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Biomedical subjects

T R Anderson

Publications and source records attributed to T R Anderson.

At least 19 recordsLinked to original sources
PubMed1993

Immunocytochemical and immunogold localization of two prolactin isoforms in the same pituitary cells and in the same granules in the tilapia (Oreochromis mossambicus).

The tilapia pituitary secretes two forms of prolactin (tPRL) and a single growth hormone (tGH). The tPRLs share only 69% sequence identity and are designated tPRL177 and tPRL188 to indicate the number of amino acid residues in each isoform. Our aim was to develop specific antisera for detection of these three related polypeptides. Ten peptides corresponding to unique epitopes on the tPRLs and two peptides of tGH were synthesized using solid-phase methods, conjugated to carrier proteins, and used as immunogens for antibody production in rabbits. Select antisera for the tPRLs were highly specific, exhibiting only 1% cross-reactivity to the alternate tPRL under noncompetitive ELISA conditions at dilutions used in immunocytochemical analysis. The anti-tGH specifically bound to cells in the proximal pars distalis. Production of both tPRLs by a single cell type was indicated by the binding of both anti-tPRL177 and anti-tPRL188 to the same cells in the rostral pars distalis. Ultrastructural analysis of PRL-producing cells stained sequentially using the two different anti-tPRL antibodies labeled with immunogold of two size classes indicated that both tPRLs appear in the same granules. These findings suggest that the biological significance of two forms of PRL in the adult tilapia is not a function of differential regulation of two different classes of PRL cells or differential release of unique secretory granules.

Amino Acid Sequence↗
PubMed1992

Induction by beta-estradiol of vitellogenin in striped bass (Morone saxatilis): characterization and quantification in plasma and mucus.

Striped bass (Morone saxatilis) were implanted with beta-estradiol to induce the production of vitellogenin, the egg yolk precursor produced by the liver. Electrophoretic analysis revealed that beta-estradiol caused marked production of a plasma protein of apparent molecular mass 170 kDa. Size exclusion chromatography suggested that the estradiol-induced protein circulated as a dimer. This protein was purified from the plasma of estradiol-treated fish by DEAE-agarose column chromatography and used to induce antibodies in rabbits and goats. Western blots revealed that the antiserum bound to the putative vitellogenin in plasma from estradiol-treated fish and adult females, but not with any proteins in male plasma. Western blot of ovarian extract revealed several smaller immunoreactive protein bands and supported the identity of the purified protein as vitellogenin. A competitive ELISA was developed with sensitivity in a range from 8 to 1000 ng/ml. Plasma concentrations of adult females during their spawning migration ranged from 100 to 600 micrograms/ml. Western blot of mucus extract revealed the presence of a 170-kDa protein in vitellogenic female fish along with several minor bands ranging from 50 to 110 kDa. Positive immunoreactivity was present in the surface mucus of all females and in none of the males collected during a spawning migration in the Hudson River.

Animals↗
PubMed1989

Ultrastructural localization of tartrate-resistant, purple acid phosphatase in rat osteoclasts by histochemistry and immunocytochemistry.

The intracellular localization of the tartrate-resistant purple acid phosphatase in osteoclasts of developing rat bone has been determined immunocytochemically using an antiserum to the purified bone-derived purple acid phosphatase. The localization of the immunoreactivity was compared with the results of enzyme histochemistry using p-nitrophenylphosphate as substrate and 10 mM tartrate. Both methods revealed the presence of the enzyme in numerous vesicles of various sizes up to 2-3 microns in diameter and in granules. There was no immunoreactivity in the Golgi apparatus, and tartrate completely inhibited the histochemical activity of this organelle. No consistent extracellular activity could be detected, nor was any reaction product observed at the ruffled border. The localization of the tartrate-resistant purple acid phosphatase in osteoclasts is consistent with an intracellular function for this enzyme.

Acid Phosphatase↗
PubMed1989

Pseudomonas aeruginosa flagellar antibodies in patients with cystic fibrosis.

