Biomedical subjects
A Smith
Publications and source records attributed to A Smith.
Yeast PKA represses Msn2p/Msn4p-dependent gene expression to regulate growth, stress response and glycogen accumulation.
Yeast cAMP-dependent protein kinase (PKA) activity is essential for growth and antagonizes induction of the general stress response as well as accumulation of glycogen stores. Previous studies have suggested that the PKA effects on the two latter processes result in part from transcription repression. Here we show that transcription derepression that accompanies PKA depletion is dependent upon the presence of two redundant Zn2+-finger transcription factors, Msn2p and Msn4p. The Msn2p and Msn4p proteins were shown previously to act as positive transcriptional factors in the stress response pathway, and our results suggest that Msn2p and Msn4p also mediate PKA-dependent effects on stress response as well as glycogen accumulation genes. Interestingly, PKA activity is dispensable in a strain lacking Msn2p and Msn4p activity. Thus, Msn2p and Msn4p may antagonize PKAdependent growth by stimulating expression of genes that inhibit growth. In agreement with this model, Msn2p/Msn4p function is required for expression of a gene, YAK1, previously shown to antagonize PKA-dependent growth. These results suggest that Msn2p/Msn4p-dependent gene expression may account for all, or at least most, of the pleiotropic effects of yeast PKA, including growth regulation, response to stress and carbohydrate store accumulation.
Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.
The propagation of embryonic stem (ES) cells in an undifferentiated pluripotent state is dependent on leukemia inhibitory factor (LIF) or related cytokines. These factors act through receptor complexes containing the signal transducer gp130. The downstream mechanisms that lead to ES cell self-renewal have not been delineated, however. In this study, chimeric receptors were introduced into ES cells. Biochemical and functional studies of transfected cells demonstrated a requirement for engagement and activation of the latent trancription factor STAT3. Detailed mutational analyses unexpectedly revealed that the four STAT3 docking sites in gp130 are not functionally equivalent. The role of STAT3 was then investigated using the dominant interfering mutant, STAT3F. ES cells that expressed this molecule constitutively could not be isolated. An episomal supertransfection strategy was therefore used to enable the consequences of STAT3F expression to be examined. In addition, an inducible STAT3F transgene was generated. In both cases, expression of STAT3F in ES cells growing in the presence of LIF specifically abrogated self-renewal and promoted differentiation. These complementary approaches establish that STAT3 plays a central role in the maintenance of the pluripotential stem cell phenotype. This contrasts with the involvement of STAT3 in the induction of differentiation in somatic cell types. Cell type-specific interpretation of STAT3 activation thus appears to be pivotal to the diverse developmental effects of the LIF family of cytokines. Identification of STAT3 as a key transcriptional determinant of ES cell self-renewal represents a first step in the molecular characterization of pluripotency.
Gene expression of monocyte chemoattractant protein-1 in giant cell tumors of bone osteoclastoma: possible involvement in CD68+ macrophage-like cell migration.
Giant cell tumor of bone (GCT) is one of a few neoplasms in which the macrophage/osteoclast precursor cells and osteoclast-like giant cells infiltrate the tumor mass. Monocyte chemoattractant protein 1 (MCP-1) is a potent chemotactic factor specific for monocytes. In search of relevant cytokines that may enhance the recruitment of these reactive cells, we evaluated the localization and regulation of MCP-1 mRNA and protein in GCT by using Northern blot analysis, in situ hybridization and immunohistochemistry. We also determined whether conditioned medium obtained from GCT cultures can recruit human peripheral blood monocytes (CD68+) in an in vitro chemotactic assay. Using Northern blot analysis, we detected the specific gene transcript for MCP-1 in all GCT samples tested. In situ hybridization and immunohistochemistry revealed that both MCP-1 gene transcript and protein were consistently present in the cytoplasm of stromal-like tumor cells of GCT. Treatment of mononuclear cells from GCT at third passage with TGF-beta1 for 24 h increased the level of MCP-1 mRNA in a dose-dependent manner, with the maximum effect at 1 ng/ml. Conditioned media from GCT cultures promoted the chemotactic migration of CD68+ peripheral monocytes, an activity which was abolished by the addition of MCP-1 antibody to the conditioned medium. Thus, the results of this study suggest that recruitment of CD68+ macrophage-like cells may be due to the production MCP-1 by stromal-like tumor cells. These CD68+ cells may originate from peripheral blood and could have the capability of further differentiating into osteoclasts in the tumor.
The mitogen-activated protein kinase phosphatase-3 N-terminal noncatalytic region is responsible for tight substrate binding and enzymatic specificity.
We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3DeltaC) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3DeltaN) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPKbeta. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3DeltaN than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3DeltaN and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPKbeta. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.
