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C L Smith

Publications and source records attributed to C L Smith.

At least 145 records · Page 8Linked to original sources
PubMed1995

Protein translation components are colocalized in granules in oligodendrocytes.

The intracellular distribution of various components of the protein translational machinery was visualized in mouse oligodendrocytes in culture using high resolution fluorescence in situ hybridization and immunofluorescence in conjunction with dual channel confocal laser scanning microscopy. Arginyl-tRNA synthetase, elongation factor 1a, ribosomal RNA, and myelin basic protein mRNA were all co-localized in granules in the processes, veins and membrane sheets of the cell. Colocalization was evaluated by dual channel cross correlation analysis to determine the correlation index (% colocalization) and correlation distance (granule radius), and by single granule ratiometric analysis to determine the distribution of the different components in individual granules. Most granules contained synthetase, elongation factor, ribosomal RNA and myelin basic protein mRNA. These results indicate that several different components of the protein synthetic machinery, including aminoacyl-tRNA synthetases, elongation factors, ribosomes and mRNAs, are colocalized in granules in oligodendrocytes. We propose that these granules are supramolecular complexes containing all of the necessary macromolecular components for protein translation and that they represent a heretofore undescribed subcellular organization of the protein synthetic machinery. This spatial organization may increase the efficiency of protein synthesis and may also provide a vehicle for transport and localization of specific mRNAs within the cell.

Amino Acyl-tRNA Synthetases↗
PubMed1995

Technetium-99m labeling of DNA oligonucleotides.

UNLABELLED: Single-stranded RNA and DNA oligonucleotides may be useful as radiopharmaceuticals for antisense and other in vivo applications if convenient methods for stably attaching radionuclides such as 99mTc can be developed. METHODS: To radiolabel DNA with 99mTc, we have used the hydrazino nicotinamide (SHNH) moiety developed elsewhere. The diethylenetriaminepentacetic acid (DTPA) chelate was used to label DNA with 111In for comparison. Complementary 22-base, single-stranded oligonucleotides were obtained, each with a primary amine attached to either 3' or 5' end with a biotin moiety on the opposite end. The DNA was conjugated with SHNH by a N-hydroxysuccinimide derivative with DTPA by the cyclic anhydride. RESULTS: Reversed-phase HPLC analysis showed that essentially complete conjugation was achieved in both cases. The purified SHNH-DNA was radiolabeled with 99mTc by transchelation from glucoheptonate at labeling efficiencies of up to 60% and DTPA-DNA with 111In acetate at up to 100% efficiency. After labeling, the ability of the DNAs to bind to streptavidin through the biotin moieties and to hybridize with their complementary DNA in saline was retained for both radiolabels as determined by size-exclusion HPLC analysis. HPLC radiochromatograms of serum incubates showed a shift to 99mTc, but not 111In, to a high molecular weight, strongly suggesting serum protein binding in the former case only. Low-molecular weight degradation products were seen with 111In, but not with 99mTc and may be related to the use of phosphodiester-linked oligonucleotides. As a further measure of label stability, the DNAS were bound to streptavidin-conjugated magnetic beads and incubated in fresh 37 degrees C human serum. Less than 4% of 99mTc and 14% of 111In was lost in 24 hr. CONCLUSION: Amino-modified, single-stranded DNA can be stably radiolabeled with 99mTc by the SHNH moiety without loss of function.

Animals↗
PubMed1994

Oligonucleotide-directed self-assembly of proteins: semisynthetic DNA--streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates.

Modified biomolecules were used for the non-covalent assembly of novel bioconjugates. Hybrid molecules were synthesized from short single-stranded DNA and streptavidin by chemical methods using a heterobispecific crosslinker. The covalent attachment of an oligonucleotide moiety to streptavidin provides a specific recognition domain for a complementary nucleic acid sequence, in addition to the four native biotin-binding sites. These bispecific binding capabilities allow the hybrid molecules to serve as versatile connectors in a variety of applications. Bifunctional constructs have been prepared from two complementary hybrid molecules, each previously conjugated to biotinylated immunoglobulin G or alkaline phosphatase. The use of nucleic acid sequences as a template for the formation of an array of proteins is further demonstrated on two size scales. A macroscopic DNA array on a microtiter plate has been transformed into a comparable protein chip. A nano-scale array was made by hybridizing DNA-tagged proteins to specific positions along a RNA or DNA sequence. The generation of supramolecular bioconjugates was shown by quantitative measurements and gel-retardation assays.

