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K Pritchard

Publications and source records attributed to K Pritchard.

At least 37 records · Page 2Linked to original sources
PubMed1996

Changes in nuclear morphology in the human endometrial glandular epithelium in women with unexplained infertility.

In the light and electron microscopical study reported here, we documented the structure of the nucleus, nucleolus and nuclear channel system (NCS) in the uterine glandular epithelium in both fertile and infertile women during the early luteal phase. Nuclear volume was found to be larger in the infertile group at day 5 after the luteinizing hormone surge (LH+5) compared to day-matched fertile subjects. A two-way analysis of variance performed on nucleolar volume data from fertile and infertile women biopsied on days LH+5, +5, and +6 revealed a significant effect of condition but no effect of day or interaction. Nucleolar volume decreased from day LH+2 to day LH+6 in the fertile group, the sharpest decrease occurring between day LH+3 and day LH+4. The largest mean volume of the NCS was found at day LH+5 in the fertile group and day LH+6 in the infertile group. The results suggest a delay in the development of this organelle in the infertile women. The present study has documented alterations in the nuclei of uterine glandular cells from infertile patients. In these infertile women, there is also a delayed elaboration of the secretory apparatus and this delay correlates well with the delayed/reduced expression of a luteal-specific glyco-protein.

Analysis of Variance↗
PubMed1996

Metabolic rate of sleeping infants.

AIM: To measure the sleeping metabolic rate (SMR) of healthy infants in the first year of life. METHODS: The SMR was measured on 73 infants aged 1 to 12 months in a special nursery using indirect calorimetry. One hundred satisfactory observations were made. The room air and radiative temperatures, humidity, and amount of insulation were measured. Parents chose the clothing and bedding that they judged their infant needed. RESULTS: The mean (SD) SMR was 2.4 (0.4) watts (W)/kg or 45 (10) W/m2. The mean SMR of infants aged 1-2 months was 38 compared with 44 W/m2 in infants of 8-12 months; the difference was not significant. There were no obvious differences in SMR between boys and girls. But there were wide differences in SMR between apparently similar infants, range 1.4 to 3.5 W/kg. Most parents selected insulation between 1 and 3 togs, and this was weakly negatively correlated with air temperature. CONCLUSION: These wide variations in SMR mean that it is impossible to give specific guidelines on the amount of clothing and bedding a particular infant will need for thermal comfort in a given room temperature.

Bedding and Linens↗
PubMed1996

Individual variation in sleeping metabolic rates in infants.

AIM: To measure the variation in sleeping metabolic rate (SMR) of healthy infants in the first year of life. METHODS: Attempts were made to measure SMR in the home of one group of infants at monthly intervals over the first year of life and of another group in the 1 to 12 age range, over three consecutive days. Sixty one infants were recruited and 181 measurements made. In 11 infants six or more satisfactory measurements were made a monthly intervals and in another 11 infants, three consecutive daily measurements were obtained. Parents chose the clothing and bedding that they judged their infant needed to fall asleep. The room air and radiative temperatures, humidity, and amount of insulation were recorded. RESULTS: The mean (SD) SMR was 2.4 (0.4) watts (W)/kg or 45 (10) W/m2. The rate was the same for infants in the age groups 0.25, 0.5, 0.75, and 1 years; individual infants did not show a systematic change with age. There was wide variation. Ninety eight per cent of infants had a SMR between 1.8 and 3.1 W/kg. The mean SMR for the infants measured on three consecutive days varied from 1.7 to 3.1 W/kg, and SMR varied within infants, from 0.06 to 0.86 W/kg. The variation within six to 11 measurements on infants over the first year of life ranged from 0.38 to 1.05 W/kg. CONCLUSION: Infants in the first year of life exhibit wide variations in SMR from day to day and from month to month. As the range is wide, guidance on clothing and bedding for thermal comfort can be given only in broad terms.

Bedding and Linens↗
PubMed1996

Reduced gene expression of vascular endothelial NO synthase and cyclooxygenase-1 in heart failure.

