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Biomedical subjects

J Lawrence

Publications and source records attributed to J Lawrence.

At least 19 recordsLinked to original sources
PubMed1995

Proton release from HeLa cells and alkalization of cytoplasm induced by diferric transferrin or ferricyanide and its inhibition by the diarylsulfonylurea antitumor drug N-(4-methylphenylsulfonyl)-N'-(4-cholorophenyl) urea (LY181984).

Proton release from HeLa cells was stimulated by an external oxidant, potassium ferricyanide, or by the growth factor diferric transferrin. This stimulated proton release was inhibited by the antitumor sulfonylurea LY181984 [N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea] over the concentration range 10 nM to 1 microM. The antitumor-inactive sulfonylurea analog LY181985 [N-(4-methylphenylsulfonyl)-N'-(phenyl)urea] was without effect at 1 microM and required 10-100 microM concentrations to inhibit proton release. Diferric transferrin-induced alkalization of the cytoplasm estimated by BCECF [2',7'-bis(2-carboxyethyl)-5,(and 6)-carboxyfluorescein] fluorescence also was inhibited by 1 microM LY181984 but not by 1 microM LY181985. The inhibited component appeared to be amiloride resistant. The proton release induced by either ferricyanide or diferric transferrin was inhibited by about 35% at a near optimal amiloride concentration of 0.2 mM or at a dimethylamiloride concentration of 0.075 mM. However, the induced proton release was inhibited further by LY181984. Conversely, when proton release was inhibited fully by LY181984 at a near optimal concentration of 10 microM (50% inhibition), increasing concentrations of amiloride or dimethylamiloride resulted in additional inhibitions of 16 and 23%, respectively. However, the inhibitions by LY181984 and the amilorides were additive, suggesting that amiloride and the sulfonylureas may act independently. Evidence for an action of the sulfonylurea in inhibiting proton efflux differently from that of the amilorides came from measurements of sodium uptake either by fluorometry or by direct measurement with 22Na+. Sodium uptake was not inhibited by either LY181984 or LY181985 in HeLa cells at concentrations of LY181984 sufficient to inhibit proton efflux by 80% or more. The results show LY181984 to be a potent inhibitor of diferric transferrin- or ferricyanide-induced proton efflux and cytoplasmic alkalization in HeLa cells and that the inhibition may involve a component of proton transport that is resistant to amiloride.

Amiloride↗
PubMed1995

Identification of antitumor sulfonylurea binding proteins of HeLa plasma membranes.

Plasma membranes of cultured HeLa S cells bound the tritiated antitumor sulfonylurea [3H]LY181984 with high affinity (Kd of about 25 nM). The number of binding sites, estimated to represent 30 to 35 pmol/mg protein, would represent a low abundance protein of the total plasma membrane proteins. The binding proteins appeared to contain one or more thiols in the binding site as high affinity binding of [3H]LY181984 was reduced by treatment with the covalent thiol blocking reagent, N-ethylmaleimide (NEM), or by oxidation with dilute hydrogen peroxide but was protected by glutathione or dithiothreitol. Elimination of binding of [3H]LY181984 by NEM was prevented by excess unlabeled LY181984 (an active sulfonylurea) but less so by excess LY181985 (an inactive sulfonylurea). The binding proteins were specifically labeled with thiol reagents following reaction of unprotected thiols with unlabeled thiol reagents. Binding proteins at ca. 34 kDa were labeled. Plasma membrane proteins after solubilization with SDS under strongly reducing conditions still bound sulfonylurea. [3H]LY181984 binding to plasma membrane proteins resolved on SDS-PAGE correlated as well with proteins in the 30-40 kDa range.

Antineoplastic Agents↗
PubMed1995

Photoaffinity labeling of mitochondrial proteins with 2-azido [32P]palmitoyl CoA.

A long-chain fatty acyl CoA photolabel, 2-azido [32P]palmitoyl CoA, was synthesized and its covalent interaction with mitochondrial membrane proteins examined. On binding of 2-azido [32P]palmitoyl CoA to beef heart mitochondria, two polypeptides were primarily labeled, the 30 kDa ADP/ATP carrier and a 41 kDa protein of unknown identity. Carboxyatractyloside and palmitoyl CoA completely protected against labeling of the 30 kDa protein indicating that it was the ADP/ATP carrier. With inverted submitochondrial particles, only the 30 kDa polypeptide was labeled by 2-azido [32P]palmitoyl CoA. The labeling was inhibited by bongkrekic acid and palmitoyl CoA but not carboxyatractyloside, providing evidence that the ADP/ATP carrier was covalently bound from the matrix side of the membrane. In brown adipose tissue mitochondria, 2-azido [32P]palmitoyl CoA photolabeled the ADP/ATP carrier and the 32 kDa uncoupling protein with some minor labeling of 36 and 68 kDa polypeptides. The results indicated that this physiological photolabeling reagent with the azido group on the CoA portion of the molecule interacts like 2-azido ADP with nucleotide binding sites of a number of important enzymes in cell metabolism. Moreover, the evidence strongly supports the hypothesis that long chain fatty acyl CoA esters are natural ligands for key nucleotide binding proteins.