Bacterial Proteins↗
PubMed1994

Top-down construction of an ordered Schizosaccharomyces pombe cosmid library.

A very rapid and efficient method for sorting and ordering large numbers of clones is presented. This top-down mapping approach divides the entire ordering problem into many smaller tasks and analyzes in parallel a gridded membrane array of clones by hybridization with probe pools. The strategy was tested on a 15-fold-coverage Schizosaccharomyces pombe cosmid library. About 1600 clones were assigned to chromosomes and to regions defined by the Not I and Sfi I restriction maps. Then, the clones were ordered into 20 contigs, which is consistent with statistical expectations for the degree of genome coverage used. The parallel ordering of clones and the computer-based analysis of digitized images make this approach very efficient; it is about 8-fold faster than existing methods. Only 61 hybridizations were needed to order 1600 clones.

Base Sequence↗
PubMed1994

Enhanced DNA sequencing by hybridization.

An enhanced version of DNA sequencing by hybridization (SBH), termed positional SBH (PSBH), has been developed. PSBH uses duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target should provide enhanced stringency in distinguishing perfectly matched 3' sequences. A second enhancement is the use of enzyme-catalyzed steps, instead of pure physical hybridization. The feasibility of this scheme has been investigated using biotinylated duplex probes containing single-stranded 5-base 3' overhangs, immobilized on streptavidin-coated magnetic beads. Ligation of a single-stranded target, hybridized to the single-stranded region of the duplex probes, provided enhanced discrimination of perfectly matched targets from those containing mismatches. In distinction to the serious complications caused by base composition effects in ordinary SBH, there was little effect of base composition in PSBH. The hardest mismatch to discriminate was the one furthest from the phosphodiester bond formed by ligation. However, mismatches in this position were efficiently discriminated by 3' extension of the duplex probe using a template-dependent DNA polymerase. These results demonstrate that PSBH offers considerable promise to facilitate actual implementations of SBH.

Base Sequence↗
PubMed1994

Ventralizing signal determined by protease activation in Drosophila embryogenesis.

Specification of dorsal-ventral cell fate during Drosophila embryogenesis is mediated by a signal transduction pathway. Asymmetry of cell fates arises through the spatially restricted production of a ligand in an extracellular compartment called the perivitelline space. The snake and easter genes are required for the production of the ligand and they encode the proenzyme form of secreted extracellular serine proteases. We have examined the effect of producing a preactivated form of the snake protease on the generation of dorsal-ventral polarity. SP6 RNA microinjection experiments reveal that different cell fates acquired at cellular blastoderm can be specified by the amount and spatial distribution of activated snake protein. Our results support a protease cascade model in which localized activation of uniformly distributed protease proenzymes leads to the spatially restricted production of ligand in the perivitelline space on the ventral side of the embryo.

Animals↗
PubMed1994

Reproduction in llamas and alpacas: a review.

In this review we attempt to compile and summarize the diverse and often contradictory material presented on the reproduction of llamas and alpacas (hereafter referred to as lamoids). Lamoids have recently gained international popularity, and theriogenologists are often asked to intervene in clinical management of reproductive problems of these animals. We therefore present a discussion of the reproductive anatomy, physiology, and behavior of llamas as well as the follicular dynamics as observed with ultrasonography. The nonsurgical embryo transfer procedure and the nutrient requirements of llamas are also discussed.

Journal Article↗
PubMed1994

Analysis of regulatory regions in the COL1A1 gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression in osteoblastic cells.