Cancer predisposition: where's the phosphate?
Mutations in a protein phosphatase and a protein kinase cause hamartomatous polyposis syndromes, which are characterised by the formation of multiple benign polyps and an increased susceptibility to some types of cancer.
Depressed heart rate variability and arterial baroreflex in conscious transgenic mice with overexpression of cardiac Gsalpha.
Recently, we developed a transgenic mouse with cardiac-specific Gsalpha overexpression (TG mouse), which exhibits enhanced postsynaptic beta-adrenergic receptor signaling, ultimately developing a cardiomyopathy. The goal of the present study was to determine whether cardiac Gsalpha overexpression alters autonomic cardiovascular control, which could shed light on the mechanism responsible for the later development of cardiomyopathy. Mean arterial pressure was increased (P<.05) in conscious, chronically instrumented TG mice (123+/-1 mm Hg) compared with age-matched wild-type (WT) control mice (103+/-1 mm Hg). Respiratory frequency was increased (P<.05) in TG mice (269+/-26/min) compared with WT mice (210+/-20/min). By use of telemetric techniques, baseline heart rate (HR) was elevated (P<.05) in conscious, untethered TG mice (696+/-13 bpm) compared with WT mice (568+/-28 bpm). Intrinsic HR, after propranolol and atropine or after ganglionic blockade with hexamethonium, was not different between TG and WT mice. Both the normal minute-to-minute and circadian variations of HR observed in WT mice were markedly blunted in TG mice. HR variability was assessed by the time-domain and frequency-domain methods. At baseline, time-domain analysis indices were reduced (P<.05) in TG mice compared with WT mice. Although the low frequency (LF) component was higher (P<.05) than the high frequency (HF) component in WT mice, the LF component was less (P<.05) than the HF component in TG mice. In addition, arterial baroreflex regulation of HR was markedly blunted in TG mice in response to both nitroglycerin-induced hypotension and phenylephrine-induced hypertension. The reduced LF/HF ratio in TG mice was surprising in view of enhanced beta-adrenergic signaling and may be due to reduced neural tone secondary to the elevated arterial pressure or alterations in arterial baroreflex control. Dobutamine infusion in WT mice also resulted in depressed HR variability. The combination of elevated baseline HR, arterial pressure, and respiratory frequency suggests that enhanced beta-adrenergic signaling in TG mice results in reduced HR variability, in terms of both minute-to-minute variability and the lack of circadian variations in HR. The lack of normal HR variability in general and the failure of HR to decline, even during sleep, may actually be critical mechanisms contributing to the ultimate development of cardiomyopathy in these animals.
Microchimerism and HLA-compatible relationships of pregnancy in scleroderma.
BACKGROUND: Fetal cells can be found in the maternal circulation in most pregnancies. Fetal progenitor cells have been found to persist in the circulation of women many years after childbirth. We tested the hypothesis that microchimerism is involved in the pathogenesis of scleroderma. Scleroderma is of interest because of a strong female predilection, an increased incidence in the years after childbearing, and clinical similarities between scleroderma and chronic graft-versus-host disease after allogeneic bone-marrow transplantation. We also investigated whether HLA-compatibility of a child was associated with later development of scleroderma in the mother. METHODS: We enrolled 40 women who had previously given birth to at least one son--16 healthy controls, 17 scleroderma patients, and seven healthy sisters of patients. We used quantitative PCR to amplify a Y-chromosome-specific sequence in whole peripheral blood from these women. Also 32 controls with 58 children, and 21 scleroderma patients with 47 children were HLA genotyped. FINDINGS: The mean number of male cell DNA equivalents among controls was 0.38 cells per 16 mL whole blood (median 0 [range 0-2]) and 11.1 (6.0 [0-61]) among scleroderma patients (p = 0.0007). Controls' youngest sons were born a mean of 15.4 years previously, and scleroderma patients' sons 18.5 years previously. Some scleroderma patients had concentrations of male DNA higher than those found in most pregnant women. HLA-class II compatibility of a child from the mother's perspective was more common among scleroderma patients than among controls, but was not essential for persistence of male DNA in maternal peripheral blood. INTERPRETATION: Low concentrations of male DNA can be detected in healthy women decades after the birth of a son. Microchimerism in scleroderma patients could be secondary to the underlying disease. However, the finding that HLA class II compatibility of a child was more common for scleroderma patients than for controls, supports the possibility that microchimerism may be involved in the pathogenesis of scleroderma.
Audit commission tackles anaesthetic services.
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Profile of human macrophage transcripts: insights into macrophage biology and identification of novel chemokines.