An enzyme-linked immunosorbent assay specific for flagellum type (a or b) of Pseudomonas aeruginosa was used to detect serum immunoglobulin antibodies in 98 random outpatients and 14 colonized cystic fibrosis patients. Antibodies were detected to both types of flagella in addition to M-2 lipopolysaccharide. Titers to both flagellar antigens (FlAg) were 10 to 100 times higher in cystic fibrosis patients than in random outpatients of a comparable age group. Mean antibody titers against b-type FlAg were 454 for outpatients (ages newborn to 21 years), whereas the mean titer for cystic fibrosis patients (ages 6 to 21 years) was 51,520. Titers against a-type FlAg were generally lower, with mean outpatient titers of 68 and mean cystic fibrosis patient titers of 34,323. Differences were also seen in antibody titer against M-2 lipopolysaccharide, but these differences did not correspond to M-2 FlAg titers. In 98 random outpatients (ages newborn to 86 years), FlAg titers generally increased with age. To demonstrate further specificity of the enzyme-linked immunosorbent assay for flagellum antibody, Western blots were performed with selected high-titer cystic fibrosis patient sera. Sera that had a high titer (greater than 25,600) for b- or a-type FlAg showed a corresponding reactive band. These results demonstrate that flagellum antibodies are produced in humans in response to P. aeruginosa infection.

Adolescent↗
PubMed1989

Flagellar antibody stimulated opsonophagocytosis of Pseudomonas aeruginosa associated with response to either a- or b-type flagellar antigen.

Pseudomonas aeruginosa exhibit one of two flagella types: a homogeneous b type, with molecular weight of 53,000, or a heterogeneous a type (subtypes a0, a1, a2, a3, and a4), with molecular weights ranging from 45,000 to 52,000. Pseudomonas aeruginosa flagellar antiserum was shown to promote uptake of radiolabeled bacteria by mouse polymorphonuclear leukocytes. Bacteria were detected directly associated with washed leukocytes and visualized, by electron microscopy, internalized in polymorphonuclear leukocytes. Phagocytosis was specific for the flagella type (a or b) in that homologous flagella serum enhanced uptake three to four times greater than heterologous serum or normal rabbit serum. An a-type antiserum was shown to enhance phagocytosis of four different a-type strains with varying subantigen types, indicating the presence of a common cross-reactive a0 antigen in this flagella type. Phagocytic killing of internalized bacteria was not seen with the addition of only flagellar antiserum.

Animals↗
PubMed1988

Enzyme-linked immunosorbent assay for detection of Pseudomonas aeruginosa H (flagellar) antigen.

An enzyme-linked immunosorbent assay (ELISA) with goat anti-rabbit IgG conjugated to peroxidase was used to test for the two antigen types of Pseudomonas aeruginosa: b (homogeneous) and a (heterogeneous) which contains the common subantigen (a0) and combinations of subtypes (a, a2 a3 a4). Preparations of b-type flagellar antigen could be distinguished from a-type by using b-adsorbed antisera titers as reciprocals of endpoint dilutions exceeding one million. Extracts from nonflagellated bacteria or purified lipopolysaccharides from the same strain were used as controls, which showed only background activity. Unknown flagellar antigen was determined using both isolated antigen preparations and formalin-killed bacterial cells. The ELISA procedure proved much more sensitive than the slide agglutination procedure: whereas nine of 18 strains tested did not react in the slide agglutination procedure all 18 strains were definitively typed as a or b strains with the ELISA. The ELISA also revealed the presence of a dominant, cross-reacting epitope (a0) in the heterogeneous a-type flagella using either isolated antigen or intact cells.

Agglutination Tests↗
PubMed1987

Interactions among prolactin, epidermal growth factor, and proinsulin on the growth and morphology of the pigeon crop-sac mucosal epithelium in vivo.