Acyl Coenzyme A↗
PubMed1995

Comparison of ulnar and radial arterial blood-flow at the wrist.

Historical descriptions of the ulnar artery as the dominant vessel to the hand appear to be inconsistent with clinical experience. Anatomical dissections and radionucleotide flow studies of the ulnar and radial arteries at the wrist were performed. These failed to demonstrate any difference between the anatomical dimensions of these vessels, but the radial artery was shown to have a statistically greater blood flow compared to the ulnar artery. This finding suggests that, contrary to popular opinion, the radial artery is the dominant vessel to the hand.

Blood Flow Velocity↗
PubMed1995

Contrast-enhanced magnetic resonance imaging of the lumbosacral roots in the dysimmune inflammatory polyneuropathies.

The diagnosis of acute Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy is based on clinical characteristics, abnormalities on nerve conduction studies, and nerve biopsy specimens indicating demyelination. Inflammation and edema are also common findings in nerve specimens. Immunotherapy is helpful in these dysimmune conditions. Occasionally the diagnosis is difficult to make, particularly when electrophysiological testing or nerve biopsy findings are not characteristic. The authors found contrast enhancement of lumbosacral roots in patients with chronic inflammatory demyelinating polyradiculoneuropathy and Guillain-Barré syndrome, but not in those with other demyelinating neuropathies. Contrast-enhanced magnetic resonance imaging could be a useful tool in the diagnosis of the dysimmune inflammatory neuropathies.

Adult↗
PubMed1994

Cyclic AMP-plus ATP-dependent modulation of the NADH oxidase activity of porcine liver plasma membranes.

Plasma membranes of porcine liver, highly purified by aqueous two-phase partition, oxidized NADH in the absence of added external acceptors. The oxidation was resistant to cyanide and responded to nanomolar concentrations of ATP alone or ATP in the presence of cyclic AMP. Both the Km for NADH and the long-term activity of the oxidase were affected. Upon incubation at 37 degrees C with cyclic AMP (0.1-10 nM) and ATP (1-100 nM), the NADH oxidase activity was inhibited. The inhibition was complex and due to an approx. 5-fold increase in the Km for NADH compared to the NADH oxidase of membranes incubated in the absence of cyclic AMP + ATP. The response to cAMP + ATP was rapid and occurred within seconds of ATP addition. The response was inhibited by the selective inhibitor of cyclic AMP-dependent protein kinase, H-89. Neither cyclic AMP alone nor ATP alone at nanomolar concentrations elicited a rapid response. However, 10 nM ATP alone did result in similar alteration of Km and Vmax as did ATP + 0.1 nM cyclic AMP. The response to ATP alone or in preparations depleted of cyclic AMP required higher ATP concentrations than with cAMP present or occurred more slowly with a lag of 1-2 min. The NADH oxidase activity of porcine plasma membranes after cyclic AMP + ATP treatment retained high activity with storage at 4 degrees C, whereas that of unincubated or sham-incubated plasma membranes was reduced with time of storage at 4 degrees C. In some but not all instances, NADH oxidase activity inactivated by incubation with NADH at 37 degrees C or after storage at 4 degrees C could be reactivated by incubation with cyclic AMP plus ATP. As with the alteration in Km, cyclic AMP alone was without effect and ATP alone was much less effective than the combination. The results demonstrate ATP-dependent modulation of the NADH oxidase activity of isolated plasma membranes at physiological concentrations of ATP. This modulation may have functional significance in mediating the hormone and growth factor responsiveness of the plasma membrane NADH oxidase activity.

Adenosine Triphosphate↗
PubMed1994

Experimental basis for separation of membrane vesicles by preparative free-flow electrophoresis.

