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PubMed1985

Hydrogen ion buffering during complete brain ischemia.

As a first step to quantify [H+] changes in brain during ischemia we used H+-selective microelectrodes and enzyme fluorometric techniques to describe the relationship between interstitial [H+] ([H+]o) and peak tissue lactate after cardiac arrest. We found a step function relationship between [H+]o and tissue lactate rather than the linear titration expected in a homogeneous protein solution. Within a blood glucose range from 3-7 mM, brain lactate rose from 8-13 mmol/kg along with a rise in [H+]o of 99 +/- 6 nM(0.44 +/- 0.02 pH). At higher blood glucose levels (17-80 mM), brain lactate accumulated to levels of 16-31 mmol/kg; concurrently [H+]o rose by 608 +/- 16 nM (1.07 +/- 0.02 pH). The unchanging level of [H+]o between 8-13 and 16-31 mmol/kg lactate implies that [H+]o is at a steady-state, but not equilibrium with respect to [H+] in other brain compartments. We propose that ion-transport characteristics of astroglia account for the observed relationship of [H+]o to tissue lactate during complete ischemia and suggest that brain infarction develops after plasma membranes in brain cells can no longer transport ions to regulate [H+].

Animals↗
PubMed1986

Molecular mechanisms of drug-induced hearing loss.

Although the ototoxic actions of a variety of drugs have long been documented, the biochemical mechanisms underlying such toxicity largely remain to be established. For example, recent advances have provided us with information about the actions of salicylates (aspirin) and diuretics (furosemide) but we are not yet able to specify the mechanisms by which these drugs damage the cochlea. On the other hand, the considerable amount of biochemical and pharmacological data on the effects of aminoglycosides (streptomycin, neomycin, gentamicin and related compounds) has enabled us to formulate a rational hypothesis of their mechanism of action. We have previously presented evidence for an involvement of polyphosphoinositides in the ototoxic actions of aminoglycosides. Recent electrophysiological and pharmacokinetic studies have shown in addition that aminoglycosides occupy at least two distinct compartments in the course of their actions. Further studies of drug uptake in vitro and of drug toxicity in cochlear perfusions suggested the involvement of an active (energy-requiring) aminoglycoside transport system. These and other data are compatible with the following multi-step model of aminoglycoside toxicity: The initial step in the reaction sequence is an electrostatic interaction of aminoglycosides with the plasma membrane. The resulting displacement of calcium accounts for acute effects but the action is reversible and antagonized by divalent cations. An energy-dependent uptake process is required for the expression of toxicity. It can be prevented by select metabolic blockers. A crucial step in subsequent intracellular drug actions is the binding of aminoglycosides to phosphatidylinositol bisphosphate inhibiting its hydrolysis and preventing its physiological function.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoglycosides↗
PubMed1983

Blood neutrophil function in primary myelodysplastic syndromes.

Ten different tests of blood neutrophil function were studied in 20 patients with primary myelodysplastic syndromes (PMDS). The patients were selected according to the new diagnostic criteria for PMDS of the FAB-cooperation group. Impairments of granulocyte functions were found in all patients. Moreover, several steps in the mobilization of granulocytes at the site of injury seemed to be affected: decreased adhesion (P less than 0.05), deficient chemotaxis (P less than 0.05), decreased enzyme content (P less than 0.001), 'slower' chemiluminescence (P less than 0.005), decreased phagocytosis (P less than 0.05) and impaired microbicidal capacity (P less than 0.025). No significant correlation between disease category and severity of granulocyte dysfunction was discerned, though an increasing number of blasts was associated with more severe granulocytic disability. Results in seven patients with abnormal karyotypes were not significantly different from 13 others with normal karyotypes. Our results indicate that defects in blood neutrophil function are a common feature in PMDS and might account for the increased frequency of infection in these patients.

Adolescent↗
PubMed1990

Genetic epidemiology of ethanol metabolic enzymes: a role for selection.