Endothelium-dependent responses are depressed in coronary and peripheral blood vessels after the onset of pacing-induced heart failure in dogs and heart failure of various etiologies in humans. The present study was designed to examine whether these responses were due to decreases in the expression of endothelial cell NO synthase (ecNOS) and cyclooxygenase-1 (COX-1). After 1 month of left ventricular pacing, 8 mongrel dogs were monitored for heart failure as defined by clinical signs and left ventricular end diastolic pressures > 25 mm Hg. Total RNA and protein were isolated from endothelial cells scraped from the thoracic aorta and analyzed by Northern and Western blotting, respectively. Blots probed with 32P-labeled cDNAs for ecNOS and COX-1 were quantified densitometrically, and results were normalized against GAPDH or von Willebrand factor (vWF). In arbitrary units, the ratios of ecNOS to GAPDH were 2.66 +/- 0.77 (mean +/- SEM, n = 17) and 1.12 +/- 0.37 (n = 6 and the ratios of COX-1 to GAPDH were 1.52 +/- 0.52 and 0.56 +/- 0.15 before and after heart failure, respectively. These represent 56% to 64% (P < .05) reductions in ecNOS and COX-1 gene expression. There was no change in the ratios of either COX-1 or ecNOS to vWF. There was also a marked reduction in ecNOS protein after heart failure, estimated at 70%. A marked reduction in nitrite production, a measure of enzyme activity, from thoracic aortas in response to stimulation by either acetylcholine or bradykinin also occurred. To determine whether ecNOS and COX-1 could be independently regulated, an orally active NO-releasing agent, CAS 936, was given to 7 normal dogs for 7 days, and aortic ecNOS and COX-1 mRNAs were analyzed. The ratio of ecNOS to GAPDH was depressed by 52% (P < .05) in aortas from these dogs, whereas the ratio of COX-1 to GAPDH was unchanged. Similar results were found when data were normalized to vWF. These results suggest that at least two endothelial vasodilator gene products are reduced in heart failure, as opposed to a selective defect in NO synthase gene expression.

Animals↗
PubMed1995

Caldesmon mRNA splicing and isoform expression in mammalian smooth-muscle and non-muscle tissues.

The recent determination of the genomic sequence of human caldesmon indicates that eight caldesmon mRNA species could be generated by selection of exon 1 or 1', exon 3a or 3ab and/or exon 4. We used reverse transcriptase PCR to determine which transcripts were produced in human, rabbit and sheep artery, vein, lung, intestine, kidney and liver. In all tissues the same three transcripts were present: exons 1'-2-3a-5-6...13, exons 1'-2-3a3b-5-6-...13 and exons 1'-2-3a3b-4-5-6...13. Exon 1 was not present and exon 4 was only present when exon 3b was also present. Three protein isoforms of caldesmon can be distinguished by electrophoresis on high-porosity 6% polyacrylamide gel: 130 kDa, 120 kDa and 70 kDa. The 70 kDa isoform lacks the sequence encoded by exon 3b. We investigated whether the two high-molecular-mass isoforms correspond to the presence and absence of exon 4 using an antiserum specific to the sequence encoded by exon 4. Western-blotting and immunoprecipitation experiments showed that both the 130 kDa and the 120 kDa isoforms were expressed with and without the exon 4 sequence. We therefore propose that the molecular-mass heterogeneity arises from additional first exons, possibly with separate promoter regions, which have not yet been characterized in the genomic sequence.

Animals↗
PubMed1994

Location of two contact sites between human smooth muscle caldesmon and Ca(2+)-calmodulin.

We measured Ca(2+)-calmodulin binding to expressed human caldesmon fragments by three techniques: tryptophan fluorescence enhancement, change in fluorescence of TA-calmodulin, and cosedimentation with calmodulin-Sepharose. Ca(2+)-calmodulin bound with similar affinity to peptide M73 (C714SMWEKGNVFSSPGF727, N terminus of domain 4b), to all the fragments of caldesmon containing this peptide, and also to H9 (Thr726-Val793), which did not contain this peptide (Kd = 0.2-0.8 microM). We conclude that Ca(2+)-calmodulin binds at two sites on caldesmon; site A is the sequence 715MWEKGNVFS723 previously identified by Zhan et al. (Zhan, Q., Wong, S. S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814), and site B is located nearer the C terminus of caldesmon. Ca(2+)-calmodulin binding at site B is coupled to reversal of caldesmon inhibition of actin-tropomyosin activated myosin MgATPase, while calmodulin binding at site A has no detectable function. H9 did not displace M73 from Ca(2+)-calmodulin, while the other fragments did. High concentrations of M73 (> 1000 x Kd) could not displace H9 bound to Ca(2+)-calmodulin-Sepharose. Thus sites A and B in calmodulin are functionally separate. Analysis of overlapping expressed fragments indicates that site B is located in the sequence Thr726-Leu767, which includes Trp749. The minimal Ca(2+)-calmodulin binding sequence could be 744SRINEWLTK752.

Actins↗
PubMed1994

Filamin and gelsolin influence Ca(2+)-sensitivity of smooth muscle thin filaments.

