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Biomedical subjects

C J Smith

Publications and source records attributed to C J Smith.

At least 235 records · Page 13Linked to original sources
PubMed1989

Control and manipulation of gene expression during tomato fruit ripening.

Ripening is a complex developmental process involving changes in the biochemistry, physiology and gene expression of the fruit. It is an active process characterised by changes in all cellular compartments. cDNA cloning has been used as an approach to analyse changes in gene expression during fruit ripening. This has revealed that several genes are switched on specifically during fruit ripening, including one encoding polygalacturonase (PG), a major cell wall protein. These cDNA clones have been used to study the expression of the genes in normal and ripening mutant fruits, and under environmental stress conditions. The PG gene has been isolated and it has been demonstrated that 1450 bases 5' of the coding region are sufficient for the tissue- and development-specific expression of a bacterial marker gene in transgenic tomatoes. Antisense RNA techniques have been developed to generate novel mutant tomatoes in which the biochemical function of this enzyme and its involvement in fruit softening has been tested.

Cloning, Molecular↗
PubMed1989

The response of large and small luteal cells from the pregnant rat to substrates and secretagogues.

Large (greater than 22 microns) and small (12-21 microns) luteal cells from Day 8 pregnant rats were separated by elutriation after enzyme dissociation. Aliquots of cells were incubated for 4 h at 37 degrees C in Medium 199 alone (control) or with medium containing dibutyryl cyclic adenosine 3', 5'-monophosphate (cAMP) at 0.5 mM or 5 mM; rat luteinizing hormone (LH) at doses of 1, 10, 100, or 1000 ng/ml; 10 micrograms/ml 25-OH-cholesterol; or 10 ng/ml testosterone. Production of progesterone, testosterone, and estradiol was measured by radioimmunoassay. Both cell types showed a similar increase in estradiol synthesis when stimulated with LH (1 microgram/ml) or dibutyryl cAMP (5 mM); however, large luteal cells aromatized exogenous testosterone, whereas small luteal cells did not. Large luteal cells produced increased amounts of progesterone at lower doses of dibutyryl cAMP (0.5 mM) and LH (10 ng/ml), compared to small cells, which required 5 mM dibutyryl cAMP or 1 microgram/ml LH for minimal stimulation. Dibutyryl cAMP (5 mM) also resulted in an increase of testosterone release from small luteal cells. Progesterone synthesis in both cell types was enhanced by 25-OH-cholesterol. These results suggest that the two cell types differ functionally with respect to steroidogenesis during pregnancy, and that the large luteal cells appear to be the primary site of progesterone and estradiol production at this stage of pregnancy.

Animals↗
PubMed1989

Role of hormone-sensitive low Km cAMP phosphodiesterase in regulation of cAMP-dependent protein kinase and lipolysis in rat adipocytes.

The time-courses of isoproterenol activation of rat adipocyte particulate low Km cAMP phosphodiesterase (PDE) activity, cAMP-dependent protein kinase (A-kinase), and glycerol production were measured in the presence and absence of insulin. Isoproterenol (100 nM) alone rapidly activated A-kinase 8- to 10-fold and increased particulate cAMP PDE by approximately 100%. A-kinase and PDE activity remained relatively constant for at least 25 to 30 min. Kact values for isoproterenol activation of PDE and lipolysis were similar. In comparison with isoproterenol, insulin (0.1-0.3 nM) alone increased particulate cAMP PDE at a slower rate and to a lesser extent (by approximately 50% within 12 to 16 min) and without any change in A-kinase. With insulin plus isoproterenol there was a rapid, transient, and synergistic activation of particulate cAMP PDE, which temporally correlated with a decrease in A-kinase and reduction in lipolysis. These and other data suggest the following: 1) there is a close concentration-dependent and temporal relationship in isoproterenol activation of adenylate cyclase, of A-kinase, and of particulate cAMP PDE; 2) isoproterenol and insulin activate particulate cAMP PDE by two distinct mechanisms; 3) the temporal changes in PDE and A-kinase in the presence of insulin and isoproterenol suggest that insulin activation of the PDE does not require, but may be enhanced by, elevated cAMP and is important in the antilipolytic action of insulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Effect of a new macrolide antibiotic (tilmicosin) on pneumonia experimentally induced in calves by Mycoplasma bovis and Pasteurella haemolytica.

