0 mM Ba2+, and 6 x 10(-6) M nifedipine, respectively. RESULTS: Under control conditions, small multicellular preparations were approximately two orders of magnitude less sensitive to bath applied acetylcholine than single cells. However, after block of acetylcholinesterase by eserine in small multicellular preparations the sensitivities of the two types of preparation were approximately the same. Block of i(f) either had no discernible effect or increased the chronotropic effect of bath applied acetylcholine on single cells or small multicellular preparations, whereas partial block of iK,ACh reduced it substantially. Similarly, block of i(f) did not suppress the initial slowing of spontaneous action potentials by vagal stimulation, whereas partial block of iK,ACh reduced it. The hyperpolarisation of the arrested sinoatrial node in response to vagal stimulation was also substantially reduced by block of iK,ACh. Partial block of iCa caused large decreases in the action potential amplitude and maximum diastolic potential, but little decrease in the rate of spontaneous action potentials, and therefore did not mimic the effect of acetylcholine. CONCLUSIONS: The chronotropic effects of bath applied acetylcholine and vagal stimulation are not principally the result of a suppression of i(f) or iCa, whereas the activation of iK,ACh may play an important role.

Cell lineage analysis of pattern formation in the Tubifex embryo. II. Segmentation in the ectoderm.

Ectodermal segmentation in the oligochaete annelid Tubifex is a process of separation of 50-microm-wide blocks of cells from the initially continuous ectodermal germ band (GB), a cell sheet consisting of four bandlets of blast cells derived from ectoteloblasts (N, O, P and Q). In this study, using intracellular lineage tracers, we characterized the morphogenetic processes that give rise to formation of these ectodermal segments. The formation of ectodermal segments began with formation of fissures, first on the ventral side and then on the dorsal side of the GB; the unification of these fissures gave rise to separation of a 50-microm-wide block of approximately 30 cells from the ectodermal GB. A set of experiments in which individual ectoteloblasts were labeled showed that as development proceeded, an initially linear array of blast cells in each ectodermal bandlet gradually changed its shape and that its contour became indented in a lineage-specific manner. These morphogenetic changes resulted in the formation of distinct cell clumps, which were separated from the bandlet to serve as segmental elements (SEs). SEs in the N and Q lineages were each comprised of clones of two consecutive primary blast cells. In contrast, in the O and P lineages, individual blast cell clones were distributed across SE boundaries; each SE was a mixture of a part of a more anterior clone and a part of the next more posterior clone. Morphogenetic events, including segmentation, in an ectodermal bandlet proceeded normally in the absence of neighboring ectodermal bandlets. Without the underlying mesoderm, separated SEs failed to space themselves at regular intervals along the anteroposterior axis. We suggest that ectodermal segmentation in Tubifex consists of two stages, autonomous morphogenesis of each bandlet leading to generation of SEs and the ensuing mesoderm-dependent alignment of separated SEs.

The Role of ORAI1 in the Odontogenic Differentiation of Human Dental Pulp Stem Cells.

Pulp capping, or placing dental materials directly onto the vital pulp tissues of affected teeth, is a dental procedure that aims to regenerate reparative dentin. Several pulp capping materials are clinically being used, and calcium ion (Ca(2+)) released from these materials is known to mediate reparative dentin formation. ORAI1 is an essential pore subunit of store-operated Ca(2+) entry (SOCE), which is a major Ca(2+) influx pathway in most nonexcitable cells. Here, we evaluated the role of ORAI1 in mediating the odontogenic differentiation and mineralization of dental pulp stem cells (DPSCs). During the odontogenic differentiation of DPSCs, the expression of ORAI1 increased in a time-dependent manner. DPSCs knocked down with ORAI1 shRNA (DPSC/ORAI1sh) or overexpressed with dominant negative mutant ORAI1(E106Q) (DPSC/E106Q) exhibited the inhibition of Ca(2+) influx and suppression of odontogenic differentiation and mineralization as demonstrated by alkaline phosphatase (ALP) activity/staining as well as alizarin red S staining when compared with DPSCs of their respective control groups (DPSC/CTLsh and DPSC/CTL). The gene expression for odontogenic differentiation markers such as osteocalcin, bone sialoprotein, and dentin matrix protein 1 (DMP1) was also suppressed. When DPSC/CTL or DPSC/E106Q cells were subcutaneously transplanted into nude mice, DPSC/CTL cells induced mineralized tissue formation with significant increases in ALP and DMP1 staining in vivo, whereas DPSC/E106Q cells did not. Collectively, our data showed that ORAI1 plays critical roles in the odontogenic differentiation and mineralization of DPSCs by regulating Ca(2+) influx and that ORAI1 may be a therapeutic target to enhance reparative dentin formation.

