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Biomedical subjects

G James

Publications and source records attributed to G James.

At least 19 recordsLinked to original sources
PubMed1992

FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains.

Myogenin belongs to a family of myogenic helix-loop-helix (HLH) proteins that activate muscle transcription through binding to a conserved DNA sequence associated with numerous muscle-specific genes. Fibroblast growth factor (FGF) inhibits myogenesis by inactivating myogenic HLH proteins. We show that activated protein kinase C (PKC) can substitute for FGF and inhibit transcriptional activity of myogenic HLH proteins. In transfected cells, FGF induces phosphorylation of a conserved site in the DNA-binding domain of myogenin. This site is phosphorylated by PKC in vivo and in vitro and mediates repression of the myogenic program through a loss in DNA binding activity. A myogenin mutant lacking the PKC phosphorylation site is not repressed by FGF, confirming this site as a molecular target for FGF-dependent repression of muscle transcription. These results establish a direct link between the signal transduction pathways that inhibit myogenesis and the transcription factors directly activating muscle-specific genes.

Amino Acid Sequence↗
PubMed1992

Protein turnover in 3T3 cells transformed with the oncogene c-H-ras1.

We have examined protein turnover, growth, DNA synthesis and proliferation in three independent clones of 3T3-NR6 cells transformed with the oncogene c-H-ras1. We find that, firstly, the half-maximum concentration of serum and insulin regulating protein turnover in ras-transformed cells is significantly reduced from 0.5 to 0.3% for serum and from 4 nM to 0.5 nM for insulin, and, secondly, ras-transformed cells consistently have lower rates of protein degradation. The catabolic effect of conditioned medium or serum withdrawal is attenuated in transformed lines by maintaining lower basal rates of protein breakdown and higher basal rates of DNA and protein synthesis. Serum stimulation of growth in transformed cells is achieved in the short term by lower rates of protein breakdown rather than higher rates of protein synthesis: rates of protein synthesis become significantly higher 24 h after serum stimulation. Therefore transformed cells have higher rates of proliferation and grow to higher densities, but display characteristics common to normal cells because rates of protein synthesis decrease and protein degradation increase as a function of cell density. We conclude that higher basal rates of protein synthesis and growth with retention of the normal proliferative response to serum result from the pleiotropic nature of ras transformation, whereas lower rates of protein degradation and increased sensitivity to serum and insulin imply a direct regulatory role for ras.

3T3 Cells↗
PubMed1992

The radiological assessment of vertebral osteoporosis.

The traditional skeletal X-ray is of little help in quantifying osteopenia in the spine, and indirect criteria, such as increased lucency, loss of horizontal trabeculae and reduction in end-plate thickness with relatively increased opaqueness, can be misleading. However, the clinical expression of osteoporosis is fracture, which can be identified by radiography. Any reduction in the anterior, middle, or total height of the vertebral body should be classified as vertebral fracture. In mild cases, such fractures are not easily detectable, particularly when previous radiographs are not available for comparison. Thus several objective methods for fracture identification have been developed. Most of these methods are based on the ratio between posterior and anterior or middle height (wedge or biconcave deformity, respectively) and on the ratio between posterior height of adjacent vertebrae or with a single vertebral reference (T4). Some of these indices are referred to normal ranges in order to take into account intervertebral and interindividual variability. In 36 women with postmenopausal osteoporosis we have compared the lateral radiographs of lumbar and thoracic spine to similar X-ray pictures taken by chance at least five years before menopause. By defining a fracture as any decrease in vertical height above 1 mm, we found 77 deforming events in 29 out of 36 patients. We then applied some of the methods to identify objectively fractures in our postmenopausal radiographs: the sensitivity of the various systems ranged from 50 to 100%; however there was a large overlap between false positives and false negatives and the methods with the highest sensitivity lack specificity and vice versa.

Body Height↗
PubMed1992

Deletion of the regulatory domain of protein kinase C alpha exposes regions in the hinge and catalytic domains that mediate nuclear targeting.