Two gnotobiotic calves were treated once with tilmicosin (20 mg kg-1) six hours before they were infected by the intratracheal route with Mycoplasma bovis and Pasteurella haemolytica serotype 1. This treatment prevented colonisation of the lungs by P haemolytica and considerably reduced colonisation by M bovis, and the clinical scores and the extent of pneumonic consolidation, compared with two untreated gnotobiotic calves, both of which had to be killed in extremis for humanitarian reasons within 24 hours of infection. In a second experiment, 10 conventionally reared calves were similarly exposed to infection and, at the onset of clinical disease, five were treated once with tilmicosin (20 mg kg-1). Colonisation by P haemolytica and M bovis, the clinical scores and extent of pneumonic consolidation were suppressed or greatly reduced in the treated compared with the untreated calves, one of which had to be killed in extremis two days after infection. It was concluded that tilmicosin had a beneficial effect.

Animals↗
PubMed1988

Interaction of 9-amino-1,2,3,4-tetrahydroaminoacridine (THA) with human cortical nicotinic and muscarinic receptor binding in vitro.

Tetrahydroaminoacridine (THA) has recently been reported to be more useful in the treatment of Alzheimer's disease than physostigmine. A comparison of the effects of these two anticholinesterase agents on in vitro enzyme and receptor activities of human cerebral cortex (obtained at autopsy) revealed similarities in their interactions with acetylcholinesterase (AChE) but striking differences in their ability to displace both nicotinic and muscarinic radioligands from membrane preparations. IC50 values (the concentration required to reduce enzyme activity by 50%) for the inhibition of total tissue AChE were 7.9 x 10(-7) M and 4.5 x 10(-8) M for THA and physostigmine, respectively, and similar values were also obtained for individual molecular forms of AChE (monomer G1, dimer G2 and tetramer G4) separated by sucrose density gradient centrifugation. In contrast, IC50 values for [3H]nicotine displacement (a measure of nicotinic cholinergic receptor binding) differed 1000-fold for THA (2 x 10(-5) M) and physostigmine (2 x 10(-2) M) and 100-fold for [3H]N-methylscopolamine displacement (a measure of muscarinic cholinergic receptor binding). Differences were also noted in the inhibition of carbachol stimulated polyphosphoinositide (PI) hydrolysis (a measure of muscarinic receptor induced second messenger activity) in isolated rat cortical miniprisms. It is suggested that variations in clinical efficacy of THA and physostigmine may be related less to their anticholinesterase properties and more to their interactions with other activities such as cholinergic receptors.

Acetylcholinesterase↗
PubMed1988

Steroid profiles of the female tilapia, Oreochromis mossambicus, and correlation with oocyte growth and mouthbrooding behavior.

Plasma levels of progesterone, 17 alpha-OH-progesterone, testosterone, and estradiol-17 beta were measured by radioimmunoassay during the ovarian cycles of two groups of female tilapia, Oreochromis mossambicus. One group included females that successfully mouthbrooded fry, while the other group consisted of females in which the zygotes were either removed or swallowed within 1 day after spawning. The mouthbrooders had a longer ovarian cycle (about 40 days) and were sampled 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, and 40 days after spawning. The non-mouthbrooders had an ovarian cycle of about 25 days. They were sampled 3, 5, 7, 10, 15, 20, and 25 days after spawning. Initial peaks in levels of testosterone, estradiol-17 beta, and progesterone occurred later in the cycle of mouthbrooders. The first peak of testosterone and estradiol-17 beta occurred at 15 days after spawning. While estradiol-17 beta levels remained high, testosterone levels fell at 25 days after spawning, and increased again just prior to spawning. In the latter phase of mouthbrooding (15-25 days after spawning), the oocytes in the ovary did not increase in size, and testosterone and estradiol levels were high. During this time, estradiol may have a function other than stimulating vitellogenesis, such as an involvement (with testosterone?) in parental behavior, or protecting the oocytes from atresia. In non-mouthbrooders, testosterone, estradiol-17 beta, and progesterone levels initially peaked at 10 days after spawning, then dropped at 15 days after spawning. At the end of the cycle, testosterone and estradiol-17 beta levels increased again. The drop in estradiol levels is contrary to the profile seen in mouthbrooders. Also in mouthbrooders, progesterone levels did not rise until 25 days after spawning, then decreased and peaked again towards the end of the cycle. 17 alpha-OH-progesterone concentrations were low, with a single peak at 7 days after spawning in non-mouthbrooders, and at 40 days after spawning in mouthbrooders. There appears to be a relationship between the delayed initial peaks of the steroid hormones measured, oocyte growth arrestment, and longer-lived postovulatory follicles in mouthbrooding female tilapia.