The synthesis of type I collagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D3. Earlier work from our laboratories has indicated that vitamin D regulation is at the level of transcription, based on results from both nuclear run-off assays and functional promoter analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloramphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D-mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the HindIII-Pstl restriction fragment between nucleotides -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone-mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides -2256 and -2216) contains a sequence which is very similar to vitamin D-responsive elements identified in the osteocalcin gene. Extracts from cultured cells which express a high level of vitamin D receptor contain a hormone:receptor complex that binds specifically to this 41 bp fragment, as demonstrated by bandshift analysis. However, deletion of this vitamin D receptor binding region from either a -3.5 kb or a -2.3 kb promoter fragment did not abolish vitamin D responsiveness. These results indicate that a vitamin D response element similar to that described for other vitamin D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D3.

3T3 Cells↗
PubMed1994

Large-scale structure conservation along the entire long arm of human chromosome 21.

Complete, contiguous NotI restriction maps were constructed for the long arm of human chromosome 21 contained in nine different cell lines. These maps span about 43 Mb and consist of about 60 distinct NotI fragments, about 80 markers, and 11 chromosomal breakpoints. Although some differences among the maps were noted, the overall large-scale structure of the chromosomes is remarkably similar, which strongly validates the use of cell lines for mapping studies. Remarkably few serious discrepancies were found between the order of markers on this NotI restriction map and various genetic and physical maps. However, when compared with the NotI physical map, both the genetic map and the radiation hybrid map of chromosome 21 appear to expanded near the centromere and especially near the telomere.

Animals↗
PubMed1994

Serum distribution of two contraceptive progestins: 3-ketodesogestrel and gestodene.

A cross-over study of two oral contraceptive formulations, containing 30 micrograms ethinylestradiol in combination with 150 micrograms desogestrel (Marvelon) or 75 micrograms gestodene (Femovan), has been performed to compare the serum distribution and pharmacokinetics of gestodene and the active metabolite of desogestrel, namely 3-ketodesogestrel. Serum concentrations of both sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) were also measured and were increased more than 3-fold and 2-fold, respectively, on day 21 of the treatment cycle, with no statistically significant difference between treatment groups. In addition, 35 days after ingestion of either oral contraceptive had ceased, the serum SHBG and CBG concentrations were similar to the pretreatment values. During treatment cycles, increased serum SHBG levels were associated with a redistribution of 3-ketodesogestrel and gestodene such that the non-protein-bound (NPB) and albumin-bound fractions were reduced in concert with an increase in the relative proportions bound to SHBG. The proportion of gestodene bound to SHBG was consistently higher than that observed for 3-ketodesogestrel, and this undoubtedly reflects the higher affinity of SHBG for gestodene (Kd = 1.2 nM at 37 degrees C) when compared to 3-ketodesogestrel (Kd = 4.7 nM at 37 degrees C). It also probably accounts, in part, for the much higher total serum levels of gestodene (8.58 nmol/L) when compared to 3-ketodesogestrel (2.37 nmol/L) during the treatment cycles. Consequently, the absolute amounts of NPB, non-SHBG-bound, and SHBG-bound gestodene are significantly higher than those measured for 3-ketodesogestrel. It is concluded that ethinylestradiol-induced increases in serum SHBG levels during treatment with Marvelon or Femovan, influenced the distribution and total amount of 3-ketodesogestrel and gestodene in serum, respectively, and that this, combined with the higher affinity of SHBG for gestodene, results in a greater amount of bioavailable gestodene compared to 3-ketodesogestrel, despite the smaller dose of gestodene administered.

Adult↗
PubMed1994

Effects of stepwise cryodilution prior to freezing and stepwise post-thaw rehydration on viability of ovine embryos.

