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Biomedical subjects

L C Smith

Publications and source records attributed to L C Smith.

At least 127 records · Page 7Linked to original sources
PubMed1991

Nutritional assessment and indications for nutritional support.

The goal of nutritional assessment is to identify prospectively all those patients who would develop a nutrition-related complication. In practical terms, there is no single test capable of achieving this goal. At present, the best method of nutritional assessment is an organized step-by-step multifactorial approach. This involves assessment of the primary illness, the patient history, and the prognosis. A nutritional status examination is done, and the current intake is compared with the nutritional goals. A decision is then made whether to force feed. If forced feeding is initiated, the effectiveness of the therapy must be monitored frequently until the patient recovers and is able to be sustained by volitional oral intake.

Enteral Nutrition↗
PubMed1991

Human immunodeficiency virus (HIV) transcripts identified in HIV-related psoriasis and Kaposi's sarcoma lesions.

Persons with HIV infection sometimes develop aggressive psoriasis or Kaposi's sarcoma (KS) not usually seen in other immunosuppressed patients. However, a specific and direct pathophysiological role for HIV-1 in these AIDS-associated disorders remains unclear since HIV has not been easily detected in these skin lesions. By combining in situ hybridization with the sensitive detection technique of confocal laser scanning microscopy, we have demonstrated HIV RNA transcripts in 5 of 15 lesional skin biopsies from HIV-infected psoriasis patients, and in 3 of 8 Kaposi's sarcoma biopsies from HIV-infected patients. HIV transcripts were not detected in normal appearing skin from HIV-infected patients or in psoriatic and normal skin biopsies from uninfected individuals (P = 0.006). Although previous attempts to demonstrate viral sequences in psoriasis and KS lesions have been unsuccessful, in situ hybridization with confocal microscopy has shown the presence of HIV RNA transcripts predominantly within CD4+, Factor XIIIa positive dermal dendrocytes. HIV or cytokines produced by infected cells in skin lesions may therefore play a direct role in the pathogenesis of HIV-associated psoriasis and KS.

Adult↗
PubMed1990

In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion.

Detailed structure-function information about human lipoprotein lipase (LPL) is unavailable because it is difficult to purify large amounts of the enzyme for study. To circumvent this problem, we constructed an in vitro LPL expression vector. Human LPL cDNA was cloned and inserted into the expression vector p91023(B). After transfection of COS M-6 cells with the human LPL cDNA construct, LPL enzyme activity was detected in cell extracts and culture medium. Purified human apolipoprotein C-II caused a 5-fold stimulation of the recombinant human LPL expressed in vitro. Using site-specific mutagenesis, Ala residues were substituted for Asn residues at two potential N-linked glycosylation sites (positions 43 and 359) and at a third unrelated Asn (position 257) in the LPL cDNA. RNA blot analysis demonstrated the presence of a single mRNA species in COS cells transfected with wild-type and mutant LPL expression vectors. Intracellular and secreted LPL activity was absent in the construct containing an Ala for Asn mutation at position 43, whereas the same substitutions at positions 257 and 359 did not appreciably affect activity. LPL activity was also absent in another construct containing a Gln for Asn mutation at position 43. Quantitation of LPL protein mass concomitant with measurement of enzyme activity showed that substitution of Ala or Gln for Asn at position 43 resulted in the production of an enzymatically inactive protein which accumulated intracellularly but was not secreted into the culture medium. Our report represents an initial documentation of the expression of cloned human LPL in vitro and of the importance of Asn-43 for both enzyme activity and secretion.

Amino Acid Sequence↗
PubMed1990

Confocal microscopic detection of human immunodeficiency virus RNA-producing cells.

