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Biomedical subjects

J Ingram

Publications and source records attributed to J Ingram.

At least 37 records · Page 2Linked to original sources
PubMed1993

Rat endopeptidase-24.18 alpha subunit is secreted into the culture medium as a zymogen when expressed by COS-1 cells.

Endopeptidase-24.18 (EC 3.4.24.18, E-24.18) is an oligomeric Zn-ectoenzyme. The alpha and beta subunits have been cloned from both rat and mouse kidneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of the astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C-terminal hydrophobic domain. In order to verify the mode of anchoring of the rat E-24.18 alpha subunit and to test the functionality of the astacin-like domain in the alpha subunit when expressed alone, COS-1 cells were transfected with a cloned cDNA for rat alpha subunit. Despite the presence of its putative transmembrane domain, the alpha subunit was not anchored in the plasma membrane but rather secreted as a dimer into the culture medium. When the enzymatic activity of the secreted recombinant protein was tested in the azocasein degradation assay, the alpha subunit was found to be inactive. Activity could, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu-157 in the active site by Val. Taken together our results suggest that the alpha subunit of Endopeptidase-24.18 contains a latent astacin-like Zn metallopeptidase activity which could be secreted as a soluble enzyme by kidney and intestine.

Animals↗
PubMed1993

Hydrolysis of human and pig brain natriuretic peptides, urodilatin, C-type natriuretic peptide and some C-receptor ligands by endopeptidase-24.11.

Endopeptidase-24.11 (E-24.11, EC 3.4.24.11) is widely believed to play a physiological role in metabolizing atrial natriuretic peptide (ANP). Since the discovery of ANP, new natriuretic peptides have been isolated and other peptides synthesized as receptor ligands. The hydrolysis in vitro of six related peptides by the endopeptidase has been studied, mainly by h.p.l.c. The initial attack on the 32-residue form of pig brain natriuretic peptide (pBNP-32) was shown to be at the Ser20-Leu21 bond, as had been previously shown for the 26-residue form. In contrast, human brain natriuretic peptide-32 (hBNP-32), which differs in ten residues from pBNP-32, was attacked first at the Met4-Val5 bond, releasing the N-terminal tetrapeptide, and only later at bonds within the ring: at Arg17-Ile18 and subsequently at four other sites. Urodilatin, which has a four-residue extension at the N-terminus compared with alpha-human atrial natriuretic peptide-28 (alpha-hANP), was degraded at about half the rate of the latter, though the C-terminal Phe-Arg-Tyr was released at the same rate. The 22-residue C-type natriuretic peptide was hydrolysed more rapidly than alpha-hANP, as were two C-receptor ligands (peptides with deletions within the ring): C-ANP4-23 (rANP4-23 des-Gln18,Ser19,Gly20,Leu21,Gly22) and SC 46542 (hANP5-28 des-Phe8,Gly9,Ala17,Gln18). Angiotensin-converting enzyme failed to hydrolyse pBNP-32, hBNP-32 or 125I-rat (r) ANP, even after prolonged incubation. Km and kcat values were determined for the hydrolysis of alpha-hANP, porcine BNP-26, porcine BNP-32 and 125I-rANP by E-24.11. Ki values were determined for six peptides, alpha-hANP, urodilatin, hBNP-32, C-type natriuretic peptide (CNP), SC 46542 and C-type natriuretic peptide (C-ANP4-23), in radiometric assays of E-24.11 with either [125I] insulin B chain or [125I] rANP as substrate. The Ki values (2.5-13 microM) for CNP were the lowest of any of the group, whereas those for hBNP-32 (151-172 microM) were the highest. The physiological significance of these results is discussed, especially in regard to the relative resistance of hBNP-32 to attack and the ability of the C-receptor ligands to compete with natriuretic peptides for hydrolysis by E-24.11.

Amino Acid Sequence↗
PubMed1993

The pharmacokinetics, tolerability and pharmacodynamics of tucaresol (589C80; 4[2-formyl-3-hydroxyphenoxymethyl] benzoic acid), a potential anti-sickling agent, following oral administration to healthy subjects.