Ovine embryos were exposed to 3 methods of stepwise cryodilution (1, 3 or 5 steps) prior to deep freezing. After 6 to 10 mo of frozen storage, the embryos were thawed and rehydrated using a reversal of each stepwise prefreezing method to remove the oryoprotectant. All embryos were cultured for 48 h, and survival was microscopically evaluated at 0, 14, 24, 38 and 48 h. Survival of embryos in culture was assessed by progressive normal development such as increased cell mass and integrity of morula or blastulation and hatching. There was no significant difference in post-thaw survival and development between 1 and 3-step treatment groups. Embryos frozen and thawed using the 5-step method had lower viability (P < 0.05) at 24 h of culture than either the 1- or 3-step method.

Journal Article↗
PubMed1994

Pioglitazone promotes insulin-induced activation of phosphoinositide 3-kinase in 3T3-L1 adipocytes by inhibiting a negative control mechanism.

Activation of phosphoinositide 3-kinase (PI 3-kinase) is an early event in insulin signal transduction that is blocked completely in adipocytes from insulin-resistant KKAy mice. Treatment of KKAy mice with pioglitazone, an anti-diabetic thiazolidinedione, partially restores insulin-dependent changes in PI 3-kinase. The mechanism of this effect of pioglitazone was investigated using murine 3T3-L1 cells as an experimental model. Insulin and insulin-like growth factor I (IGF-I) each elicited rapid (within 2 min) and large (2-5-fold) increases in PI 3-kinase activity that could be immunoprecipitated using anti-phosphotyrosine (pY) antibodies. Maximal insulin-induced activity of PI 3-kinase in pY-immunoprecipitates was similar in 3T3-L1 adipocytes and mouse adipocytes, but the kinetics of activation differed. Insulin- and IGF-I-induced changes in PI 3-kinase were each half-maximal at 3-5 nM of hormone and were not additive. Increases in both insulin-induced and IGF-I-induced pY-immunoprecipitable PI 3-kinase activity were observed when 3T3-L1 fibroblasts became confluent and when they adopted the adipocyte phenotype. Pioglitazone (10 microM), administered either acutely or chronically to either 3T3-L1 adipocytes or 3T3-L1 fibroblasts, did not greatly alter the kinetics, magnitude or sensitivity of changes in PI 3-kinase elicited by either insulin or IGF-I. In contrast, the attenuation by isoproterenol of insulin-induced changes in PI 3-kinase was prevented in cells pretreated with pioglitazone. This effect of pioglitazone did not involve inhibition of isoproterenol-elicited accumulation of cyclic AMP. Pioglitazone also prevented attenuation of insulin induced changes in PI 3-kinase by cell penetrating analogs of cyclic AMP. Pioglitazone, therefore, has no direct effect on insulin-stimulated PI 3-kinase activity, but interferes with a cyclic AMP-dependent mechanism that normally antagonizes this action of insulin. These data support the proposition that the facilitation of insulin action by pioglitazone involves, at least in part, an inhibition of a negative control mechanism.

1-Phosphatidylinositol 4-Kinase↗
PubMed1994

Pioglitazone promotes insulin-induced activation of phosphoinositide 3-kinase in 3T3-L1 adipocytes by inhibiting a negative control mechanism.

Activation of phosphoinositide 3-kinase (PI 3-kinase) is an early event in insulin signal transduction that is blocked completely in adipocytes from insulin-resistant KKAy mice. Treatment of KKAy mice with pioglitazone, an anti-diabetic thiazolidinedione, partially restores insulin-dependent changes in PI 3-kinase. The mechanism of this effect of pioglitazone was investigated, using murine 3T3-L1 cells as an experimental model. Insulin and insulin-like growth factor I (IGF-I) each elicited rapid (within 2 min) and large (2- to 5-fold) increases in PI 3-kinase activity that could be immunoprecipitated using anti-phosphotyrosine (pY) antibodies. Maximal insulin-induced activity of PI 3-kinase in pY-immunoprecipitates was similar in 3T3-L1 adipocytes and mouse adipocytes, but the kinetics of activation differed. Insulin- and IGF-I-induced changes in PI 3-kinase were each half-maximal at 3-5 nM of hormone and were not additive. Increases in both insulin-induced and IGF-I-induced pY-immunoprecipitable PI 3-kinase activity were observed when 3T3-L1 fibroblasts became confluent and when they adopted the adipocyte phenotype. Pioglitazone (10 microM), administered either acutely or chronically to either 3T3-L1 adipocytes or 3T3-L1 fibroblasts, did not alter greatly the kinetics, magnitude or sensitivity of changes in PI 3-kinase elicited by either insulin or IGF-I. In contrast, the attenuation by isoproterenol of insulin-induced changes in PI 3-kinase was prevented in cells pretreated with pioglitazone. This effect of pioglitazone did not involve inhibition of isoproterenol-elicited accumulation of cyclic AMP. Pioglitazone also prevented attenuation of insulin induced changes in PI 3-kinase by cell penetrating analogs of cyclic AMP. Pioglitazone, therefore, has no direct effect on insulin-stimulated PI 3-kinase activity, but interferes with a cyclic AMP-dependent mechanism that normally antagonizes this action of insulin. These data support the proposition that the facilitation of insulin action by pioglitazone involves, at least in part, an inhibition of a negative control mechanism.