The local pigeon crop-sac assay was used to test the direct effects of epidermal growth factor (EGF) and several other growth factors and hormones on the growth of mucosal epithelial cells in vivo. Insulin, relaxin, multiplication-stimulating activity, proinsulin, and platelet-derived or fibroblast growth factors had no direct stimulatory activity by themselves. Human insulin-like growth factor I and EGF caused dose-related stimulation, but were less effective than ovine (o) PRL. Insulin, platelet-derived growth factor, fibroblast growth factor, and multiplication-stimulating activity did not affect the proliferative response to oPRL when injected along with the hormone. Proinsulin augmented the direct mitogenic action of oPRL, but not that of EGF. When EGF was injected locally with PRL no interaction occurred even though both hormones were independently mitogenic. A single sc injection of a high dose of oPRL (0.5 mg) given at a site distant from the crop-sac had no effect on the mucosal epithelial cells, but it caused a significant increase in their response to the direct action of oPRL. However, the systemically acting PRL did not affect the direct local effect of EGF. The gross pattern of mucosal cell proliferation induced by PRL (parallel ridges resembling gastric rugae) differed from that produced by EGF (usually irregular patches), and PRL, but not EGF, promoted the accumulation of lipid droplets in the stimulated mucosal epithelial cells. Furthermore, the crop mucosal cells in the midline of the organ are unresponsive to PRL, but were highly responsive to EGF. These results indicate that although EGF and PRL are both mitogenic to crop mucosal epithelial cells, the former does not mimic the latter. They produce a different growth pattern, and EGF fails to promote differentiation of the resultant daughter cells. Moreover, EGF is a less specific mitogen than is PRL, and the two hormones do not interact in their mitogenic effects.

Animals↗
PubMed1986

The human growth hormone gene is negatively regulated by triiodothyronine when transfected into rat pituitary tumor cells.

Human growth hormone (hGH) gene expression and regulation by thyroid and glucocorticoid hormones were examined after its stable introduction into rat anterior pituitary tumor cells. Transcripts from the hGH gene were correctly initiated and could be positively regulated by glucocorticoids. In contrast to the endogenous rat GH gene and a transfected, modified rat GH gene, the hGH gene was negatively regulated by triiodothyronine. Analysis of hGH gene 5'-flanking deletions indicated that a distal regulatory element is required for efficient transcription. Thus promoter, glucocorticoid, and thyroid hormone control elements are present on the transfected hGH gene, whose expression may be negatively controlled by thyroid hormone.

Animals↗
PubMed1986

Purification and characterization of purple acid phosphatase from developing rat bone.

Tartrate-resistant acid phosphatase active on nucleoside di- and triphosphate substrates was isolated from developing rat bone and purified 2500-fold. The enzyme concentration had a purple coloration and activity that was sensitive to reducing agents. Mild reducing agents such as ferrous ion and ascorbic acid caused loss of purple color and increased activity toward substrates severalfold; however, a strong reductant such as dithionite caused loss of both color and activity which were partially restored by addition of ferrous ion and ascorbic acid. Enzyme activity was homogeneous with protein during the final gel permeation steps of chromatography and gave an apparent molecular size of about 40,000 Da. Determination of iron in the most pure preparation revealed the presence of 1.3 atoms of iron per molecule of the tartrate-resistant enzyme E2. Other properties of the purified enzyme include a pI of approximately 9.5 and sensitivity to inhibition by ions of copper, zinc, fluoride, and molybdate. Antibody prepared to the pre-concanavalin A (Con A)-Sepharose purified enzyme reacted with all protein from the Con A step, but it did not react with tartrate-sensitive acid phosphatase from rat bone or with potato acid phosphatase. Purple acid phosphatase from rat bone has many properties that parallel the iron-containing purple acid phosphatases from rat spleen, bovine spleen, and pig uterine secretions.

Acid Phosphatase↗
PubMed1986

The 5'-flanking sequences of the human growth hormone gene contain a cell-specific control element.