High throughput partial sequencing of randomly selected cDNA clones has proven to be a powerful tool for examining the relative abundance of mRNAs and for the identification of novel gene products. Because of the important role played by macrophages in immune and inflammatory responses, we sequenced over 3000 randomly selected cDNA clones from a human macrophage library. These sequences represent a molecular inventory of mRNAs from macrophages and provide a catalog of highly expressed transcripts. Two of the most abundant clones encode recently identified CC chemokines. Macrophage-derived chemokine (MDC) plays a complex role in immunoregulation and is a potent chemoattractant for dendritic cells, T cells, and natural killer cells. The chemokine receptor CCR4 binds MDC with high affinity and also responds by calcium flux and chemotaxis. CCR4 has been shown to be expressed by Th2 type T cells. Recent studies also implicate MDC as a major component of the host defense against human immunodeficiency virus.
Expression of the protective proteins hemopexin and haptoglobin by cells of the neural retina.
The blood-retinal barrier, consisting of retinal pigment epithelial cells and retinal endothelial cells, prevents hemopexin and haptoglobin, anti-oxidant protective plasma proteins normally synthesized by the liver, from entering the neural retina. If present, these proteins must, therefore, be made locally. The cell types within the retina in which hemopexin and haptoglobin mRNAs are made have been investigated. RNA was extracted from both the neural retina and pigment epithelium obtained by dissection of human donor eyes as well as from cultured pigment epithelial and photoreceptor cells. The mRNAs for both haptoglobin and hemopexin were detected, using reverse-transcriptase polymerase chain reaction, in the neural retina and cultured photoreceptors but not in pigment epithelial cells. The cellular location of these mRNAs was determined using in situ hybridization of sections of human retina which revealed that haptoglobin mRNA was located principally in the photoreceptor cells, cells of the inner nuclear layer and some cells of the ganglion cell layer. Hemopexin mRNA, previously shown to be made in the human neural retina (Hunt et al., 1996. Journal of Cellular Physiology 168: 71-80), is expressed by most of the cells of neural retina including the photoreceptors and, notably, the ganglion cells.
The use of transgenic animals to assess the role of metabolism in target organ toxicity.
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Differential stability of a human mini-chromosome in mouse cell lines.
A 4 Mb human mini-chromosome, DeltaDelta2, was transferred from Chinese hamster ovary (CHO) cells into a mouse L cell line. The mini-chromosome could be transferred intact into the L cells, with 112/119 clones maintaining a mini-chromosome of the same size as the original. Ten clones were grown for 30 days in continuous culture. The mini-chromosomes were maintained stably with or without selection at a copy number of 1-2 per cell and none experienced any size alterations, as determined by pulsed-field gel electrophoresis. Thus DeltaDelta2 is structurally and mitotically stable in L cells. This contrasts with results in embryonic stem cells, in which DeltaDelta2 is highly unstable. These findings indicate that established somatic cell lines, such as L cells and CHO cells, have less stringent controls over centromeric function than do normal embryonic cells.
UBE3A "mutations" in two unrelated and phenotypically different Angelman syndrome patients.
Angelman syndrome (AS) is a rare neurodevelopmental disorder. Recently, several mutations have been found in the E6-AP ubiquitin protein ligase gene (UBE3A) in a group of patients who are nondeleted and do not have uniparental disomy or imprinting defects. Most of the reported mutations cluster within exons 9 or 16 of the UBE3A gene, and nearly all are predicted to give rise to truncated E6-AP ligases. Here, we describe two AS patients with dissimilar phenotypes. At the molecular level, they are both nondeleted, do not display uniparental disomy, and have normal imprint patterns. One has the typical AS phenotype and carries the previously reported 1344delAG de novo mutation involving a functionally significant region of UBE3A. The other expresses an atypical phenotype in that she has less severe ataxia, no inappropriate laughing, or epilepsy, and her EEG was normal at an early age. A 14-bp deletion in the 3' untranslated region of exon 16 (3'UTRdel14) adjacent to the poly(A) signal was identified. Further investigation revealed that the DNA change was a neutral polymorphism. Haplotype analysis indicated that both the AS patient and her normal sibling had inherited the same maternal UBE3A gene and its 5' flanking region. Although the 14-bp change has no functional significance, it assists with counseling to determine future risks of recurrence in this family.
Evaluation of methylation analysis for diagnostic testing in 258 referrals suspected of Prader-Willi or Angelman syndromes.
Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurodevelopmental disorders with interrelated genetic mechanisms because genomic imprinting within the chromosome 15q11-13 region affects both the PWS and the AS locus. Methylation analysis is one method of distinguishing between the maternally and paternally inherited chromosome 15. Here we present clinical and molecular data on a large series of 258 referred patients, evaluated with methylation analysis: 115 with suspected PWS and 143 with suspected AS. In these patients, the clinical phenotype was graded into three groups: classical (group 1); not classical but possible (group 2); not classical and unlikely (group 3). For PWS, a fourth group consisted of hypotonic babies. DNA methylation analysis confirmed the diagnosis of PWS in 30 patients (26%) and AS in 28 patients (20%). For 21 PWS patients the mechanism was established: 15 had deletions, 4 had uniparental disomy (UPD) and 2 a presumed imprinting defect. Clinically all those with an abnormal methylation pattern had the classical phenotype and none of those with a normal methylation pattern had classical PWS. For 23 AS patients in whom a mechanism was established, 17 had a deletion, 3 had UPD and 3 had a presumed imprinting defect. There was greater clinical overlap in AS, with 26 classical AS patients having a normal methylation pattern while an abnormal methylation pattern was seen in one patient from group 2. In addition, there were a further 40 patients with a normal methylation pattern in whom AS was still a possible diagnosis. Our conclusion is that methylation analysis provides an excellent screening test for both syndromes, providing approximately 99% diagnosis for PWS and for AS, a 75% diagnostic rate, supplemented for the remaining 25% with an essential basic starting point to further investigations.
Histochemical and ultrastructural modification of mucosal mast cell granules in parasitized mice lacking the beta-chymase, mouse mast cell protease-1.
The soluble beta-chymases mouse mast cell protease-1 (mMCP-1) and rat mast cell protease-II are predominantly expressed by intestinal mucosal mast cells (IMMCs) and may promote mucosal epithelial permeability when released during intestinal allergic hypersensitivity responses. To study the function of these chymases, we generated mice with a homozygous null mutation of the mMCP-1 gene and investigated their response to infection with the intestinal nematode Nippostrongylus brasiliensis. Whereas mMCP-2, -4, and -5 were transcribed normally, there was no transcription of the mMCP-1 gene in null (-/-) mice, nor was mature mMCP-1 protein detected in (-/-) jejunal mucosa. In contrast, levels of mMCP-1 in wild-type (+/+) jejunal mucosa increased 200- to 350-fold from 0.66 microg mMCP-1/g wet weight in uninfected mice to 129 and 229 microg/g wet weight on days 8 and 10 of infection, respectively. The kinetics of IMMC recruitment differed in -/- mice compared with +/+ controls on days 8 (P < 0.05) and 10 (P < 0.03) of infection. The IMMCs in infected -/- mice stained poorly, if at all, for esterase with naphthol AS-D chloroacetate compared with the intense staining observed in +/+ controls. Ultrastructurally, the prominent crystal intragranular structures that are found in intraepithelial +/+ IMMCs were absent from -/- IMMCs. These data show that disruption of the mMCP-1 gene leads to profound histochemical and ultrastructural changes in IMMC granules.
A case of chronic myelomonocytic leukemia with isochromosome 14q.
A 72-year-old man with increased monocytes and eosinophils in the bone marrow was found to have an isochromosome, i(14q), as the sole abnormality on bone-marrow cytogenetics. Follow-up over 12 months with repeat cytogenetic testing of bone marrow specimens on three further occasions showed an additional abnormality consisting of an apparently balanced translocation, t(6;21)(p21.1; q22). This abnormality, together with other features, indicated disease progression. A review of seven reported cases of i(14q) revealed limited follow-up information.
Effects of the common cold on mood and performance.
Previous research has shown that both experimentally-induced and naturally occurring upper respiratory tract illnesses (URTIs) influence mood and mental functioning. None of the previous studies of naturally occurring colds has conducted appropriate virological assays to determine the nature of the infecting agent. This is an essential methodological step in studies of malaise associated with URTIs. The aim of this research was to investigate the effects of naturally occurring colds on mood and objective measures of performance. This was done by first conducting a cross-sectional comparison of 37 healthy people and 158 volunteers with colds and then a longitudinal study in which 100 volunteers developed colds and 87 remained healthy. Virological techniques were used to identify infecting agents and comparisons made across the different groups. The results showed that having a cold was associated with reduced alertness and slowed reaction times. These effects were observed both for colds where the infecting virus was identified and those where it was not. Similar effects were obtained for both rhinovirus and coronavirus colds. One may conclude that upper respiratory tract illnesses lead to a reduction in subjective alertness and impaired psychomotor functioning. This was true for both illnesses where the infecting agent was identified and for those clinical illnesses where no virus was detected. It is now important to identify the mechanisms linking infection and illness with the behavioural changes. Similarly, the impact of these effects on real-life activities such as driving needs examining. Finally, methods of treatment need to be developed which not only treat the local symptoms of the illnesses but remove the negative mood and the performance impairments.