1. Tucaresol (589C80; 4[2-formyl-3-hydroxyphenoxymethyl] benzoic acid) interacts stoichiometrically with haemoglobin to increase oxygen affinity. By decreasing the proportion of insoluble deoxy sickle haemoglobin at capillary oxygen concentrations, tucaresol may be of therapeutic benefit in sickle cell anaemia. 2. In this study, which involved the first administration to man, the pharmacokinetics and pharmacodynamics of tucaresol were studied in healthy male volunteers following oral doses of 200-3600 mg. 3. Peak drug concentrations in plasma and erythrocytes were linearly related to dose; mean (s.d.) values were 95.8 (26.1) and 1035 (67) micrograms ml-1, respectively, at the highest dose. Median tmax in plasma was 6.5 h and in erythrocytes 24.5 h, when approximately 60% of the administered dose was in the target tissue. Plasma drug concentrations fell biexponentially with commencement of the apparent terminal elimination phase at approximately 24 h. The terminal elimination half-life from plasma increased with dose (r = 0.77; P < 0.0001) from 133-190 h at 400 mg to a mean (s.d.) of 289 (30) h at 3600 mg. Erythrocyte drug concentrations declined mono-exponentially with a half-life that was always shorter than the apparent terminal half-life in plasma: overall mean (95% CI) of t1/2 erythrocyte/t1/2 plasma ratio was 0.57 (0.53, 0.61). The erythrocyte AUC/plasma AUC ratio increased with dose (r = 0.67; P < 0.001). 4. The proportion of haemoglobin modified to a form with high oxygen affinity (%MOD) increased in a dose-related manner above doses of 800 mg reaching 19-26% after the 3600 mg dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1993

Using incentives to motivate women to seek prenatal care: an effective outreach strategy.

Expanded Medicaid eligibility and case-managed care have contributed to improved birth outcomes and reduced Medicaid expenditures in Alabama. In 1990, 26.5 percent of all women delayed entry to care until the fourth month of pregnancy or later. Additionally, more than 1,000 women in the state received no care at all. In many of these cases, women perceived prenatal care as unimportant or unnecessary until later in pregnancy. As a result, the Alabama Medicaid Agency developed Healthy Beginnings, an incentive and awareness program which utilizes coupons to motivate women to seek prenatal care. Pregnant women can receive the coupon book (worth about $300) simply by dialing a toll-free number. To take advantage of the free gifts and discounts, expectant mothers must visit a private physician or health clinic and have their coupons validated monthly. The project was initiated in August, 1990, and already there is clear evidence that the use of incentives represents a viable outreach strategy to motivate poor and uninsured women to seek care. In the program's first year, more than 20,000 women received the coupons, of which 78 percent were Medicaid recipients, uninsured or in a presumptive (Medicaid) eligibility period. Focus group studies, field visits and a survey were used to evaluate the effectiveness of the new program. Preliminary data analysis has revealed that the incentive program may be a significant factor in motivating women to seek early and continuous prenatal care.

Alabama↗
PubMed1992

Molecular cloning of the alpha-subunit of rat endopeptidase-24.18 (endopeptidase-2) and co-localization with endopeptidase-24.11 in rat kidney by in situ hybridization.

Endopeptidase-24.18 (endopeptidase-2, EC 3.4.24.18, E-24.18) is a Zn-ectoenzyme of rat renal and intestinal microvillar membranes exhibiting an oligomeric structure, alpha 2-beta 2. The primary structure of the alpha-subunit of E-24.18 has been defined by molecular cloning and its expression mapped in rat kidney by in situ hybridization. A 2.9-kb cDNA coding for the alpha-subunit was isolated and sequenced. It had an open reading frame of 2,244 base pairs coding for a type I membrane protein of 748 amino acids. The deduced amino acid sequence showed 87% identity with that of meprin A, a mouse metallo-endopeptidase, sharing common properties with the rat enzyme, and 85% identity with the human intestinal enzyme, 'PABA-peptide hydrolase'. Northern blot analysis revealed the alpha-subunit to be encoded by a single mRNA species of 3.2-kb. In situ hybridization performed on rat kidney showed a co-localization of E-24.18 with endopeptidase-24.11 in proximal tubules of juxtamedullary nephrons, suggesting that the two enzymes have similar or complementary physiological functions in kidney.