3T3 Cells↗
PubMed1994

The initiation of neurite outgrowth by sympathetic neurons grown in vitro does not depend on assembly of microtubules.

Neurite formation by dissociated chick sympathetic neurons in vitro begins when one of the many filopodia that emanate from the cell body of a neuron is invaded by cytoplasm containing microtubules and other components of axoplasm (Smith, 1994). This study was undertaken to determine whether this process depends on assembly of microtubules. To inhibit microtubule assembly, neurons were grown in medium containing nocodazole or colchicine. In one series of experiments, neurons first were exposed to the microtubule-stabilizing drug, taxol, so that existing microtubules would remain intact while assembly of new microtubules was inhibited. The ability of neurons to form neurites was assessed by time-lapse video microscopy. Neurons subsequently were stained with antibodies against the tyrosinated and acetylated forms of alpha-tubulin and examined by laser confocal microscopy to visualize microtubules. Neurons were able to form short processes despite inhibition of microtubule assembly and they did so in a way that closely resembled process formation in control medium. Processes formed by neurons that had not been pretreated with taxol were devoid of microtubules. However, microtubules were present in processes of taxol-pretreated neurons. These microtubules contained acetylated alpha-tubulin, as is typical of stable microtubules, but not tyrosinated alpha-tubulin, the form present in recently assembled microtubules. These findings show that the initial steps in neurite formation do not depend on microtubule assembly and suggest that microtubules assembled in the cell body can be translocated into developing neurites as they emerge. The results are compatible with models of neurite formation which postulate that cytoplasm from the cell body is transported into filopodia by actomyosin-based motility mechanisms.

Animals↗
PubMed1994

Comparison of serum calcium levels between junior high school children with high-normal and low-normal blood pressure. The child and adolescent blood pressure program.

The objective of this study was to compare serum calcium levels, dietary calcium intake, and urinary calcium excretion between junior high students with high-normal and low-normal blood pressure. The study was conducted in 11- to 14-year-old children recruited after blood pressure screening of 5th to 8th grade Minneapolis and St. Paul Public School students. Comparisons were made between a group of 243 children selected from the upper 15 percentiles of the blood pressure distribution (high-normal group) and 40 children randomly selected from the lowest 10% of the blood pressure distribution (low-normal group). Blood samples were obtained from the participants at clinic visits conducted after school. Calcium measurements were based on the principle that serum calcium is found in three forms: 1) an ionized fraction; 2) a fraction complexed with organic anions such as citrate, phosphate, and lactate; and 3) a protein-bound fraction. Dietary calcium intake was determined from food diaries, and urinary electrolytes were determined in 24-h urine collections. Serum total calcium levels were not significantly different between groups. However, serum ultrafilterable, true ionized, ionized normalized for pH, and complexed calcium levels were significantly greater in the low-normal group. There was no significant difference in 24-h intake of calcium or other nutrients between the groups. The low-normal group excreted significantly more calcium than the high-normal group, but there were no significant differences in sodium, potassium, or chloride excretion. This report of the relation between calcium and blood pressure represents the first study in children or adolescents to include serum, dietary, and urine data.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Anti-neutrophil cytoplasmic antibodies (ANCA) in myelodysplasia and other haematological disorders.