Sheep aorta thin filaments were prepared by ultracentrifugation of an ATP-containing extract in the presence of different concentrations of ethanediol. Thin filaments prepared without ethanediol contained small quantities of tropomyosin (0.027 Tm:actin) and caldesmon (0.017 CD:actin) and activated the MgATPase of skeletal myosin independently of Ca2+. Ultracentrifugation in the presence of 10-20% ethanediol resulted in preparation of thin filaments with increased content of tropomyosin (0.17 Tm:actin) and caldesmon (0.04 CD:actin). These thin filaments possessed high Ca(2+)-sensitivity in activation of skeletal muscle myosin ATPase. Besides actin, tropomyosin and caldesmon, thin filaments contained gelsolin and filamin. Gelsolin content (0.007 gelsolin:actin) was independent of the presence of ethanediol. The filamin content decreased from 0.015 to 0.007 mol:mol actin when the ethanediol concentration was increased from 0 to 20%, and was negatively correlated with the Ca2+ sensitivity of thin filaments. In a reconstituted system, pure filamin or gelsolin affected caldesmon regulation of actomyosin ATPase. Gelsolin (0.01:actin) reduced the inhibition of actomyosin ATPase caused by caldesmon and increased the potency of Ca(2+)-calmodulin in reversing this inhibition. Filamin (0.007:actin) also decreased the inhibitory action of caldesmon on actin-activated myosin ATPase and also potentiated the reversal of this inhibition by calmodulin. We conclude that minor components of smooth muscle thin filaments (gelsolin and filamin) significantly modify caldesmon mediated regulation of actomyosin ATPase. We suggest a tropomyosin-mediated mechanism by which filamin or gelsolin could exert similar effects.

Actin Cytoskeleton↗
PubMed1994

Changes in the caldesmon isoform content and intimal thickening in the rabbit carotid artery induced by a silicone elastomer collar.

The presence of a silicone elastomer collar around one carotid artery of a rabbit induces thickening of the tunica intima. We used immunoblotting to study quantitatively changes in the isoforms of caldesmon, a protein implicated in the regulation of contractility in smooth muscle, while also monitoring the histological changes during 28 days after collaring. Control rabbit carotid arteries (n = 28) contained 245 +/- 6.4 nmol/g protein of the larger isoform of caldesmon (CDh) and 68.3 +/- 3.6 nmol/g protein of the smaller isoform (CD1). Four days after collaring, intimal thickening was slight, but 44% of arterial CDh had been lost; this loss of CDh was therefore from the tunica media. At 10 days, CDh fell to 37% of the control level. Immunofluorescence using CDh-specific antibodies showed that the CDh level was diminished but remained uniform across the wall of collared arteries. At 14 days, when intimal thickening was maximal, there was 30% more CD1 than in controls. At 28 days, the neointima had thinned, and CD1 had fallen to below control levels. Thus, CD1 levels reflected the development and regression of neointima. Changes in caldesmon isoforms showed that smooth muscle cell phenotypic changes occurred throughout the arterial wall.

Animals↗
PubMed1994

Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression.

Recently, we have shown that chronic exercise increases endothelium-derived relaxing factor (EDRF)/nitric oxide (NO)-mediated epicardial coronary artery dilation in response to brief occlusion and acetylcholine. This finding suggests that exercise can provide a stimulus for the enhanced production of EDRF/NO, thus possibly contributing to the beneficial effects of exercise on the cardiovascular system. Therefore, the purpose of the present study was to examine whether chronic exercise could influence the production of NO (measured as the stable degradation product, nitrite) and endothelial cell NO synthase (ECNOS) gene expression in vessels from dogs after chronic exercise. To this end, dogs were exercised by running on a treadmill (9.5 km/h for 1 hour, twice daily) for 10 days, and nitrite production in large coronary vessels and microvessels and ECNOS gene expression in aortic endothelial extracts were assessed. Acetylcholine (10(-7) to 10(-5) mol/L) dose-dependently increased the release of nitrite (inhibited by nitro-L-arginine) from coronary arteries and microvessels in control and exercised dogs. Moreover, acetylcholine-stimulated nitrite production was markedly enhanced in large coronary arteries and microvessels prepared from hearts of dogs after chronic exercise compared with hearts from control dogs. One potential mechanism that may contribute to the enhanced production of nitrite in vessels from exercised dogs may be the induction of the calcium-dependent ECNOS gene. Steady-state mRNA levels for ECNOS were significantly higher than mRNA levels for von Willebrand's factor (vWF, a specific endothelial cell marker) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a constitutively expressed gene) in exercised dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1993

The Ca(2+)-sensitizing component of smooth muscle thin filaments: properties of regulatory factors that interact with caldesmon.