17-alpha-Hydroxyprogesterone↗
PubMed1988

Weight, corticosterone and glucose: changes with time of day after food deprivation.

The effects of constant food deprivation, terminated at different times of day were examined with respect to percent body weight loss (%BWL), plasma corticosterone (PC) and plasma glucose (PG). A 19-hour food deprivation paradigm schedule staggered around varying times of the day-night cycle was used. Patterns of %BWL related to nocturnal lipogenesis and diurnal lipolysis showed the greatest loss (10%) occurring at 0700 hr, while in evening hours, there was an increasing pattern of weight loss, with the greatest amount (7%) occurring at 2200 hr. A pattern suggestive of neuroregulatory cycles of glucocorticoid release was evident for PC levels; maximum levels for PC (23.2 micrograms%) were reported at 0700 hr and at 1900 hr (16.6 micrograms%). Similar patterns were also noted in PG levels, with respective maximum levels of 138.0 mg% and 125.8 mg% occurring at 0700 hr and 1900 hr. These results indicate that the time of day that a deprivation schedule is initiated and terminated is an important consideration due to the impact of circadian photoperiodism. Time of day should be an essential consideration when utilizing deprivation paradigms.

Animals↗
PubMed1988

Early psychological aspects of severe hand injury.

We investigated the incidence and nature of psychological symptoms occurring during the first two months after severe hand injuries. 94% of patients had significant symptoms at some point early in rehabilitation, including nightmares (92%), flashbacks (88%), affective lability (84%), preoccupation with phantom limb sensations (13%), concentration/attention problems (12%), cosmetic concerns (10%), fear of death (5%), and denial of amputation (3%). Two months later, flashbacks (63%) remained pronounced. Nightmares (13%), affective lability (48%), concentration/attention problems (5%), fear of death (0%), and denial of amputation (0%) declined markedly, while cosmetic concerns (17%) and preoccupation with phantom limb sensations (17%) increased. Based on these findings, we believe that psychological treatment should often be given as part of the rehabilitation process.

Affective Symptoms↗
PubMed1988

Muscarinic cholinergic receptor subtypes in hippocampus in human cognitive disorders.

Total muscarinic receptor levels, the levels of the subtypes exhibiting high and low affinity for pirenzepine, and the high- and low-affinity agonist states of the receptor were investigated in hippocampal tissue obtained at autopsy from mentally normal individuals and the following pathological groups: Alzheimer's disease, Parkinson's disease, Down's syndrome, alcoholic dementia, Huntington's chorea, and motor-neurone disease. A moderate decrease in the density of both high-affinity pirenzepine and high-affinity agonist subtypes was found in Alzheimer's disease, whereas a trend towards an increase in the overall muscarinic receptor density was apparent in the parkinsonian patients without dementia, mainly due to an increase in the low-affinity agonist state; the differences between the Alzheimer's disease and nondemented parkinsonian cases were highly significant. As previously reported, the levels of both choline acetyltransferase and acetylcholinesterase were markedly reduced in both Alzheimer's disease and Parkinson's disease--with a greater loss of both enzymes in the demented subgroup of parkinsonian patients. Activities of the cholinergic enzymes were also extensively reduced in Down's syndrome, accompanied by a loss of high-affinity pirenzepine binding. There were no significant receptor or enzyme alterations in the other groups studied. These observations suggest that in the human brain, extensive degeneration of cholinergic axons to the hippocampus, as indicated by a loss of cholinergic enzymes, is not necessarily accompanied by extensive muscarinic receptor abnormalities (as might be expected if a major subpopulation were presynaptic). Moreover, the opposite changes in muscarinic binding in Parkinson's and Alzheimer's diseases may be related to the greater severity of dementia in the latter disease.

Acetylcholinesterase↗
PubMed1988

Selective cytotoxicity of hydroquinone for melanocyte-derived cells is mediated by tyrosinase activity but independent of melanin content.