A central anomaly in the pathogenesis of AIDS is that few actively infected CD4+ cells (1 in 10(4)-10(5) have been detected in the peripheral blood, even though dramatic depletion (often greater than 90%) of CD4+ cells is the hallmark of disease progression. A sensitive, 35S-based human immunodeficiency virus (HIV) RNA in situ hybridization technique was coupled with a new detection method, confocal laser scanning microscopy, to examine transcriptionally active HIV-infected cells from individuals at different disease stages. In 35 symptomatic HIV-infected individuals (AIDS and AIDS related complex), an average of 1 in 350 mononuclear cells produced HIV RNA. In contrast, in an asymptomatic group of 30 individuals, an average of 1 in 2000 mononuclear cells produced HIV RNA. These data, obtained using this improved detection method, suggest there are more HIV RNA-producing cells in HIV-infected individuals than previously reported. In addition, increased numbers of HIV transcribing cells were found to correlate with declining clinical condition as assessed by Karnofsky performance score. These data suggest that viremia per se may account for the pathologic consequences in HIV infection.

AIDS-Related Complex↗
PubMed1990

Retinol-binding protein: a major secretory product of the pig conceptus.

Pig conceptuses and endometrial explants recovered from gilts between Days 10 and 15 of pregnancy were cultured in leucine-deficient or methionine-deficient medium supplemented with 3H-leucine or 35S-methionine, respectively, for 30 h. Conceptus and endometrial tissues from Day 15 of pregnancy were fixed in Bouin's fixative for immunocytochemistry and light microscopy. Conceptus culture medium from Day 15 of pregnancy was pooled, dialyzed, and fractionated by anion exchange and gel filtration chromatography. A family of 3-5 low molecular weight (Mr) acidic (Mr = 19,000-22,000; pI = 5.6-6.5) 3H-leu-labeled proteins were isolated and identified by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), electroblotting, and fluorography. The two major proteins (pCSP-1 and pCSP-2) were excised from a polyvinylidene difluoride transfer membrane, and NH2-terminal amino acids were sequenced. One peptide was sequenced through 33 amino acids and the second, which shared 100% homology, was sequenced through 22 amino acids. Analysis of the larger sequence indicated that it shared 93.9% and 90.9% homology with the first 33 amino acids of human and rabbit plasma retinol-binding protein (RBP), respectively. Analyses of culture medium from pig conceptus incubations by 2D-PAGE and immunoprecipitation with rabbit anti-human RBP serum indicated that immunoreactive RBP was produced between Days 10 and 15 of pregnancy and was present in Day 30 allantoic fluid. Western blotting of enriched fractions of Day 15 conceptus RBP followed by immunostaining indicated that five isoforms of radiolabeled RBP were present. Immunoreactive RBP was detected in trophectoderm and yolk sac of conceptuses and endometrial surface and glandular epithelium at Day 15 of pregnancy. Results from this study demonstrate that pig conceptuses secrete RBP prior to onset of conceptus elongation and throughout the peri-implantation period, which suggests that RBP and associated retinoids influence conceptus development.

Amino Acid Sequence↗
PubMed1990

Control of first cleavage in single-cell reconstituted mouse embryos.

Karyoplasts derived from mouse embryos at the initial and final stages of the first or second mitotic interphase were fused to early and late enucleated 1-cell embryos. The time of cleavage of reconstituted and control embryos was recorded at 1-h or 8-h intervals after manipulation. This enabled assessment of nuclear and cytoplasmic control over the mitotic apparatus of the 1-cell embryo. Early nuclei from 1- or 2-cell embryos fused to late enucleated embryos delayed cleavage but for only a few hours. However, late nuclei fused to early enucleated embryos were unable to advance the cytoplasmic timing of the next cleavage division. Furthermore, these reconstituted embryos stayed in interphase longer than did controls and many embryos with nuclei derived from late 2-cell embryos failed to cleave. These findings suggest that, allowing for a short period, early nuclei can synchronize with late cytoplasm with no major damage to the cleavage apparatus. It is proposed that this period is required for the completion of DNA synthesis by the early nuclei. However, late nuclei cannot induce mitosis before the expected cytoplasmic time, and, with 2-cell karyoplasts, this interaction causes many embryos to 'block' in interphase, without cleaving, suggesting incompatible nucleo-cytoplasmic interactions between late 2-cell karyoplast and early 1-cell stage cytoplasm.

Animals↗
PubMed1989

Pyrene-labeled lipids: versatile probes of membrane dynamics in vitro and in living cells.