Sex hormones and antiandrogens influence in vitro growth of dermal papilla cells and outer root sheath keratinocytes of human hair follicles.

Anagen hair bulb papillae, interfollicular dermal fibroblasts, and interfollicular keratinocytes isolated from fronto-parietal scalp biopsies as well as outer root sheath keratinocytes from plucked anagen hairs were separately grown in subculture for 14 d. The effect of different concentrations (2.4 nM-17.3 microM) of testosterone, dihydrotestosterone, and the antiandrogens cyproterone acetate or 17 alpha-propylmesterolone on growth behavior of the mesenchymal and epithelial cell types of the hair follicle were comparatively studied by means of growth curves, cell doubling times, and 3H-thymidine incorporation. For control, all cell lines were subcultured in hormone-free medium. Testosterone and dihydrotestosterone (345 nM) significantly reduced proliferation of papilla cells compared with dermal fibroblasts (p < 0.01) and outer root sheath keratinocytes compared with interfollicular keratinocytes (p < 0.01), as well as compared with cells cultured in control medium. Low concentrations of 17 beta-estradiol were ineffective, whereas doses of 180 nM 17 beta-estradiol increased the growth velocities of all cell types, especially of papilla cells, compared with dermal fibroblasts. Low doses of either cyproterone acetate (24 nM) or 17 alpha-propylmesterolone (29 nM) induced a growth enhancement, especially of papilla cells and outer root sheath keratinocytes, whereas high doses of cyproterone (1.20 microM) and 17 alpha-propylmesterolone (1.45 microM) had opposite effects. These changes were significant between papilla cells and dermal fibroblasts as well as between outer root sheath keratinocytes and interfollicular keratinocytes. Applying increasing doses of androgens to cyproterone acetate (24 nM)- or 17 alpha-propylmesterolone (29 nM)-containing media neutralized the growth-stimulating effect of antiandrogens, particularly in papilla cells and outer root sheath keratinocytes. However, minor differences between testosterone and dihydrotestosterone effects on cell growth were found. The data clearly demonstrate that the changes of in vitro growth of hair follicle cells depend on the concentrations of androgens and antiandrogens, as higher doses of both antiandrogens tested retarded the cell proliferation similar to testosterone or dihydrotestosterone. The papilla cells and outer root sheath keratinocytes reacted more sensitively to the hormones tested, thereby confirming the concept of a distinct androgen sensitivity of these specialized hair follicle cells.

Segmental cell kinetics of the human anagen hair: a DNA-flow cytometric analysis.

The cell kinetics of anagen scalp hair taken by punch biopsies from 70 healthy male volunteers were determined at nine different defined bulbar and follicular hair segments using microdissection and DNA-flow cytometry. The highest mean proliferative activity (S-phase) was measured within the lowermost bulbar segment (14.0%), but decreased to 7.6% at Auber's segment and to 5.9% at the follicle isthmus. Notably, the S-phase data of the upper follicular segments (subdermal 2.4%, infundibular 2.4%) were found to be similar to those of the epidermis (2.5%). This study supporting and supplementing former autoradiographic investigations on human hair matrix epithelium clearly demonstrates the main proliferative activity of the anagen hair follicle being localized in the bulbar segments below Auber's level. Moreover, the method described is well suited for studying the effects of agents influencing cell growth (e.g., hormones or drugs) on the cell kinetics of different anagen hair compartments.

Glycosaminoglycans inhibit degradation of insulin-like growth factor-binding protein-5.