BACKGROUND: Anti-neutrophil cytoplasmic antibodies (ANCA) are typically associated with small vessel vasculitides. They are also found in situations where other autoantibodies are common, sometimes after infections and possibly in individuals who have received multiple blood transfusions. AIMS: The aim of this study was to determine the incidence of ANCA in a variety of haematological disorders, where these predisposing factors may be at work. METHODS: Sera from patients with myelodysplasia (n = 26), acute myeloid leukaemia (AML) (n = 3), and myeloproliferative (n = 25) or lymphoproliferative syndromes (n = 16) were screened for ANCA using a crude neutrophil cytoplasmic extract ELISA and indirect immunofluorescent examination of normal peripheral blood neutrophils. Positive results were confirmed by ELISAs for anti-proteinase 3, anti-myeloperoxidase or anti-elastase antibodies. RESULTS: ANCA were demonstrated in two patients with myelodysplasia, both with chronic myelomonocytic leukaemia and greater than 5% blasts in the bone marrow. Both of these individuals were infected at the time that ANCA were demonstrated and other autoantibodies were present. One of these individuals had never had evidence of any vasculitis; the other probably developed myelodysplasia after treatment with cyclophosphamide for Wegener's granulomatosis. ANCA were demonstrated in one individual with AML secondary to myelodysplasia. ANCA were also found in a patient with lymphoma in whom autoantibodies against red cells and platelets were already noted. ANCA were demonstrated in one further individual with lymphomatoid granulomatosis, a condition that resembles Wegener's granulomatosis clinically and histologically, but which is treated as a lymphoma. No ANCA were present in any of the patients with myeloproliferative syndromes. DISCUSSION: ANCA probably occur secondary to immune dysregulation in myelodysplasia and the lymphoproliferative conditions and they are not necessarily associated with the presence of a vasculitis.

Adult↗
PubMed1994

Squirrel monkey corticosteroid-binding globulin: primary structure and comparison with the human protein.

Squirrel monkey (Saimiri sciureus) corticosteroid-binding globulin (CBG) is the product of a 1.6-kilobase mRNA in the liver. Analyses of two overlapping cDNAs revealed that the squirrel monkey CBG precursor comprises 406 amino acids, the first 22 residues of which exhibit 91% identity with the human CBG leader sequence. The mature form of squirrel monkey CBG, therefore, very likely comprises 384 amino acids and has a polypeptide mol wt of 42,854. Compared to human CBG, the squirrel monkey protein contains an additional residue (threonine) at position 144, and the two proteins exhibit 86% sequence identity if this is taken into account. Squirrel monkey CBG contains five consensus sites for N-glycosylation, four of which are located in analogous positions in human CBG, and has two cysteine residues in the same relative positions as the cysteines in human CBG. Unlike CBG in most other species, squirrel monkey CBG appears to circulate as a dimer, and its affinity for glucocorticoids is remarkably low. We, therefore, expressed cDNAs for human and squirrel monkey CBGs in Chinese hamster ovary (CHO) cells and compared the physico-chemical properties of the products with those of the corresponding serum proteins. Squirrel monkey CBG is produced by CHO cells as a dimer, and its subunit size heterogeneity is similar to that associated with CBG in serum. In addition, the cortisol-binding affinity of squirrel monkey CBG produced by CHO cells is similar to that of the natural protein and is 5- to 8-fold lower than that of natural or recombinant human CBG. Mutants in which a threonine at position 144 was either added to human CBG or subtracted from squirrel monkey CBG were also expressed in CHO cells. This demonstrated that this additional amino acid in the squirrel monkey CBG sequence may actively contribute to its propensity for spontaneous dimerization, but does not account for its relatively low steroid-binding affinity.

Amino Acid Sequence↗