Pyrene-labeled analogs of fatty acids have been studied as probes of lipid metabolism in vitro and in cultured cells. Procedures for the synthesis of complex pyrenyl lipids and the analytical methods for their separation and quantification are described. Pyrenyl-lipids have been used to quantify the relationship between lipid structure and the rates of spontaneous lipid transfer. Modifications of these methods have also been used to monitor protein-mediated lipid transfer, lipolysis and lipid translocation across bilayer membranes. According to several criteria, pyrene dodecanoic acid has been identified as a good analog of some naturally occurring fatty acids. Digital imaging microscopy has been used to monitor the rate of accumulation of pyrenyl lipids in living cells.

Animals↗
PubMed1989

Validation of an imaging system: steps to evaluate and validate a microscope imaging system for quantitative studies.

The processing methods described in this article are general and can be used with any computer with adequate software without reference to a specific experimental apparatus. To achieve appropriately corrected images, the operations must be performed correctly and in a specific order to extract meaningful information from digital images. The operations are subtraction of the dark current, a shading correction after low pass filtering of the shading mask, a geometric correction with a reference grid obtained at the longest wavelength of interest, and finally registration. Since the digital data sets are too large for manual evaluation, statistical criteria must be used to define the analytical capabilities and error limits of the system before the more interesting cellular studies can be undertaken.

Algorithms↗
PubMed1989

Influence of nuclear and cytoplasmic activity on the development in vivo of sheep embryos after nuclear transplantation.

Reconstituted sheep embryos have been produced by electrofusion-mediated nuclear transplantation in which single cells derived from embryos at the 16-cell (Day 4) and the inner cell mass (ICM) of early blastocyst stage (Day 6) were fused to unfertilized enucleated secondary oocytes. Electrofusion rates were higher with 16-cell blastomeres (82%) than with ICM cells (47%) and when an alternating current (a.c.) preceded the direct current (d.c.) fusing pulse (88% vs. 47%). The addition of cytochalasin B (7.5% micrograms/ml) to the medium for 1 h after electrofusion significantly improved the development to morula-blastocyst stage of reconstituted embryos derived from both 16-cell blastomeres (11% vs. 35%) and ICM cells (0% vs. 56%), indicating that the cytoskeletal mechanisms operating at the time of oocyte activation may affect the developmental potential of exogenous nuclei. Transplantation of 22 reconstituted morulae and blastocysts from both groups into the uteri of recipient ewes led to the development of four lambs (18%) with the phenotype of the nuclear donor breed. These findings indicate that at least some nuclei derived from transcriptionally active embryos are totipotent and able to be reprogrammed to support full-term development when fused to enucleated secondary oocytes.

Animals↗
PubMed1989

Physical stability of a total nutrient admixture for total parenteral nutrition.

The stability of a total nutrient admixture (TNA) has been postulated to be less than 7 days in refrigerated storage. When a TNA destabilizes, lipid particles coalesce and enlarge. Liposomes larger than 6 microns can obstruct pulmonary capillaries. A TNA containing 1500 ml of 7% Vamin, 1000 ml of 50% dextrose and 500 ml of 10% Intralipid, including the usual electrolytes, minerals and vitamins, was studied. Liposome size was measured in the original Intralipid and the TNA at intervals up to 14 days at 4 degrees C followed by 2 days at 22 degrees C. There was a small increase in liposome size up to 16 days. However, the number of particles larger than 6 microns was insignificant (by light microscopy, 3.9 +/- 2.4 [+/- SD] per 20 high-power fields; by Coulter counter, 99.8% smaller than 1.9 microns, with 0% larger than 6 microns; and by electron microscopy, 100% smaller than 2.0 microns). The osmolality and pH of the TNA were 1472 +/- 31 mOsm/kg and 5.5 +/- 0.1 respectively (mean +/- SD), with no significant change during the study times. The authors concluded that this TNA remains physically stable when refrigerated for 14 days and at room temperature for a further 2 days.

Amino Acids↗
PubMed1989

Inhibitors of sterol synthesis. Chromatography of acetate derivatives of oxygenated sterols.