Members of the protein kinase C (PKC) family are characterized by an NH2-terminal regulatory domain containing binding sites for calcium, phosphatidylserine, and diacylglycerol (or tumor-promoting phorbol esters), a small central hinge region and a COOH-terminal catalytic domain. We have constructed fusion proteins in which the regulatory domain of PKC alpha was removed and replaced by a 19-amino acid leader sequence containing a myristoylation consensus or by the same sequence in which the amino-terminal glycine was changed to alanine to prevent myristoylation. The goal was to generate constitutively active mutants of PKC that were either membrane bound, due to their myristoylation, or cytoplasmic. Western blotting of fractions from COS cells transfected with plasmids encoding wild-type and mutant proteins revealed that PKC alpha resided entirely in a Triton X-100 soluble (TS) fraction, whereas both the myristoylated and nonmyristoylated mutants were associated primarily with the nuclear envelope fraction. A similar mutant that lacked the 19 amino acid leader sequence was also found almost entirely in the nuclear envelope, as was a truncation mutant containing only the regulatory domain, hinge region, and a small portion of the catalytic domain. However, an additional truncation mutant consisting of only the regulatory domain plus the first one-third of the hinge region was almost entirely in the TS fraction. A nonmyristoylated fusion protein containing only the catalytic domain was also found in the nuclear envelope. Immunostaining of cells transfected with these constructs revealed that both the myristoylated and nonmyristoylated mutants were localized in nuclei, whereas wild-type PKC alpha was primarily cytoplasmic and perinuclear. Phorbol dibutyrate treatment of PKC alpha-transfected cells resulted in increased perinuclear and nuclear staining. The results are consistent with a model in which activation of PKC, by phorbol esters or by deletion of the regulatory domain, exposes regions in the hinge and catalytic domains that interact with a PKC "receptor" present in the nuclear envelope, and may explain the ability of wild-type PKC to be translocated to the nucleus under certain conditions.

Animals↗
PubMed1992

Ventricular size, cognitive function and depression in patients with multiple sclerosis.

The purpose of this study was to explore further the hypothesis that changes in cognitive function may occur in the mild stages of multiple sclerosis (MS) by determining whether ventricular enlargement was related to cognitive function. Ten measures of ventricular size were made in a sample of 123 MS patients with mild disability and 60 well-matched healthy controls. In addition, sixteen tests of cognitive function and the Beck Depression Inventory were administered. For the MS group, there were significant correlations between the ventricular measures and cognitive performance but not for the normal controls. Scores on the Beck Depression Inventory were not correlated with either cognitive performance or ventricular enlargement. These findings suggest that for the MS group cognitive impairment was related to the disease process but not to the level of depression.

Adult↗
PubMed1991

Comprehensive school health programs in Texas public schools.

One goal of the Texas Comprehensive School Health Initiative (TCSHI) involves assessing the status of comprehensive school health (CSH) programs in Texas public schools. This project used a case study approach to establish baseline information regarding CSH programs in selected school districts across the state. Eight researchers were trained to use a case study instrument and procedures for conducting interviews. Seven school districts participated in the survey during fall 1990. Results indicated considerable concern and support for CSH programs but little coordination of programs was evident. Six districts were not organized to address health comprehensively. Generally, school districts were overwhelmed with other responsibilities, so CSH programming was not a priority. Results will be used to revise the survey instrument and further expand the data base.

Adolescent↗
PubMed1990

Fatty acylated proteins as components of intracellular signaling pathways.