Human ethanol consumption has a profound impact on nutritional status, causing major alterations in intermediary metabolism and critical deficiencies of vitamins and trace elements. The major enzyme systems responsible for the principal steps in ethanol metabolism have been characterized and the genes cloned, and significant functional polymorphisms have been identified. An inactive allele of the mitochondrial ALDH is associated with flushing and reduced alcohol intake. This allele may also confer greater sensitivity to some of ethanol's toxic effects. In populations not possessing this variant, twin and adoptive studies have revealed that heritability for alcoholism is greater than 50%. The occurrence of three functional polymorphisms in the ethanol metabolic pathway, including two mutations which are conserved across populations, suggests a role for selection in their maintenance. The two general categories of selective forces to maintain these polymorphisms are food toxins and infectious diseases. Of the infectious agents, amoebi and other anaerobic and microaerophilic organisms of the gut are the most logical candidates.

Alcoholism↗
PubMed1990

The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing.

The first cleavage in mammalian pre-rRNA maturation occurs near the 5' end within the 5' external transcribed spacer. Using mouse cell extracts, we show that this processing is abolished by micrococcal nuclease pretreatment. Autoantibodies that recognize the U3, U8, and U13 snRNPs (anti-fibrillarin) deplete processing activity from the extract and selectively immunoprecipitate both rRNA substrates and processing products from the reaction. Specific involvement of the U3 snRNP is demonstrated by native gel electrophoresis of the processing reaction followed by Northern blotting and by oligonucleotide-directed RNAase H abolition of processing activity. Our identification of U3 function is discussed with respect to the molecular basis of pre-rRNA recognition by the U3 snRNP, possible roles of U3 and other nucleolar snRNPs in rRNA processing, and the morphological organization of the nucleolus and the ribosomal transcription complex.

Animals↗
PubMedDate not supplied

Use of colloidal gold cytochemistry in the study of the basic cell biology of cancer.

We are currently investigating the morphologic aspects of two areas of the basic cell biology of cancer: tumor-specific surface antigens as targets for immunotoxins, and the phenomenon of multidrug resistance in chemotherapy of human tumors. Colloidal gold cytochemistry has provided a useful method for the electron-microscopic cytochemical detection of materials endocytosed by cells in culture. This technique has been used to study the internalization pathway of ligands bound to the surface of cancer cells, particularly antibodies for use as immunologic targeting reagents for the construction of immunotoxins. These colloidal gold conjugates with monoclonal antibodies have demonstrated the internalization of these immunologic reagents through coated pits and receptosomes, which is a necessary step in the delivery of immunotoxins into the cell where they can mediate their cell-killing functions. Morphologic methods have been employed for the screening and selection of monoclonal antibodies reactive with the surface of human ovarian cancer cells for use as immunotoxins and have demonstrated the in vivo activity of immunotoxins made with these antibodies and Pseudomonas exotoxin in a nude mouse model system. In other studies, we have employed such reagents for the immunocytochemical detection of the surface expression of P170, the cell-surface efflux pump protein responsible for the phenotype of multidrug resistance in tumor cells, and to investigate the distribution of this protein by using immunocytochemistry in normal human tissues. These results have suggested a role for P170 in normal cell membrane transport of metabolites in various organ systems.

Clathrin↗
PubMed1988

Large scale purification of factor X by hydrophobic chromatography.

Factor X is a critical enzyme in the blood coagulation cascade, however, in recent years the coagulation zymogen factor X has received additional interest as a selective proteinase to allow production of functional eukaryotic proteins in a prokaryotic expression system. Traditional factor X purification schemes suffer from low yields, low capacity, lengthy dialysis steps, and contamination by the autoproteolytic activated enzyme factor Xa. By incorporating a reversible inhibitor of factor X activation, we were able to recover 67% of the factor X present without any detectable activated enzyme. Six liters of plasma could be processed onto a 50 mL phenylalanine-Sepharose hydrophobic chromatography column without saturating the matrix. The final product is devoid of detectable proteolytic activity. At time of use, the zymogen is specifically activated with a Sepharose-bound activating enzyme isolated from Russell's Viper Venom, resulting in factor Xa free of other detectable proteinases.

Chromatography↗
PubMed1991

1H NMR spectroscopic imaging of the monkey brain using binomial water suppression in a stimulated-echo sequence.