In practice it has been possible to separate membrane particles of different origins but of similar chemical composition by preparative free-flow electrophoresis. Examples include the vacuolar (tonoplast) and plasma membranes of plants and membranes derived from the cis and trans regions of the rat liver Golgi apparatus. Yet, when analyzed for intrinsic molecules that might contribute to significant differences in surface charge, the separated membranes were surprisingly similar. As more information was generated, it became apparent that the membranes with greatest electrophoretic mobility (i.e. lysosomes, rightside-out tonoplast vesicles and membranes from the trans region of the Golgi apparatus), where those membranes with an inherent ability to acidify their interiors. By so doing, the vesicles generate a membrane potential, negative outside, which might serve as a basis for enhanced electrophoretic mobility. To test the hypothesis, tonoplast membranes were incubated with ATP to drive proton import or with monensin to dissipate the ATP-supported proton gradient. With ATP, mobility was enhanced. Also, when ATP-treated vesicles were analyzed in the presence of monensin, the ATP effect on mobility was reversed. Similarly with Golgi apparatus, mobility of the most electrophoretically mobile portions of the separation was enhanced by ATP and the ATP effect was reversed with monensin. A trans origin of the vesicles was verified by assay of the trans Golgi apparatus marker, thiamine pyrophosphatase. Finally, incubation with ATP (and reversal by monensin) was employed as an aid to the free-flow electrophoretic separation of kidney endosomes from complex mixtures. These lysosomal derivatives also are capable of acidification of their interiors in an ATP-dependent process and of generating, at the same time, a negative (outside) membrane potential. The findings provide both an experimental basis to enhance membrane separations by preparative free-flow electrophoresis and, at the same time, a theoretical basis to help explain why certain membranes of very similar overall chemical composition may be separated by electrophoretic methods.

Adenosine Triphosphatases↗
PubMed1994

Analysis of protein transmembrane helical regions by a neural network.

Neural networks were used to generalize common themes found in transmembrane-spanning protein helices. Various-sized databases were used containing nonoverlapping sequences, each 25 amino acids long. Training consisted of sorting these sequences into 1 of 2 groups: transmembrane helical peptides or nontransmembrane peptides. Learning was measured using a test set 10% the size of the training set. As training set size increased from 214 sequences to 1,751 sequences, learning increased in a nonlinear manner from 75% to a high of 98%, then declined to a low of 87%. The final training database consisted of roughly equal numbers of transmembrane (928) and nontransmembrane (1,018) sequences. All transmembrane sequences were entered into the database with respect to their lipid membrane orientation: from inside the membrane to outside. Generalized transmembrane helix and nontransmembrane peptides were constructed from the maximally weighted connecting strengths of fully trained networks. Four generalized transmembrane helices were found to contain 9 consensus residues: a K-R-F triplet was found at the inside lipid interface, 2 isoleucine and 2 other phenylalanine residues were present in the helical body, and 2 tryptophan residues were found near the outside lipid interface. As a test of the training method, bacteriorhodopsin was examined to determine the position of its 7 transmembrane helices.

Amino Acid Sequence↗
PubMed1994

Critical care issues in the patient with hematologic malignancy.

Patients with lymphomas, multiple myeloma, and leukemia are often at risk for life-threatening complications. Complications include viral infections (eg, herpes zoster, herpes simplex, cytomegalovirus) and hemolytic anemia, which are related to the hematologic origin of the malignancy. Life-threatening disorders related to amount of tumor burden are leukostasis and hyperviscosity. Complications related to therapy include pulmonary capillary leak syndrome and tumor lysis syndrome. Good assessment skills assist in early identification of individuals at risk and initiation of preventive measures.

Critical Care↗
PubMed1994

Detection of Mycobacterium avium-intracellulare complex in bone marrow specimens of patients with acquired immunodeficiency syndrome.

Thirty-seven bone marrow core biopsy specimens from 21 human immunodeficiency virus-infected patients with Mycobacterium avium-intracellulare complex bacteremia were stained using rabbit polyclonal antibodies against Mycobacterium bovis strain Bacillus-Calmette-Guerin (BCG) and Mycobacterium duvalii, as well as Kenyon and Fite stains, to compare sensitivities of these techniques and evaluate possible response to therapy. The patients in this study had participated in a phase I/II trial of liposome-encapsulated gentamicin therapy. Two biopsy specimens had inadequate tissue for evaluation. Thirty-two specimens demonstrated bacilli with anti-M duvalii, 33 with anti-BCG, 20 with Kenyon, and 23 with Fite. Two were negative with all stains. Fifteen biopsy specimens had epithelioid granulomas, 12 had histiocytic granulomas, and 1 had a granuloma of indeterminate type. The remaining seven biopsy specimens had no granulomas. Four of these seven demonstrated bacilli with anti-M duvalii, 5 with anti-BCG, 1 with Kenyon, and 2 with Fite. The number of M avium-intracellulare organisms per milliliter of blood decreased in 14 of 21 patients after liposome-encapsulated gentamicin therapy. However, none of the 11 patients whose pre- and post-therapy bone marrow core biopsy specimens were both evaluable demonstrated a reduction in the number of M avium-intracellulare organisms. The authors concluded that anti-M duvalii and anti-BCG are more sensitive than acid-fast stains for identifying M avium-intracellulare infection in bone marrow core biopsy specimens of patients who have acquired immunodeficiency syndrome (AIDS) with M avium-intracellulare bacteremia. Bone marrow core biopsy specimens may provide a perspective on M avium-intracellulare infection in AIDS patients that differs from the one provided by blood cultures.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Isolation and sequence analysis of polyketide synthase genes from the daunomycin-producing Streptomyces sp. strain C5.