The separation of the acetate derivatives of a number of oxygenated sterols was achieved by medium pressure liquid chromatography on silica gel columns and by normal and reversed phase high performance liquid chromatography. We have explored the application of these chromatographic systems for the analysis of oxygenated sterols of plasma samples from two normal human subjects. The addition of highly purified [14C]cholesterol to plasma permitted the detection and quantitation of oxygenated sterols formed by autoxidation of cholesterol during processing of the samples. Special attempts to suppress autoxidation of cholesterol included the use of an all-glass closed system for saponification and extraction under argon followed by rapid removal of cholesterol from the polar sterols by reversed phase medium pressure liquid chromatography. Chromatographic analyses of the [3H]acetate derivatives of the polar sterols provided a sensitive approach for the detection and quantitation of the individual oxygenated sterols. Oxygenated sterols detected in plasma included cholest-5-ene-3 beta,26-diol, (24S)-cholest-5-ene-3 beta,24-diol, and cholest-5-ene-3 beta,7 alpha-diol. After correction for their formation by autoxidation of cholesterol during processing of the samples, very little or none of the following sterols were observed: cholest-5-ene-3 beta,7 beta-diol, 5 alpha,6 alpha-epoxy-cholestan-3 beta-ol, 5 beta,6 beta-epoxy-cholestan-3 beta-ol, and cholestane- 3 beta, 5 alpha,6 beta-triol, and the 25-hydroxy, 22R-hydroxy, 21-hydroxy, 20 alpha-hydroxy, and 19-hydroxy derivatives of cholesterol.

Acetic Anhydrides↗
PubMed1988

Geometric correction of digital images using orthonormal decomposition.

We have developed an algorithm which can be used to correct the geometric distortion of digital images. The method uses an orthonormal decomposition and a two-dimensional Horner's scheme to construct and evaluate a polynomial equation of arbitrary degree in two independent variables. This numerical scheme for geometric correction combines several methods selected on the basis of their computation efficiency and numerical stability. The differences and advantages of this numerical scheme are compared with methods found in the image processing literature. The algorithm presented here has a reduced number of mathematical operations, is flexible and numerically stable. Based on the least-squares criteria, the algorithm provides corrected pixel positions with an accuracy equal to or better than the pixel size.

Algorithms↗
PubMed1988

Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos.

Nuclei were transplanted from embryos of mice at different stages of the 1st and 2nd cell cycle to oocytes enucleated at various times after fertilization. After transfer of pronuclei, a greater proportion of embryos developed to blastocysts if donor and recipient embryos were at the same stage of the cell cycle (synchronous transfer = 94%, asynchronous transfer = 76%). By contrast, when 2-cell blastomere nuclei were fused to the cytoplasm of enucleated zygotes, there was a significant effect of both cytoplast and karyoplast cell cycle stage on the development of the reconstituted embryos. Karyoplasts and cytoplasts derived from embryos at later stages of the cell cycle had greater potential to support development to blastocysts in vitro. It is suggested that the secretion of stage-specific messengers and the timing of nuclear membrane breakdown are the main factors causing the karyoplast and cytoplast effects, respectively.

Animals↗
PubMed1987

Monoclonal antibodies as probes of high-density lipoprotein structure: identification and localization of a lipid-dependent epitope.

Eight stable murine monoclonal antibodies (mabs) were raised against human high-density lipoproteins (HDL). Three different antibody reactivities were demonstrated by immunoblotting. A group of five antibodies were specific for apolipoprotein A-I (apoA-I) and bound to similar or overlapping epitopes. The second type of reactivity, shown by mab-32, was specific for apoA-II. In the third group, two antibodies showed high reactivity with apoA-II and slight cross-reactivity with apoA-I. The properties of two antibodies, mab M-30 specific for apoA-I and mab M-32 specific for apoAII, were characterized in detail as probes of HDL structure. The association of 125I-labeled HDL or synthetic complexes of apoA-I and phosphatidylcholine with mab M-30 was lipid dependent. Mab M-32 binding to apoA-II was independent of lipid. The lipid-dependent epitope bound by mab M-30 has been localized to an 18 amino acid synthetic apoA-I peptide. Moreover, studies with HDL2, HDL3, and immunoadsorbed HDL subfractions indicate that binding of mab M-30 to HDL is influenced by some component within the microenvironment individual HDL particles. These lines of evidence suggest that the molar ratio of apoA-I to apoA-II is the critical determinant. Binding of mab M-32 to HDL increased the reactivity of HDL to mab M-30 in a dose-dependent manner, indicating an unusual form of cooperativity between two mabs that recognize different proteins in HDL. These monoclonal antibodies will be valuable in studies of the metabolic significance of protein-protein and lipid-protein interactions in HDL.