Amino Acid Sequence↗
PubMed1992

Children's self-allocation and use of classroom curricular time.

A class of 9-10 year-olds (N = 12) in a British primary school were observed as it moved over a one-year period through three types of classroom environment, traditional directive, transitional negotiative and established negotiative. Each environment offered the children a differing relationship with curricular time, its control and allocation, moving from teacher-allocated time to child allocation. Pupil self-report and classroom observation indicated differences in the balance of curricular spread and allocated time on curricular subject in relation to the type of classroom organisation and who controlled classroom time. These differences were at both class and individual child level. The established negotiative environment recorded the most equitable curricular balance, traditional directive the least. While individual children responded differently within and across the three classroom environments, the established negotiative where time was under child control recorded preference for longer activity periods compared to where the teacher controlled time allocations.

Achievement↗
PubMed1991

Complications of indwelling venous access lines in the pediatric hematology patient: a prospective comparison of external venous catheters and subcutaneous ports.

Since 1984, 316 subcutaneous ports (SP) and 339 external venous catheters (EC) [Roko Catheter, The Hospital for Sick Children (HSC)] have been inserted in hematology/oncology patients at HSC. During a 22-month period (July 1987 to April 1989), a committed central line nurse (J.I.) prospectively collected clinical and microbiologic data on 144 consecutive SPs and 130 consecutive ECs. Children with the SP had 0.6 infected lines and 0.7 infectious episodes per 1,000 patient days compared to 2.9 infected lines and 4.3 infectious episodes per 1,000 patient days with the EC (p less than 0.001). This lower infectious complication rate with SP was demonstrated in the entire group of unselected patients and in a cohort of children with acute lymphoblastic leukemia (ALL) receiving intensive chemotherapy, and it was evident in all age groups. In view of the other advantages of SP--normal activity, absence of the need for home maintenance, improved body image, less expense--these data suggest that SPs are the preferred device in pediatric patients and provide effective venous access with acceptable complication rates.

Adolescent↗
PubMed1990

Varieties of curricular experience: backmarkers and frontrunners in the primary classroom.

In the first study a class of 10-year-olds was observed for the first and last five weeks of the autumn term. On both a class and individual child basis there was considerable difference between teacher-believed and child-actual timetables, and this increased over the term. In the second study, 53 children were classified into "frontrunners" or "backmarkers", depending on success in pacing the taught curriculum. For backmarkers, strategies for maintaining contact were mainly child-determined, though for frontrunners mainly teacher-determined. Although the teachers, who proved to be traditionally directive, believed their paper timetable closely represented children's true classroom experience, a picture emerged of most children correctly perceiving themselves as backmarkers, and as having responsibility for instigating their own, largely passive, coping strategies with subsequent negative feelings about themselves and their situation.

Achievement↗
PubMed1989

Proteins of the kidney microvillar membrane. Structural and immunochemical properties of rat endopeptidase-2 and its immunohistochemical localization in tissues of rat and mouse.