From the studies presented above, it is obvious that fatty acylation is a common modification among proteins involved in cellular regulatory pathways, and in certain cases mutational analyses have demonstrated the importance of covalent fatty acids in the functioning of these proteins. Indeed, certain properties provided by fatty acylation make it an attractive modification for regulatory proteins that might interact with many different substrates, particularly those found at or near the plasma membrane/cytosol interface. In the case of intracellular fatty acylated proteins, the fatty acyl moiety allows tight binding to the plasma membrane without the need for cotranslational insertion through the bilayer. For example, consider the tight, salt-resistant interaction of myristoylated SRC with the membrane, whereas its nonmyristoylated counterpart is completely soluble. Likewise for the RAS proteins, which associate weakly with the membrane in the absence of fatty acylation, while palmitoylation increases their affinity for the plasma membrane and their biological activity. Fatty acylation also permits reversible membrane association in some cases, particularly for several myristoylated proteins, thus conferring plasticity on their interactions with various signaling pathway components. Finally, although this has not been demonstrated, it is conceivable that covalent fatty acid may allow for rapid mobility of proteins within the membrane. Several questions remain to be answered concerning requirements for fatty acylation by regulatory proteins. The identity of the putative SRC "receptor" will provide important clues as to the pathways in which normal SRC functions, as well as into the process of transformation by oncogenic tyrosine kinases. The possibility that other fatty acylated proteins associate with the plasma membrane in an analogous manner also needs to be investigated. An intriguing observation that can be made from the information presented here is that at least three different families of proteins involved in growth factor signaling pathways encode both acylated and nonacylated members, suggesting that selective fatty acylation may provide a means of determining the specificity of their interactions with other regulatory molecules. Further studies of fatty acylated proteins should yield important information concerning the regulation of intracellular signaling pathways utilized during growth and differentiation.

Acetyltransferases↗
PubMed1990

Diurnal and postural variations in plasma atrial natriuretic factor, plasma guanosine 3':5'-cyclic monophosphate and sodium excretion.

1. We studied diurnal patterns of plasma atrial natriuretic factor, plasma guanosine 3':5'-cyclic monophosphate and urinary sodium excretion in normal subjects after 3 days on a 200 mmol of sodium/60 mmol of potassium diet. On the fourth day blood samples and urine were collected every 3 h. 2. Two studies were performed. In study 1, normal subjects (n = 8) were recumbent for 23 h from 09.00 hours to 08.00 hours the next day. In study 2, normal subjects (n = 10) were permitted to ambulate from 09.00 hours to 23.00 hours and then were recumbent until 08.00 hours the next day. 3. In study 1, assumption of the recumbent posture was associated with increases in plasma atrial natriuretic factor (P less than 0.01), plasma guanosine 3':5'-cyclic monophosphate (P less than 0.05) and urinary sodium excretion (P less than 0.05). 4. In contrast, in study 2 there were no significant changes in plasma atrial natriuretic factor during the day; instead, plasma atrial natriuretic factor increased overnight, reaching a peak at 24.00 hours after 1 h of recumbency (P less than 0.01). A smaller rise in plasma guanosine 3':5'-cyclic monophosphate (P less than 0.05) occurred; urinary sodium excretion decreased markedly (P less than 0.01) and there was no change in creatinine clearance. 5. In both studies, recumbency was associated with an initial drop, followed by a rise, in packed cell volume. 6. These data demonstrate that assumption of the supine position induces a rise in plasma atrial natriuretic factor and accounts for most of the observed variation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Myristoylation, phosphorylation, and subcellular distribution of the 80-kDa protein kinase C substrate in BC3H1 myocytes.