The 5'-flanking sequences of the human growth hormone (hGH) gene contain a cell-specific control element. Hybrid genes containing truncated 5'-flanking DNA fragments from the hGH gene fused to the chloramphenicol acetyl transferase (cat) gene were examined using transient transfection of rat anterior pituitary (GC) and nonpituitary cell lines (HeLa, Rat 2, and KB); preferential expression of these gene hybrids was only observed in GC cells. Deletions through the 5'-flanking sequences of the hGH gene revealed that the region containing nucleotides -230 to -180 is required for efficient cat gene expression in GC cells. This region of DNA is highly homologous to a region of the rGH gene that contains a tissue-specific control element. A hybrid gene containing the tissue-specific control element, but lacking the proximal promoter elements, of the hGH gene upstream from the promoter of the thymidine kinase gene (TKp) from herpes simplex virus ligated to the cat gene produced more CAT activity than the TKp.cat gene in GC cells but not in HeLa cells. These data suggest that the tissue-specific control element located in the 5'-region of the hGH gene can act in the presence of a heterologous promoter and is specific for expression in pituitary cells.

Acetyltransferases↗
PubMed1984

Prolactin's mitogenic action on the pigeon crop-sac mucosal epithelium involves direct and indirect mechanisms.

It has long been known that prolactin (PRL) induces proliferation of the mucosal epithelial lining of the pigeon crop-sac. This system was used to study possible interactions of other hormones with PRL's mitogenic effect directly on the crop-sac cells. Injection of a sheep pituitary powder into the loose skin between the leg and abdomen of pigeons increased the responsiveness of the crop-sac to direct, local application of PRL. Similar systemic injections of anterior pituitary hormones, ACTH and TSH (alone or together), or a combination of LH and FSH, were without effect on the local response to PRL. However, systemically injected PRL and GH resulted in a dramatic augmentation of the response of the organ to local injection of PRL. PRL was more potent than GH in this regard. These results suggest that PRL has at least two modes of action as a mitogen on this epithelium--its well-known direct action on the cells and an indirect action as well that is possibly mediated by the secretion of another factor into the blood stream. Because it was previously found that somatomedin-like molecules, including proinsulin, act synergistically with PRL to promote proliferation of these cells when coinjected locally with PRL (T. R. Anderson, J. Rodriguez , D. S. Pitts , E. M. Spencer, and C. S. Nicoll , 1983, In "Insulin-like Growth Factors/Somatomedins," de Gruyter , Berlin), The effects of systemic injections of proinsulin were also tested. Proinsulin had a potentiating effect similar to that of the pituitary powder and of GH or PRL. These results are consistent with the following interpretations: When PRL promotes crop-sac proliferation to produce crop-milk in pigeons and doves, the hormone acts through at least two mechanisms. One of these is a direct effect on the crop-sac, which sensitizes the mucosal epithelium to the mitogenic action of a somatomedin-like growth factor. The second effect is an increase in the production and/or secretion of the factor, which then acts synergistically with PRL to promote proliferation of the crop-sac mucosal cells. More direct evidence for this proposal was provided by the finding that serum from saline- or PRL-treated pigeons did not stimulate crop-sac mucosal growth when it was injected locally. However, the serum from PRL-treated birds had a much higher level of an activity that augmented the effects of PRL when the sera were locally injected in combination with PRL.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Inhibition by dithionite and reactivation by iron of the tartrate-resistant acid phosphatase in bone of osteopetrotic (ia) rats.

The staining intensity and inhibitor sensitivity of acid phosphatase activity was determined histochemically in various tissues of normal and ia rat pups by the use of freeze-dried whole body sections. Activity was determined using alpha-naphthylphosphate as substrate and hexazonium pararosaniline as coupler. Sections from ia rats (6 and 24 days old) showed markedly higher enzyme activity in bone than sections from normal littermates. However, there were no differences between ia and normal pups in acid phosphatase activity in soft tissues and developing teeth. Preincubation of sections with 1-100 mM sodium dithionite (an iron-binding agent) caused a dose-related inhibition of enzyme activity in bone of ia and normal pups, but only slight inhibition of activity in soft tissues. Partial restoration of the dithionite-inhibited activity in bone was achieved by subsequent preincubation in 1 mM FeCl2. Addition of 100 mM sodium tartrate to the staining solution of non-preincubated sections caused almost complete inhibition of activity in soft tissues and the developing teeth but no inhibition of the activity in bone that was sensitive to sodium dithionite. These data indicate a) that sodium dithionite can be used as a specific histochemical inhibitor of the tartrate-resistant acid phosphatase and b) that the source of increased acid phosphatase activity in bone from ia rats is mostly from the tartrate-resistant acid phosphatase.