A new proton, two-dimensional pulse sequence for 1H NMR spectroscopic imaging (chemical shift imaging) was tested in phantoms and in the monkey brain. The pulse sequence consisted of one binomial chemically selective pulse and two spatially selective pulses in the stimulated-echo sequence. The point-spread function (which is influenced by k-space filtering and the number of phase-encoded steps) of a 1 mm source phantom was measured using a 16 x 16 spatial matrix and was found to have a FWHM of 10 mm (100 mm field of view) with very little rippling outside the main lobe. The binomial excitation profile was measured in order to correct the NMR intensity for the variable flip angles. Spectroscopic images were measured in the monkey brain with a 15 mm slice thickness and a 16 x 16 spatial matrix. Proton spectra derived from the brain contained sharp resonances of choline, creatine and N-acetyl aspartate with minimal lipid contamination. Proton spectra derived from the subcutaneous fat and adipose tissue behind the eyes contained large lipid resonances.

Animals↗
PubMed1990

Cysteine, a chelating moiety for synthesis of 99Tcm radiopharmaceuticals. III: Functionalization of hydroxy group for 99Tcm chelation.

A method is offered for converting an organic hydroxy compound to a 99Tcm-binding ligand under mild experimental conditions. Cyclohexanol was selected as a typical hydroxy compound and to this molecule cysteine was attached through substitution at the sulphur atom by a two-step chemical synthesis. The ligand thus obtained could be successfully radiolabelled with 99Tcm. The biodistribution of the resulting radiolabelled compound had the characteristic of a mixed hydrophilic-lipophilic 99Tcm chelate and was similar to that of 99Tcm-HIDA derivatives, a well established class of 99Tcm-chelates. So it may be concluded that the above method of functionalization of a hydroxy compound does not generate any unusual physiological properties and may be recommended as a general method for radiolabelling an organic compound at the alcoholic site with 99Tcm.

Animals↗
PubMed1988

Determination of telenzepine in human serum by gas chromatography-mass spectrometry.

A method for determining telenzepine in human serum is described. Analytes are obtained from alkalinized serum by extraction of the drug using reversed-phase octadecylsilane-bonded silica cartridges. Telenzepine and a desmethyl analogue added to serum as internal standard are retained on the C18 cartridge and recovered by elution with methanol. The gas chromatographic properties of telenzepine and the internal standard are improved by a two-step derivatization involving a benzodiazepinone-benzimidazole rearrangement and simultaneous formation of a methyl ester function. The processed extract is analysed by gas chromatography-mass spectrometry with selected-ion monitoring. Quantification is linear over the range 2-40 ng/ml. Inter-day precision is within 7%, except at the detection limit of 2 ng/ml (16%). Application of this assay to routine analysis is limited by the extensive sample pretreatment essential for derivatization of telenzepine.

Adult↗
PubMed1984

Statistical properties of single sodium channels.

Single channel currents were obtained from voltage-activated sodium channels in outside-out patches of tissue-cultured GH3 cells, a clonal line from rat pituitary gland. In membrane patches where the probability of overlapping openings was low, the open time histograms were well fit by a single exponential. Most analysis was done on a patch with exactly one channel. We found no evidence for multiple open states at -25 and -40 mV, since open times, burst durations, and autocorrelation functions were time independent. Amplitude histograms showed no evidence of multiple conductance levels. We fit the gating with 25 different time-homogeneous Markov chain models having up to five states, using a maximum likelihood procedure to estimate the rate constants. For selected models, this procedure yielded excellent predictions for open time, closed time, and first latency density functions, as well as the probability of the channel being open after a step depolarization, the burst duration distribution, autocorrelation, and the distribution of number of openings per record. The models were compared statistically using likelihood ratio tests and Akaike's information criterion. Acceptable models allowed inactivation from closed states, as well as from the open state. Among the models eliminated as unacceptable by this survey were the Hodgkin-Huxley model and any model requiring a channel to open before inactivating.

Animals↗
PubMed1991

Independent performance among individuals with mental retardation: promoting generalization through self-instruction.