Numerous reports have described a phosphoprotein with an apparent molecular mass of 68-87 kDa, often referred to as the 80K protein, which serves as a major specific substrate for protein kinase C in a wide variety of cell types. This protein has been shown to be myristoylated in macrophages, apparently in a stimulus-dependent manner. In the present study, we have defined the kinetics for myristoylation of the 80K protein in BC3H1 myocytes and have examined the subcellular distribution of the [3H]myristate and 32P-labeled forms of the protein before and after activation of protein kinase C by phorbol dibutyrate (PDBu). The 80K protein was identified in BC3H1 myocytes by apparent molecular mass of 68 kDa (consistent with the previously reported size of the murine homologue), isoelectric point of 4.6-4.8, PDBu-inducible phosphorylation, peptide mapping, and labeling with [3H]myristate. Incorporation of [3H]myristate by this protein occurred through an amide linkage and was abolished completely by cycloheximide. Pulse labeling of quiescent cells with [3H]myristate revealed no alteration in myristoylation of the 80K protein in either the crude membrane or soluble fractions after PDBu-induced phosphorylation. The subcellular distribution of this protein (approximately 80% membrane, approximately 20% cytosol) also was the same in control and PDBu-stimulated cells. Phosphorylation of both the membrane-bound and soluble forms was increased approximately 6-fold upon stimulation of cultures with PDBu; the soluble form was phosphorylated to a 4-fold higher stoichiometry than its membrane-bound counterpart. Together, these data demonstrate that the 80K protein is myristoylated cotranslationally in BC3H1 cells and that protein kinase C-dependent phosphorylation of the 80K protein does not alter its subcellular distribution or degree of myristoylation. The fact that 20% of total myristoylated 80K protein resides in the cytosol also indicates that myristoylation alone is not sufficient to target this protein to the plasma membrane.

Animals↗
PubMed1989

Identification of a novel fatty acylated protein that partitions between the plasma membrane and cytosol and is deacylated in response to serum and growth factor stimulation.

Several proteins involved in transmembrane signaling have been shown previously to be modified covalently by long-chain fatty acids. Using the BC3H1 cell line, which contains a broad array of fatty acylated proteins, we have examined the possibility that acylation of certain proteins is modulated in response to mitogenic stimulation. In the present study, we describe a 64-kDa palmitoylated protein, referred to as p64, that is deacylated following stimulation of quiescent cells with fetal bovine serum, fibroblast growth factor, and phorbol dibutyrate. Western blot analysis of membrane and soluble fractions using a polyclonal antibody against p64 revealed that approximately 70% of p64 in unstimulated cells is present in the cytosol in a non-acylated form, whereas palmitoylated p64 is found exclusively in the membrane fraction. Extraction of membranes with 0.5 M sodium chloride, 0.2 M sodium pyrophosphate, or 0.2 M sodium carbonate failed to release p64, suggesting that the acylated form of this protein is tightly associated with membranes. Pulse labeling of proteins in quiescent cells with [3H] palmitate and subsequent chasing in medium containing 20% fetal bovine serum, fibroblast growth factor, or phorbol dibutyrate revealed that the fatty acid associated with p64 undergoes mitogen-stimulated turnover, whereas turnover of fatty-acid on other acylated proteins is not observed. Palmitate is the predominant fatty acid associated with p64; however, small amounts of covalent myristate are also detected. Both fatty acids are attached post-translationally to p64 through a hydroxylamine-sensitive linkage, suggesting that acylation of this protein is catalyzed by a palmitoyl transferase with relaxed specificity for fatty acid substrates. Together, these results suggest that palmitoylation may participate in the association of p64 with the plasma membrane and that mitogen-dependent deacylation might alter interactions between this protein and other membrane components.

Acylation↗
PubMed1988

Regulation of glomerular filtration rate in chronic congestive heart failure patients.

The purpose of this study was to identify the factor(s) that characterize impaired glomerular filtration rate (GFR) in congestive heart failure (CHF) patients. We studied 34 patients, measuring systemic hemodynamics, vasoactive hormones, and sodium and volume status. Renal plasma flow (para-aminohippurate) and GFR (inulin) were assessed by steady-state clearance techniques. Both linear and multiple regression analyses were performed. Impaired GFR was characterized by reduction of renal blood flow and renal fraction of cardiac output, and by an increase of renal vascular resistance. The correlation between GFR and filtration fraction (r = 0.492, P less than 0.003) indicated that individuals with greatest impairment of GFR had the lowest filtration fraction and increased overall renal vascular resistance, indicating dependence of GFR on afferent, rather than efferent vasoconstriction, under conditions of low renal perfusion. Mean vasoactive hormones and blood volume were increased, but without clear cut correlation with GFR. The greatest impairment of GFR was observed in elderly CHF patients, as renal blood flow and function demonstrated an age-dependent decline, in addition to the adverse effects of CHF. In a multiple regression model, renal blood flow and filtration fraction accounted for 69% and 25%, respectively, of the variability of GFR, with a co-linear influence of age. Thus, we have identified age-related, abnormal renal hemodynamic patterns in CHF, and the fragile nature of GFR in the elderly CHF population.