We have investigated the nature of the native Ca(2+)-sensitizing factor of smooth muscle native thin filaments. Two different types of preparation yielded protein mixtures which consistently conferred Ca(2+)-sensitivity on thin filaments reconstituted from aorta actin, tropomyosin and caldesmon. These new preparations produced a good model of native thin filaments, unlike previous systems reconstituted with calmodulin or S. 100. We conclude that the Ca(2+)-sensitizing factor is a protein of molecular weight in the range 15 kDa - 40 kDa which is heat-labile and which has properties distinct from calponin, S. 100 or calmodulin.

Actomyosin↗
PubMed1993

The fine structure of the human endometrial glandular epithelium in cases of unexplained infertility: a morphometric study.

In the present study attempts have been made to express in quantitative terms the fine structure of the endometrial glandular epithelium in well-timed biopsies from fertile women during the early luteal phase. Biopsies from women with unexplained infertility have also been examined. Certain abnormalities of glandular maturation were found in these women. The nuclear channel system was not present at day luteinizing hormone (LH) + 4 in the infertile group while at LH + 6 there were more cells containing this feature than seen in the fertile group. There also seem to be abnormalities associated with intracellular deposits of glycogen-rich material and mitochondrial elaboration in the infertile women. Changes in the secretory apparatus and rough endoplasmic reticulum were observed. The evidence from the present study suggests that the endometria in the examined group of infertile women is responsive to steroidal stimulation. This response however, may be delayed or inadequate.

Cell Nucleus↗
PubMed1993

The Canadian experience with intensive fluorouracil, epirubicin and cyclophosphamide in patients with early stage breast cancer.

A multicentre dose-finding pilot study was conducted to determine an intensive regimen of fluorouracil (F), epirubicin (E) plus cyclophosphamide (C) [FEC] that was tolerable and acceptable to patients with node-positive operable (n = 266) or locally advanced (n = 22) breast cancer. Consecutive patients were treated with fluorouracil and epirubicin administered intravenously on days 1 and 8, in addition to cyclophosphamide orally for 14 days. Chemotherapy cycles were repeated at monthly intervals for 6 months, and dosages were increased according to a predetermined protocol. End-points were hospital admissions due to febrile neutropenia and changes in cardiac function as assessed by radionuclide angiography. The first 46 patients were treated with doses of F = 375 mg/m2, E = 50 mg/m2 and C = 75 mg/m2 (level 1), then 42 patients received F = 500 mg/m2, E = 50 mg/m2 and C = 75 mg/m2 (level 2), 69 patients received F = 500 mg/m2, E = 60 mg/m2 and C = 75 mg/m2 (level 3), and 42 patients received F = 500 mg/m2, E = 70 mg/m2 and C = 75 mg/m2 with concurrent antibiotics (level 4). Rates of febrile neutropenia were 8.7% (level 1), 7.1% (level 2), 18.8% (level 3), and 31% (level 4) [p = 0.002]. Accrual to level 4 was discontinued according to study protocol and a further 89 patients were recruited at level 3 dosages with antibiotic prophylaxis (level 3a), resulting in a 5.6% rate of febrile neutropenia. The difference in febrile neutropenia rates between dosage levels 3 and 3a was statistically significant (p = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1992

A pilot study of intensive cyclophosphamide, epirubicin and fluorouracil in patients with axillary node positive or locally advanced breast cancer.

A multicentre pilot study has been conducted to determine an intensive regimen of cyclophosphamide, epirubicin, and fluorouracil which was tolerable and acceptable to patients with node positive breast cancer. Consecutive patients with operable axillary node positive breast cancer (T1-3, N1-2, M0), 266 patients, or locally advanced breast cancer (T4), 22 patients, were treated with cyclophosphamide post-operatively for 14 days and epirubicin and fluorouracil, both intravenously on days 1 and 8. Each cycle was repeated monthly for 6 months. Dosages were increased according to predetermined guidelines. Outcome measures were admission to hospital for febrile neutropenia and change in cardiac function as assessed by radionuclide angiography. The first 46 patients were treated at the doses of cyclophosphamide = 75 mg/m2, epirubicin = 50 mg/m2, fluorouracil = 375 mg/m2 (level 1), then 42 patients at cyclophosphamide = 75 mg/m2, epirubicin = 50 mg/m2 and fluorouracil = 500 mg/m2 (level 2), 69 patients at cyclophosphamide = 75 mg/m2, epirubicin = 60 mg/m2, and fluorouracil = 500 mg/m2 (level 3), and 42 patients at cyclophosphamide = 75 mg/m2, epirubicin = 70 mg/m2, and fluorouracil = 500 mg/m2 with concurrent antibiotics (level 4). The rates of febrile neutropenia were 8.7% (level 1), 7.1% (level 2), 18.8% (level 3), and 31% (level 4), respectively, P = 0.002. Accrual to level 4 was discontinued according to study guidelines and a further 89 patients were recruited at level 3 dosages with antibiotic prophylaxis (level 3a), resulting in a 5.6% rate of febrile neutropenia. The difference in febrile neutropenia rates between levels 3 and 3a was statistically significant. There were no toxic deaths and 2 cases of heart failure. In conclusion, through a careful dose-finding study in patients with operable or locally advanced breast cancer, an intensive epirubicin-containing adjuvant regimen has been established which is presently being compared with standard CMF (cyclophosphamide, methotrexate, 5-fluorouracil) chemotherapy in a randomised trial. In addition, this study suggests that antibiotic prophylaxis reduces the risk of febrile neutropenia in breast cancer patients receiving intensive chemotherapy.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1991