The full participation of individuals with mental retardation in the community requires performance of newly acquired skills in novel circumstances and across the varying demands characteristic of life in the everyday world. For example, a person who has learned how to ride a bus may have to adapt to changes in scheduling, bus routes, or fares. Or an employee who has been taught by a coworker to make salads in a fast-food restaurant will need to continue to complete orders when the coworker no longer is present. Although skill generalization is an implicit educational goal, instructional strategies rarely are employed to influence the attainment of this goal (Haring & Laitinen, in press). Self-instruction is a strategy that has been effective in promoting the independent performance of people with mental retardation. Self-instruction provides individuals with the means for guiding their own behavior in novel situations not associated with training and after assistance has been withdrawn. For example, by using self-instruction, people with mental retardation have learned, among other skills, to sequence their tasks, increase their rate of production, and solve work-related problems. This chapter evaluated studies that investigated use of self-instruction among individuals with mental retardation in community settings. The focus of the review was on identification of factors relating to generalization across people, situations, tasks, and time. The combination of these factors suggests a model for promoting independent performance among individuals with mental retardation. This model combines self-instruction with teaching multiple exemplars and comprises the following six steps: (a) Select an array of examples (responses) an individual is likely to be required to perform in an environment (step 1); (b) classify responses into teaching sets based upon a functional analysis (step 2); (c) divide items of each set into responses that will serve as training examples and those that will serve as generalization probes (step 3); (d) teach trained examples using self-instruction (step 4); (e) evaluate effect of training on trained and untrained examples (i.e., generalization probes) as well as verbalized self-instructional statements (step 5); and (f) withdraw training based upon performance criteria while evaluating the effect of withdrawal (step 6). These steps represent the best practices for promoting independent performance of individuals with mental retardation in the community.

Attention↗
PubMed1984

Macromolecule-drug conjugates in targeted cancer chemotherapy.

Although clinical trials using macromolecular conjugates of cytotoxic drugs in a treatment of malignant disease are fragmentary at best, a number of carrier systems that have been examined in vitro, in tissue culture, and in vivo animal experiments demonstrate great promise and warrant further extensive examination. The optimal drug delivery and, consequently, maximum therapeutic effect will be accomplished when all available information from diverse disciplines can be integrated. This review has attempted to point out some of the limitations of taking an optimistic view of the question of targeting drug delivery using macromolecule-drug conjugates. For example, a strategy for the development of macromolecular conjugates can be established based on characteristics of the targeted tumors and the drugs in prospect of great success. Biological and physiological features of the tumor such as a cell type, site, and the pharmacological and physicochemical properties of chemical agents such as site of action and chemical stability must be considered at the first step. Selection of the optimum carrier system will be accompanied when all these problems are carefully considered. Similarly, the optimum method of conjugation is likely to vary as a function of the carrier, the chemotherapeutic agent, and the delivery site or site of action of the agent. The stability of the bond must adopt itself to the mode and site of action of the agent, the necessity for release, and the availability of hydrolytic enzymes which break the linkage and release the agent. On the other hand, the success of such conjugates synthesized according to this strategy will depend on physicochemical properties of the conjugates such as molecular size, electric charge, and solubility; chemical and biological stability of active components of conjugates and linkages; interaction with the tumor cells; cytotoxicity in tissue culture system; pharmacokinetics in the body such as absorption profile, localization, and elimination manner; in vivo antitumor activity; and biodegradability and antigenicity of the conjugates. The practitioners of macromolecular conjugate research find themselves in the interesting, but difficult, position of being at the interface between basic information on the drugs and biological systems and an expanding clinical demand for more sophisticated therapeutic agents. Consequently, knowledge is demanded not only of the chemical, physical, and pharmaceutical properties of macromolecules, but also of pathophysiology of the condition being treated.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

The role of tissue expansion in the reconstruction of secondary burn defects.

Tissue expansion has become firmly established as an acceptable and useful modality in the treatment of various soft tissue defects. This article relates our experience with this technique for the reconstruction of secondary burn deformities in 26 patients. Details of expander placement and use are discussed, as well as the results and complications in this series. The advantages of this method, including a superior quality reconstruction, limited donor site morbidity, and cost-effectiveness, are discussed. The disadvantages of several operative steps, multiple office visits, and the objectionable appearance during expansion are enumerated. Guidelines for patient selection and technique refinements are also considered to reduce complications and to achieve an optimal functional and aesthetic result.

Adult↗
PubMed2025

Multi-step genomics on single cells and live cultures in sub-nanoliter capsules.

Single-cell sequencing methods uncover natural and induced variation between cells. Many functional genomic methods, however, require multiple steps that cannot yet be scaled to high throughput, including assays on living cells. Here we develop capsules with amphiphilic gel envelopes (CAGEs), which selectively retain cells and large analytes while being freely accessible to media, enzymes and reagents. Capsules enable high-throughput multi-step assays combining live-cell culture with genome-wide readouts. We establish methods for barcoding CAGE DNA libraries, and apply them to measure persistence of gene expression programs in cells by capturing the transcriptomes of tens of thousands of expanding clones in CAGEs. The compatibility of CAGEs with diverse enzymatic reactions will facilitate the expansion of the current repertoire of single-cell, high-throughput measurements and extension to live-cell assays.