Animals↗
PubMed1987

Mechanism and rate of permeation of cells by polycyclic aromatic hydrocarbons.

The principal mechanism of cellular uptake of benzo(a)pyrene and other polycyclic aromatic hydrocarbons (PAH) from lipoproteins into cells is spontaneous transfer through the aqueous phase (Plant, A. L., Benson, D.M., and Smith, L.C. (1985) J. Cell Biol. 100, 1295-1308). Cellular uptake of benzo(a)pyrene from low density lipoproteins followed first-order kinetics with a rate constant that was independent of the relative lipoprotein concentrations or cell number but which was 2 orders of magnitude smaller than the rate constant for benzo(a)pyrene desorption from low density lipoproteins. Moreover, identical rate constants for cellular uptake of benzo(a)pyrene were observed when the donor vehicle was high density lipoproteins, very low density lipoproteins, or single bilayer phosphatidylcholine vesicles, even though rate constants for benzo(a)pyrene transfer from these donor vehicles differed by 10-fold. When phosphatidylcholine vesicles containing benzo(a)pyrene and a nontransferable fluorescence quencher were mixed with cells in a stopped-flow system, two kinetic components were distinguished: a fast component with a rate constant corresponding to that measured for transfer of benzo(a)pyrene out of vesicles, followed by a much slower component, with a time course approximating that measured for cellular accumulation of benzo(a)pyrene by other techniques. Rate constants for desorption of a series of PAH which contained different number of aromatic rings from phosphatidylcholine vesicles differed over a 70-fold range. First-order rate constants for cell uptake of benzo(a)pyrene and five other PAH of different molecular sizes had the same 70-fold range of values, but were 2 orders of magnitude smaller than their respective rate constants for desorption from single bilayer vesicles. In addition, activation energies for cell uptake were essentially identical to the respective activation energies for desorption of PAH from phosphatidylcholine vesicles, confirming the mechanistic similarity of the two processes.

Animals↗
PubMed1987

Fluoride therapy for osteoporosis: characterization of the skeletal response by serial measurements of serum alkaline phosphatase activity.

Optimum use of fluoride therapy for osteoporosis requires a sensitive and convenient index of the skeletal response to fluoride. Since previous studies had shown that serum alkaline phosphatase activity (SALP) was increased in response to fluoride therapy, we examined serial measurements of SALP in 53 osteoporotics treated with 66 to 110 mg of sodium fluoride (NaF) for 12 to 91 months. SALP was increased in 87% of the subjects during therapy with fluoride. The increase in SALP was thought to reflect the osteogenic action of fluoride based on the findings that SALP correlated with both trabecular bone area (r = .81, P less than .001) and osteoid length (r = .67, P less than .01) in iliac crest biopsies, predicted increased bone density on spinal radiographs in response to fluoride therapy with an 87% accuracy, and predicted decreased back pain in response to fluoride with a 91% accuracy. In addition, the SALP response to fluoride was seen earlier than other therapeutic responses as indicated by the findings that the tau 1/2 for the SALP response (ie, time for 1/2 of the patients to show a significant response) was significantly less (1.2 +/- 0.3 yr) than that for the pain response (1.6 +/- 0.3 yr, P less than .05) or that for the radiographic response (3.7 +/- 0.5 yr, P less than .001). Although most patients responded to fluoride with an increase in SALP, evaluation of the kinetics of the SALP response to fluoride revealed marked interpatient variation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