A contiguous region of about 30 kbp of DNA putatively encoding reactions in daunomycin biosynthesis was isolated from Streptomyces sp. strain C5 DNA. The DNA sequence of an 8.1-kbp EcoRI fragment, which hybridized with actI polyketide synthase (PKS) and actIII polyketide reductase (PKR) gene probes, was determined, revealing seven complete open reading frames (ORFs), two in one cluster and five in a divergently transcribed cluster. The former two genes are likely to encode PKR and a bifunctional cyclase/dehydrase. The five latter genes encode: (i) a homolog of TcmH, an oxygenase of the tetracenomycin biosynthesis pathway; (ii) a PKS Orf1 homolog; (iii) a PKS Orf2 homolog (chain length factor); (iv) a product having moderate sequence identity with Escherichia coli beta-ketoacyl acyl carrier protein synthase III but lacking the conserved active site; and (v) a protein highly similar to several acyltransferases. The DNA within the 8.1-kbp EcoRI fragment restored daunomycin production to two dauA non-daunomycin-producing mutants of Streptomyces sp. strain C5 and restored wild-type antibiotic production to Streptomyces coelicolor B40 (act VII; nonfunctional cyclase/dehydrase), and to S. coelicolor B41 (actIII) and Streptomyces galilaeus ATCC 31671, strains defective in PKR activity.

Amino Acid Sequence↗
PubMed1993

NADH oxidase activity of rat liver plasma membrane activated by guanine nucleotides.

The activity of a hormone- and growth-factor-stimulated NADH oxidase of the rat liver plasma membrane responds to guanine nucleotides, but in a manner that differs from that of the classic trimeric and low-molecular-mass monomeric G-proteins. In the absence of added bivalent ions, both GTP and GDP as well as guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) but not guanosine 5'[beta-thio]diphosphate (GDP[beta-S]) stimulate the activity over the range 1 microM to 100 microM. Other di- and tri-nucleotides also stimulate, but only at concentrations of 100 microM or higher. Added bivalent ions are not required either for NADH oxidation or guanine nucleotide stimulation. Bivalent ions (Mg2+ > Mn2+ > or = Ca2+) alone stimulate only slightly at low concentrations and then inhibit at high concentrations. The inhibitions are augmented by GDP or GTP [gamma-S] but not by GTP. Although the activity is the same, or less, in the presence of 0.5 mM MgCl2, GTP at 1-100 nM and other nucleotides at 0.1 mM or 1 mM still stimulate in its presence. The NADH oxidase is activated by mastoparan but aluminum fluoride is weakly inhibitory. Cholera and pertussis toxins elicit only marginal responses. Both the Mg2+ and the GDP and GTP[gamma-S] inhibitions (but not the GTP stimulations) shift to higher concentrations when the membrane preparations are first solubilized with Triton X-100. The results suggest a role for guanine nucleotides in the regulation of plasma membrane NADH oxidase, but with properties that differ from those of either trimeric or the low-molecular-mass G proteins thus far described.

Animals↗
PubMed1993

Good laboratory practice and laboratory accreditation.

Principles of good laboratory practice (GLP) and laboratory accreditation programs, particularly as they pertain to the environmental sector, are reviewed. The multitude of programs is proving costly for many laboratories and there is mounting pressure to develop reciprocity agreements between programs and to consolidate nationally and internationally. Inclusion of GLP and laboratory accreditation requirements in government regulations is resulting in a significantly increased number of laboratories participating in these programs.

Accreditation↗
PubMed1993

Formative evaluation: implementation of primary nursing.

Staff nurses initiated the change from a total patient care to a primary nursing delivery system. The effects of the change were measured on the following: nurses' perceptions of their work environment; communication and collaboration with other health care professionals; parent satisfaction and quality of care provided, and direct and indirect nursing activities. The measurements were taken prior to, six months and twelve months post implementation. Action was taken based on the findings of this evaluation process which enabled the facilitation of the change process.

Attitude of Health Personnel↗