Journal Article↗
PubMed1992

Rapid large-scale preparation of recombinant Erwinia chrysanthemi L-asparaginase.

L-asparaginase from Erwinia provides an alternative to the enzyme from E. coli for the effective treatment of acute lymphoblastic leukaemia. A procedure was required for the large-scale partial purification of the recombinant Erwinia enzyme cloned and expressed in Erwinia. Enzyme was extracted from Erwinia at high pH and extraneous protein precipitated at low pH. S-Sepharose FF was selected as the medium of choice for the chromatography step since it was adequate for the high flow rates required (linear flow rate 315 cm h-1) and the methylsulphonate functional groups exploited the high pI of the enzyme by allowing binding of L-asparaginase at pH 4.8 while most of the other proteins passed through the column. The useful capacity of the matrix was up to 34 mg enzyme/ml matrix at a linear flow rate of 95 cm h-1 and 15.4 mg enzyme/ml matrix at a linear flow rate of 315 cm h-1. Weakly bound protein was removed by a wash at pH 6.0. The L-asparaginase was eluted by a wash at pH 6.8 (linear flow rate 95 cm h-1) and was substantially pure, only requiring polishing steps to be suitable for use as a parenteral agent. The purity of the protein was complemented by a 92% recovery of active enzyme from this cation-exchange matrix.

Asparaginase↗
PubMedDate not supplied

Regioisomeric products of propranolol metabolism. The monomethyl ethers of 3,4-dihydroxypropranolol and of 3,4-dihydroxypropranolol glycol.

Regioisomeric monomethyl ethers of the 3,4-catechol of propranolol (1) and its 3-aryloxypropane-1,2-diol (glycol) metabolite were prepared to prove the structures of these putative products of oxidative metabolism. The ring regioisomer 4-methoxy-3-hydroxypropranolol (3) was prepared from 1-acetoxy-3-acetyl-4-methoxynaphthalene (8). Baeyer-Villiger oxidation was the key step in converting the 3-acetyl functionality to the desired 3-naphthol. The ring regioisomer 4-hydroxy-3-methoxypropranolol (4) was prepared from 3-methoxy-1,4-dihydroxynaphthalene (13) by selective 1-O-acylation with trimethylacetyl chloride. 4-O-Benzylation, followed by hydrolysis, and side chain elaboration afforded the 4-O-benzyl ether of 4. Similar methods afforded glycols 5 and 6, with the side chain obtained by osmium tetroxide oxidation of an O-allyl group. GC/MS analysis using the trifluoroacetyl derivatives of these known standards showed both 3 and 4 were metabolites of 1 in the rat. From a single dose study in man, 4 was identified as a minor urinary metabolite, and both regioisomeric glycol metabolites 5 and 6 were observed. In addition, another regioisomeric hydroxymethoxyglycol metabolite was found.

Animals↗
PubMedDate not supplied

Selection for surgery and long-term results in renovascular hypertension.

From 1960 to December 1977, 251 patients with renovascular hypertension (RVH) were observed; 219 were operated upon. Long-term results were assessed in 130 patients. Selection for surgery follows three steps: 1) screening of patients with renal artery disease, which is done by angiography; 2) diagnosis of RVH, which is reached mainly by differential renal function studies and renal vein renin measurements (ancillary methods are intravenous pyelography and sequential scintigraphy; the significance of renin measurements is discussed); and 3) prediction of the results of surgery and choice of technique. An original dynamic test of the authors is employed and reconstruction of the renal artery is the procedure of choice. This is done in the majority of cases by aortorenal bypass grafts using dacron prostheses. Techniques and complications are discussed and comparison with venous autograft is made. Hospital mortality was 3.2%. Overall long-term results were favorable in 78%. Long-term mortality was 6% and occurred mainly in patients who remained hypertensive. Results in atherosclerotic patients are compared with those obtained in fibrous stenoses. Results of renal artery reconstructions were far better than those of nephrectomies and lead us to restrict indications for such a procedure.

Aorta, Abdominal↗