Acid Phosphatase↗
PubMed1982

Effects of iron and ascorbic acid on acid phosphatases of the enamel organ of rat molars.

Protein extracts from 6- to 11-day-old rat enamel organs were applied to columns of carboxymethyl-52 cellulose. Protein eluted from the columns was assayed for acid phosphatase activity with substrates para-nitrophenylphosphate (p-NPP), beta-glycerolphosphate (beta-GP), ATP and phosphocasein. A weakly-bound peak of activity (A) emerged first which was insensitive to stimulation by iron and ascorbic acid. This enzyme hydrolysed only the phosphomonoester substrates (p-NPP and beta-GP). A strongly bound peak of activity (B) emerged later and was completely separated from the first activity. It hydrolysed all substrates except beta-GP, and was stimulated at least 10-fold by 0.1 mM ferrous ion (Fe2+) in the presence of a strong reducing agent (1.0 mM ascorbic acid). Both substances were more effective as stimulators when used together than they were when each was used separately. Dependency on these co-factors for the development of full activity increased with purification, especially when phosphocasein was substrate. The results were similar for each age of rat used. These properties of enzyme B are parallel with those of the acid phosphoprotein phosphatases of liver and spleen, and the tartrate-resistant acid phosphatase of rat bone. We conclude that enzyme B requires iron and a reducing agent for full activity and has properties that distinguishes it from the classical acid phosphatases (E.C. 3.1.3.2.).

Acid Phosphatase↗
PubMed1982

Separation and partial purification of acid phosphates of the enamel organ of rat molars.

At least two types of acid phosphatases with markedly different properties were separated from the enamel organ of rat molar tooth buds. One enzyme (A) bound weakly to the CM-cellulose column and was eluted with a combined linear salt and pH gradient; another enzyme (B) bound strongly to the column and was eluted with a second linear salt gradient at constant pH. Enzyme A was identified as a phosphomonoester hydrolase (3.1.3.2) similar to the lysosomal enzyme of soft tissues and the tartrate-sensitive enzyme of bone. Enzyme B did not hydrolyse aliphatic monophosphate ester substrates but, like enzyme A, it did split the aryl monophosphate ester substrate, para-nitrophenylphosphate, as well as the phosphate esters of casein and the acid anhydride substrates, ATP and inorganic pyrophosphate. This enzyme is similar to the low molecular weight tartrate-resistant acid phosphatases of bone and soft tissues.

Acid Phosphatase↗
PubMed1982

Paraplegic use of the Orlau swivel walker: case report.

This case report describes the characteristics, biomechanics, fitting, training, advantages, and disadvantages of the Orlau swivel walker. Advantages of the walker include stability, easy mobility, and the ability of the wearer to ambulate with free hands. Disadvantages include slow velocity of locomotion, comparative metabolic inefficiency, awkwardness of getting into and from the standing position, and inability to be used on uneven surfaces. In a comparison of the metabolic costs and efficiency of the Orlau swivel walker, bilateral knee-ankle-foot orthoses, and a wheelchair, the swivel walker was least efficient (1.9ml O2/kg/m) in comparison to the knee-ankle-foot orthoses (0.9ml O2/kg/m) and the wheelchair (0.18ml O2/kg/m). However, in evaluating a means of locomotion, various factors besides metabolic costs need to be considered; these include efficiency, velocity, the physiologic and psychologic benefits of standing, and the patient's needs. When considering all these factors for an individual patient, the swivel walker may be an effective device for meeting the needs of many individuals with spinal cord injury.

Adult↗