The phosphoramidon-insensitive endopeptidase-2 in rat renal brush borders was investigated by immunochemical approaches with a rabbit polyclonal antibody raised to the purified enzyme released from the membrane by papain. An immunoaffinity column successfully purified the detergent-solubilized form of endopeptidase-2. This preparation had an apparent subunit Mr of 80,000, and did not show the two subunits, of Mr 80,000 and 74,000, consistently found in the papain-solubilized forms, indicating that the latter resulted from proteolysis by papain. SDS/polyacrylamide-gel electrophoresis of non-reduced samples of the enzyme revealed a band of Mr 220,000, confirming the presence of disulphide-bridged subunits. Treatment with endoglycosidases H and F generated smaller molecular forms, indicating that endopeptidase-2 contained about 30% asparagine-linked carbohydrate and that a few of these oligosaccharide chains were of the high-mannose type. Treatment with phosphatidylinositol-specific phospholipase indicated that the enzyme did not possess a glycolipid membrane anchor. A survey of rat tissues examined immunohistochemically and by immunoblotting revealed that only the kidney and intestinal tract expressed the antigen in significant amounts. Although some weak staining was seen in salivary glands and thyroid, other organs and tissues including brain and spinal cord were negative by both immunochemical techniques. In the kidney the antigen was confined to the lumen of the proximal tubule and was seen mainly in the population of juxtamedullary nephrons. In the gut, luminal staining was observed throughout its whole length, from duodenum to rectum. Excellent cross-reactivity of the antibody with Balb/c mouse tissues was observed. Immunohistochemistry of mouse kidney and gut revealed a distribution identical with that observed in the rat. Immunopurification of the detergent-solubilized mouse kidney antigen showed it to be a protein containing disulphide-linked subunits of Mr 90,000. It possessed endopeptidase-2-like activity, but was more efficient in hydrolysing azo-casein and less efficient in hydrolysing a model substrate than the rat enzyme. The close similarity between rat endopeptidase-2 and mouse meprin is further supported by these results.

Animals↗
PubMed1989

Possible foetotoxic effects of mercury vapour: a case report.

A thirty-year-old dental surgeon who worked until the 35th week of pregnancy in a surgery in which mercury vapour concentrations in excess of the threshold limit value (TLV) had been detected, gave birth at 42 weeks to a small-for-dates baby with severe brain damage. The possibility that this baby's development might have been harmed by mercury is discussed and the literature relevant to teratogenicity of mercury reviewed. Attention is drawn to the need for further research into the effects on health and pregnancy outcome of mercury vapour in dental surgeries.

Adult↗
PubMed1989

A case report of central nervous system lymphoma following immunosuppression.

Lymphoma is a well recognised complication of immunosuppression. A case is presented of a patient who developed CNS lymphoma thirty-three months after renal transplantation. The incidence and characteristics of lymphoma developing post-transplantation and the treatment of central nervous system (CNS) lymphomas are discussed.

Adult↗
PubMed1988

Is there a tripeptidyl peptidase in the renal brush-border membrane?

A recent claim that the renal brush border contains a tripeptidyl peptidase [Andersen & McDonald (1987) Am. J. Physiol. 253, F649-F655] was examined. In a fluorescent assay, the hydrolysis of Gly-Pro-Met-2-naphthylamide (-NH-Nap) and Gly-Pro-Leu-NH-Nap by pig kidney microvilli was strongly inhibited by amastatin or di-isopropyl phosphorofluoridate (inhibitors of aminopeptidases and dipeptidyl peptidase IV). The products formed were shown to be Gly-Pro and Met-NH-Nap (or Leu-NH-Nap) and free 2-naphthylamine. Specific antibodies to pig and rat aminopeptidase N abolished the apparent tripeptidyl peptidase activity. We conclude that these substrates are hydrolysed by the sequential attack of dipeptidyl peptidase IV and aminopeptidase N and that pig and rat brush borders lack a detectable tripeptidyl peptidase.

Aminopeptidases↗
PubMed1987

Proteins of the kidney microvillar membrane. Purification and properties of the phosphoramidon-insensitive endopeptidase ('endopeptidase-2') from rat kidney.