Human dermal fibroblasts secrete insulin-like growth factor-binding protein-3 (IGFBP-3), -4, and -5. Fibroblast-conditioned medium contains minimal intact IGFBP-5, and this form of IGFBP is predominantely a 23-kilodalton fragment, suggesting that the IGFBP-5 fragment is derived from intact IGFBP-5 by proteolysis. In this study we investigated the effects of glycosaminoglycans on IGFBP-5 degradation in fibroblast-conditioned medium. The addition of heparin, heparan sulfate, and dermatan sulfate (100 micrograms/ml) to the medium of fibroblast monolayer cultures inhibited IGFBP-5 degradation, as determined by the conversion of intact IGFBP-5 to a 23-kilodalton fragment. In contrast, hyaluronic acid, keratan sulfate, and chondroitin sulfate-A and -C had no effect. Heparin and heparan sulfate inhibited IGFBP-5 degradation at concentrations of 1 or 2.5 micrograms/ml, but 100 micrograms/ml dermatan sulfate were required. Heparin was also inhibitory in vitro, that is when conditioned medium and heparin were incubated without cells. Experiments with modified forms of heparin showed that O-sulfate groups in the 2 or 3 carbon position were required for heparin to be inhibitory. Completely desulfated heparin had no activity, and N-resulfation of desulfated heparin had only a minimal effect. Dextran sulfate, pentosan polysulfate, and fucoidan, which are composed of different saccharide units but contain O-sulfate groups in the 2 or 3 carbon positions, also inhibited IGFBP-5 degradation. These results demonstrate that heparin-like molecules are important regulators of IGFBP-5 degradation. O-Sulfation of the 2 or 3 position of the saccharide ring is required for inhibitory activity. As glycosaminoglycan side-chains are present in proteoglycans that are present in extracellular matrix and on cell surfaces, these side-chains represent a potential mechanism for regulating IGFBP-5 proteolysis in vivo.

Growth and cell kinetics of human hair papilla cells in vitro. An autoradiographic and flow cytometric study.

Hair papilla, a distinct specialized dermal compartment, plays a fundamental role in the biology of hair growth. Recently some attention has been focused on hair papilla cells (HPC) as possible targets for drugs influencing the hair growth. Isolation and cultivation of the HPC facilitates screening for such drugs. In the present work, growth and cell kinetics of human occipital scalp follicle HPC have been studied in vitro. HPC grow according to a Gompertz function, i.e. with considerable growth delay long before becoming confluent cultures, due probably to elongation of the potential doubling time (Tpot) and to a parallel increasing cell loss rate. The [3H]dT labelling index of the HPC strongly depends on the age of the subculture; the cycle time being about 4 days. A potential doubling time of about 93 h, indicative of growth fraction (GF) = 1, and a duration of S phase and G2 + M phase of about 8 h each were found by the combined application of continuous labelling with [3H]dT and DNA flow cytometry.

Modulation of AMPA receptor kinetics differentially influences synaptic plasticity in the hippocampus.

Prior studies showed that positive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators facilitate long-term potentiation (LTP) and improve the formation of several types of memory in animals and humans. However, these modulators are highly diverse in their effects on receptor kinetics and synaptic transmission and thus may differ also in their efficacy to promote changes in synaptic strength. The present study examined three of these modulators for their effects on synaptic plasticity in field CA1 of hippocampal slices, two of them being the benzamide drugs 1-(quinoxalin-6-ylcarbonyl)piperidine (CX516) and 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546) which prominently enhance synaptic transmission yet differ in their relative impact on amplitude versus duration of the synaptic response. The third drug was cyclothiazide which potently blocks AMPA receptor desensitization. Effects on plasticity were assessed by measuring (i) the likelihood of obtaining stable potentiation when using theta-burst stimulation with three instead of four pulses per burst, (ii) the maximum amount of potentiation under optimal stimulation conditions, and (iii) the effect on long-term depression (LTD). Both benzamides facilitated the formation of stable potentiation induced with three-pulse burst stimulation which is normally ineffective. CX546 in addition increased maximally inducible potentiation after four-pulse burst stimulation from about 50% to 100%. Burst response analysis revealed that CX546 greatly prolonged the duration of depolarization by slowing the decay of the response which thus presumably leads to a more continuous N-methyl-D-aspartate (NMDA) receptor activation. Cyclothiazide was ineffective in increasing maximal potentiation in either field or whole-cell recordings. CX546, but not CX516, also enhanced nearly two-fold the NMDA receptor-dependent long-term depression induced by heterosynaptic 2 Hz stimulation. Tests with recombinant NMDA receptors (NR1/NR2A) showed that CX516 and CX546 have no direct effects on currents mediated by these receptors. These results suggest that (1) modulation of AMPA receptors which increases either response amplitude or duration can facilitate LTP formation, (2) modulators that effectively slow response deactivation augment the maximum magnitude of LTP and LTD, and (3) receptor desensitization may have a minor impact on synaptic plasticity in the hippocampus. Taken together, our data indicate that AMPA receptor modulators differ substantially in their ability to enhance synaptic potentiation or depression, depending on their particular influence on receptor kinetics, and hence that they may also be differentially effective in influencing higher-order processes such as memory encoding.