Ca(2+)-dependent regulation of vascular smooth-muscle caldesmon by S.100 and related smooth-muscle proteins.

1. We have investigated the ability of bovine brain S.100, and of three related proteins from sheep aorta smooth muscle, to confer Ca(2+)-sensitivity on thin filaments reconstituted from smooth-muscle actin, tropomyosin and caldesmon. 2. At 37 degrees C in pH 7.0 buffer containing 120 mM-KCl, approximately stoichiometric amounts of S.100 reversed caldesmon's inhibition of the activation of myosin MgATPase by smooth-muscle actin-tropomyosin. The [S.100] which reversed by 50% the inhibition by caldesmon (the E.C.50) was 2.5 microM when [caldesmon] = 2-3 microM in the assay mixture. When [KCl] was decreased to 70 mM, E.C.50 = 11.5 microM; at 25 degrees C in 70 mM-KCl, up to 20 microM-S.100 had no effect. When skeletal-muscle actin rather than smooth-muscle actin was used to reconstitute thin filaments, 20 microM-S.100 did reverse inhibition by caldesmon, at 25 degrees C in buffer containing 70 mM-KCl. This dependence on conditions is also characteristic of the calmodulin-caldesmon interaction. 3. These results suggested that S.100 or a related protein might interact with caldesmon in smooth muscle. We therefore attempted to prepare such a protein from sheep aorta. Three proteins were purified: an Mr-17,000 protein (yield 16 mg/kg), an abundant Mr-11,000 protein (yield 48 mg/kg), and an Mr-9000 protein (yield 4 mg/kg). Neither of the last two low-Mr proteins had any effect on activation of myosin MgATPase by reconstituted thin filaments. The protein of Mr 17,000 had Ca(2+)-sensitizing activity, and behaved exactly like brain calmodulin in the assay system.

Actin Cytoskeleton↗
PubMed1989

Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments.

We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37 degrees C in buffer containing 120 mM-KCl, 4 microM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 microM-Ca2+-calmodulin was necessary at 25 degrees C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 10(6) M-1 at 25 degrees C (60 mM-KCl); K = 1.4 x 10(6) M-1 at 37 degrees C in 70 mM-KCl-containing buffer; K = 0.35 x 10(6) M-1 at 37 degrees C in 120 mM-KCl- containing buffer (pH 7.0). At 37 degrees C/120 mM-KCl, but not at 25 degrees C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 10(6) M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions (37 degrees C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin).

Actins↗
PubMed1989

Correlation of blood pressure in normotensive and hypertensive individuals with platelet but not lymphocyte intracellular free calcium concentrations.

1. Fifty-two normotensive and essential hypertensive subjects were studied. Intracellular free calcium concentration [( Ca2+]i) was measured in lymphocytes (37 subjects) and platelets (18 subjects) by means of the fluorescent indicators, quin 2 and fura-2. In 31 subjects, plasma ionized calcium concentration was also measured. 2. There was a positive correlation between platelet [Ca2+]i and systolic blood pressure (r = 0.485, P less than 0.05), diastolic blood pressure (r = 0.542, P less than 0.02) and mean blood pressure (r = 0.534, P less than 0.02). 3. No statistically significant relationship was observed between plasma ionized calcium and blood pressure. 4. No relationship was found between lymphocyte [Ca2+]i and blood pressure, or between lymphocyte [Ca2+]i and plasma ionized calcium. 5. There was no relationship between [Ca2+]i of lymphocytes and platelets measured simultaneously from the same subject. 6. These findings reconcile previous conflicting reports and show a relationship between platelet but not lymphocyte [Ca2+]i and blood pressure in man.

Adult↗