A second endopeptidase is present in the renal microvillar membrane of rats that can be distinguished from endopeptidase-24.11 by its insensitivity to inhibition by phosphoramidon. The purification of this enzyme, referred to as endopeptidase-2, is described. The enzyme was efficiently released from the membrane by treatment with papain. The subsequent four steps depended on ion-exchange and gel-filtration chromatography. These steps were monitored by the hydrolysis of various substrates: 125I-insulin B chain (the normal assay substrate), benzoyl-L-tyrosyl-p-aminobenzoate (Bz-Tyr-pAB), azocasein and benzyloxycarbonyl-L-phenylalanyl-L-arginine 7-amino-4-methylcoumarylamide (Z-Phe-Arg-NMec). All four assays revealed comparable stepwise increases in activity in the main stages of the purification, although it was apparent that the last-named fluorogenic assay depended on traces of aminopeptidase activity present in the preparation. The Km for 125I-insulin B chain was 16 microM and that for Bz-Tyr-pAB was 4.7 mM. Several experimental approaches confirmed that both peptides were hydrolysed by the same enzyme. The pH optimum was 7.3. Phosphate buffers were inhibitory and shifted the optimum to above pH 9. Zinc was detected in the purified enzyme; EDTA and 1,10-phenanthroline were strongly inhibitory. SDS/polyacrylamide-gel electrophoresis revealed polypeptides of equal staining intensity of Mr 80,000 and 74,000 in reducing conditions. In non-reducing conditions a single band of apparent Mr 220,000 was seen. Gel filtration yielded an Mr of 436,000. These results are consistent with an oligomeric structure in which the alpha and beta chains are linked by disulphide bridges. Endopeptidase-2 hydrolysed a number of neuropeptides. Enkephalins resisted attack, only the heptapeptide [Met]enkephalin-Arg6-Phe7 being susceptible to slow hydrolysis. Luliberin (luteinizing-hormone-releasing hormone) and bradykinin were rapidly hydrolysed. Neurotensin was shown to be slowly attacked at the Tyr3-Glu4 bond. Thus the specificity appears to be limited to the hydrolysis of bonds involving the carboxy group of aromatic residues, provided that this P1 residue is extended by additional residues, at least to the P3' position. The relationship of this membrane metalloendopeptidase to mouse meprin and human 'PABA peptidase' is discussed.

Animals↗
PubMed1986

Radiation inactivation analysis of kidney microvillar peptidases.

Five membrane peptidases were studied by radiation inactivation analysis of pig kidney microvillar membranes. One heterodimeric enzyme, gamma-glutamyl transferase, presented a target size corresponding to the dimeric Mr. The other enzymes are known to be homodimers. Three of these, aminopeptidase A. aminopeptidase N and dipeptidyl peptidase IV, gave results clearly indicating the monomer to be the target and, hence, in this group the association of the subunits was not essential for activity. The target size for endopeptidase-24.11 was intermediate between those for monomer and dimer and its functional state was not resolved by the experiments.

Animals↗
PubMed1983

Hyperthermia with implanted electrodes: in vitro and in vivo correlations.

Hyperthermia as a treatment for cancer has elicited much recent interest. However, major difficulties persist both in the technology for heating deep-seated tumors, and in thermal dosimetry. We have investigated a heating technique for deep-seated neoplasms that employs an internal implanted electrode and an external electrode to apply radiofrequency current to a tumor mass. The internal electrode consists of an array of stainless steel needles or wires which define a Faraday cage within the tumor, while the external electrode consists of a variety of electrical conductors at the skin surface. Phantom measurements have closely reproduced calculated temperature distributions. The temperature profiles within the volume enclosed by the internal electrode show relatively homogenous heating. Temperature measurements in a rat tumor model have demonstrated that significant heating within such an internal electrode array is easily obtained. The heating may extend some centimeters outside the electrode. Using a dog model we have shown that with such a treatment technique the temperature profiles obtained are reproducible both spatially and temporally. A case report of a clinical application is presented. A 5 cm bronchogenic carcinoma was easily heated without significant heating of the surrounding normal lung, and without apparent toxicity. Such a technique may be applicable to a variety of operable but unresectable neoplasms. The reproducibility and relative homogeneity of heating suggest possible usefulness in combined modality trials.

Adenocarcinoma↗
PubMed1982

Bronchial provocation tests in atopic patients with allergen-specific IgG4 antibodies.

Atopic asthmatics who produce allergen-specific IgE antibodies develop an immediate reaction to bronchial provocation; those who produce allergen-specific IgG4 antibodies develop a delayed response; and those who produce both classes of antibody against the same allergen exhibit a dual response. Individual patients showed a variable immune response to different allergens, producing bot classes of antibody against one, but only IgE or IgG4 against others. These findings suggest that allergen-specific IgG4 antibodies are important in the production of severe asthma and probably atopic eczema as well.

Adolescent↗