Adult↗
PubMed1988

Protein fatty acylation: a novel mechanism for association of proteins with membranes and its role in transmembrane regulatory pathways.

A wide range of proteins of cellular and viral origin have been shown to be modified covalently by long-chain fatty acids. Recent studies have revealed at least two distinct types of protein fatty acylation which involve different fatty acyltransferases. The abundant fatty acid, palmitate, is incorporated post-translationally through a thiol ester linkage into a variety of cell surface glycoproteins and non-glycosylated intracellular proteins. In contrast, the rare fatty acid, myristate, is incorporated co-translationally through an amide linkage into numerous intracellular proteins. Identification of proteins that contain covalent fatty acids has revealed that this modification is common to a broad array of proteins that play important roles in transmembrane regulatory pathways. For many of these proteins, the fatty acid moiety appears to play an important role in directing the polypeptide to the appropriate membrane and in mediating protein-protein interactions within the membrane. This review will summarize recent studies that define different pathways for protein fatty acylation and will consider the potential functions for this unique covalent modification of proteins.

Acylation↗
PubMed1987

Comprehensive care of patients with head injuries.

The comprehensive head injury service run by the neurosurgeons at the Hull Royal Infirmary for the surrounding population of one million was analysed. The analysis showed that all patients with either a fractured skull or a lowered level of consciousness should be admitted to a district general hospital because the associated risk of their having a major head injury is over 20%. Those patients with both a fractured skull and a lowered level of consciousness have a 60% likelihood of a major head injury and should be transferred immediately to the neurosurgical unit. Patients with compound or complicated fractures of the skull and those without fractured skulls but with neurological impairment persisting for four hours or more, should also be transferred to the neurosurgical unit. If these guidelines are followed about 200 patients/million population will be referred to the neurosurgical centre. Patients with a minor head injury and none of the clinical risk factors may safely be sent home. This should reduce the rate of admissions to hospital for head injuries by 60%.

Comprehensive Health Care↗
PubMed1987

Sequential changes in plasma luteinizing hormone and plasma prorenin during the menstrual cycle.

Prorenin, the enzymatically inactive biosynthetic precursor of renin, is secreted by the kidneys. However, the ovaries appear to be the source of the cyclical increase in plasma prorenin that occurs in the middle of the menstrual cycle. In this study we examined the temporal relationship between changes in plasma prorenin and LH in normal women to determine whether ovarian prorenin secretion might be stimulated by LH. Blood was collected from nine normal women daily for 7 days in the midcycle period and from six of them every 8 h on 6 of these days. Time zero was taken as the highest plasma LH value. The initial rise in LH (-24 h) preceded the initial rise in prorenin (-8 h) and the LH peak preceded the prorenin peak by 8-16 h. These sequential increases in plasma LH and prorenin occurred in the presence of high plasma estradiol levels. While LH fell in parallel with estradiol, the prorenin peak was more sustained and plasma prorenin remained above baseline at 40 and 48 h, at a time when both estradiol and LH had reached a new basal level. These results suggest that gonadotropins stimulate ovarian prorenin release. The timing of the changes in plasma prorenin and its presence in high concentrations in ovarian follicular fluid suggest that prorenin may be involved in the process of ovulation. The results also suggest that changes in plasma prorenin may determine the activity of an ovarian renin system that functions independently of circulating active renin.

Adult↗