In previous studies we have shown melanotic melanomas to be exquisitely more sensitive to hydroquinone (HQ) inhibition than non-melanotic cell lines in vitro. Indeed, incorporation of [H3] Urd and [H3] Thd have been shown to be respectively 80 and 35 times more sensitive to HQ inhibition. The difference between the cell lines studied was their derivation, marked by their different melanin contents. The presence of melanin was proposed as a possible explanation of the differences. However, comparative experiments reported here demonstrate that amelanotic melanoma cell lines are equally susceptible to HQ inhibition. Thus, the action of HQ is apparently independent of the melanin content of the cell. Significantly, the tyrosinase levels in the melanomas and the amelanomas were found to be comparable and markedly different from that in the non-melanoma control cell lines. Thus, the results reported here support the hypothesis put forward by other workers that hydroquinone melanotoxicity is independent of cellular melanin content but requires the presence of active tyrosinase.

Animals↗
PubMed1988

Nucleotide sequence analysis of tetracycline resistance gene tetO from Streptococcus mutans DL5.

Streptococcus mutans DL5, isolated from the dental plaque of a pig, was resistant to high levels of streptomycin (Sm, 20 mg/ml), erythromycin (Em, 1 mg/ml), and tetracycline (Tc, greater than 100 micrograms/ml), but contained no detectable plasmid DNA. The Smr and Emr determinants were cloned from cellular DNA on the self-replicating 5-kilobase-pair (kbp) EcoRI fragment of pAM beta 1 and the 4.2-kbp cryptic plasmid pVA380-1, respectively, by transformation of Streptococcus sanguis Challis. Helper plasmid cloning, with a Challis host containing pVA380-1, was required to clone the Tcr determinant of strain DL5 on this vector. A single-colony isolate of the original Tcr clone contained a hybrid plasmid, pDL421, composed of 2.6 kbp of vector DNA and 11.4 kbp of S. mutans DNA. Plasmid pDL421 did not hybridize to plasmids containing the streptococcal Tcr determinants tetL, tetM, and tetN. A shortened derivative of this hybrid plasmid, pDL422, missing a 4.9-kbp HincII fragment from the S. mutans DNA but still encoding Tcr, was obtained by subcloning in S. sanguis Challis. The Tcr gene was located in a 1,917-base-pair open reading frame (ORF) corresponding to a 72-kilodalton protein. The ORF exhibited 99.4% sequence identity with the 1,917-base-pair tetO gene from a strain of Campylobacter coli (W. Sougakoff, B. Papadopoulou, P. Nordmann, and P. Courvalin, FEMS Microbiol. Lett. 44:153-160, 1987). A 1.67-kbp NdeI fragment, internal to the ORF from strain DL5, as well as pDL421 hybridized under stringent conditions to DNA from 10 of 10 Tcr strains of C. coli and Campylobacter jejuni from human and animal sources, but not to DNA from Tcs isolates of these two species.

Amino Acid Sequence↗
PubMed1988

Lectin-stimulated lymphocyte responses: use and misuse in clinical research.

The use of mitogen stimulation assays in clinical research has increased rapidly during the last 25 years. However, little is understood about the mechanisms of action of mitogens. This, coupled with the complexity of the mitogen stimulation assay, has led to much misuse and abuse of this assay system. Data are presented that demonstrate the inherent variability of the assay system due to diurnal variation in the response of human lymphocytes to mitogen stimulation and to the variation in response between patients. It is argued that the use of the assay should be confined to rigorously controlled studies using either animal lymphocytes or paired samples from humans, one of which should be treated in vitro with test material.

Adult↗
PubMed1987

Guanine nucleotide modulation of muscarinic cholinergic receptor binding in postmortem human brain--a preliminary study in Alzheimer's disease.

The coupling of cortical muscarinic receptors to guanosine triphosphate (GTP) binding proteins, as defined by changes in agonist affinity states of the receptor in the presence of magnesium ions (Mg2+) and a GTP analogue has been investigated using carbachol in competition experiments with either N-methylscopolamine (NMS) or pirenzepine (PZ). The stability of the system with regard to autopsy delay and freezing was first established in membrane preparations from mouse brain. Applying the same methods to human autopsy tissue from the parietal cortex of Alzheimer's diseased cases and controls, matched for age and postmortem delay, there was no significant difference in the detectable coupling of the total (NMS-labelled) muscarinic receptor population. However, coupling of the 'M1' muscarinic receptor subtype, selectively labelled by PZ, appeared to be more labile than that of the receptor population as a whole and the modulation of this subtype by the GTP analogue was significantly attenuated in Alzheimer's disease.

Aged↗
PubMed1987

Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity.

Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.

Alcohol Oxidoreductases↗
PubMed1987

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.

3',5'-Cyclic-AMP Phosphodiesterases↗