Workshop on bioanalytical methods validation for macromolecules: summary report.
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Biological Assay↗
Biomedical subjects
K J Miller
Publications and source records attributed to K J Miller.
Synthesis of a low-molecular-weight form of exopolysaccharide by Bradyrhizobium japonicum USDA 110.
A novel extracellular low-molecular-weight polysaccharide was detected as a contaminant within extracellular cyclic beta-1,6-beta-1,3-glucan preparations from Bradyrhizobium japonicum USDA 110 cultures. Compositional analysis, methylation analysis, and nuclear magnetic resonance analysis revealed that this low-molecular-weight polysaccharide was composed of the same pentasaccharide repeating unit previously described for the high-molecular-weight form of the exopolysaccharide (EPS) synthesized by B. japonicum strains. Mass spectrometry analysis indicated that the size of this low-molecular-weight form of EPS was consistent with a dimeric form of the pentasaccharide repeating unit.
Bradyrhizobium↗
Inducible nitric oxide synthase in P11 cells: expression in the presence of interferon-gamma, lipopolysaccharide, and modified serum.
P11 cells, derived from the transplantahle rat pituitary tumor 7315a, have been used previously ias a model system to study the regulation of serotonin2A (5-HT2A) receptor expression. As our laboratory has been interested in characterizing the interactions between the 5-HT2A receptor and inducible nitric oxide synthase (iNOS), we have analyzed the Pl I cell line for iNOS expression. Treatment of P ll cells with interferon-gamma and lipopolysaccharide resulted in a 23-fold increase in nitrite production and induced expression of iNOS protein. The increase in nitrite levels was attenuated by the non-selective nitric oxide synthase (NOS) inhibitor N i-nitro-L-arginine methyl ester, hut not the neuronal NOS inhibitor 7-nitroindazole. Typically, Pl 11 cells have been grown in either charcoal-stripped or dialyzed serum-containing medium. We have observed that Pl 1 cells grown under these culture conditions express basal iNOS activity, as evidenced by a 5-fold increase in nitrite accumulation over a 48-hr period, compared with that in cells grown in non-modified serum, which was inhibited by the selective iNOS inhibitor L.N6-(1-iminoethyl)-lysine. Conditioned medium from Pll cells was ahle to stimulate nitrite accumulation in C6 glioma cells, suggesting that the Pl I cells may produce a pro-inflammatory-like factor. As pro-inflammatory cytokines have been shown to modify hormone secretion from the anterior pituitary, the P11 cell line may be a useful in vitro model by which to characterize the function of cells from this organ. In addition, our data suggest that alteration of the microenvironment of the anterior pituitary may result in iNOS expression, possibly altering the function of the hypothalamic-pituitary-adrenal axis.
Animals↗
Comparisons of body image dimensions by race/ethnicity and gender in a university population.
OBJECTIVE: We examined affective and cognitive components of body image related to physical appearance, weight, and health among 120 university men and women of three racial/ethnic groups: African American, European American, and Latino/a American. METHOD: Participants completed a Background Information Sheet, the Multidimensional Body-Self Relations Questionnaire (MBSRQ), the Body-Esteem Scale (BES) with additional items, and the Balanced Inventory of Desirable Responding (BIDR). We tested for effects of race/ethnicity and gender on the body image measures while controlling for age, body size, social desirability, and socioeconomic status (SES). RESULTS: African Americans reported greatest body satisfaction and least overestimation of weight. Latino/a Americans were equal to or higher than European Americans on all indices. Gender differences occurred on global body image, weight concerns, fitness, and health. There were no Gender x Race/Ethnicity interactions. DISCUSSION: This pattern of racial/ethnic and gender differences shows a need for exploring a wider range of culturally relevant body image dimensions.
Adolescent↗
Effects of metformin on intestinal 5-hydroxytryptamine (5-HT) release and on 5-HT3 receptors.
Nearly 30% of patients treated with metformin experience gastrointestinal side effects. Since release of 5-hydroxytryptamine (5-HT) from the intestine is associated with nausea, vomiting, and diarrhea, we examined whether metformin induces 5-HT release from the intestinal mucosa. In 40% of tissue biopsy specimens of human duodenal mucosa, metformin (1, 10, and 30 microM) caused an increase in 5-HT outflow by 35, 70, and 98%, respectively. Peak increases in 5-HT outflow were observed after 10-15 min exposure to metformin, returning to baseline levels after 25 min. Tetrodotoxin (1 microM) reduced by about 50% the metformin-evoked increase in 5-HT outflow (P<0.05). Metformin-evoked release was not affected by scopolamine + hexamethonium, propranolol, the 5-HT3 receptor antagonist dolasetron, naloxone, or the NK1 receptor antagonist L703606. In the presence of tetrodotoxin (1 microM), somatostatin (1 microM) further reduced metformin-induced 5-HT release by 15-20%. In view of the 5-HT releasing effects of selective 5-HT3 receptor agonists to which metformin (N-N-dimethylbiguanide) is structurally related, we investigated whether metformin directly interacts with 5-HT3 receptors. Receptor binding (inhibition of [3H]-GR65630 binding) and agonist effects (stimulation of [14C]-guanidinium influx) at 5-HT3 receptors were studied in murine neuroblastoma N1E-115 cells, which express functional 5-HT3 receptors. Metformin up to 0.3 mM failed to inhibit [3H]-GR65630 binding and to modify displacement of [3H]-GR65630 binding induced by 5-HT. 5-HT (3 microM) stimulated the influx of [14C]-guanidinium in intact N1E-115 cells. Metformin up to 1 mM failed to modify basal influx, 5-HT-induced influx, and 5-HT+ substance P-induced influx of [14C]-guanidinium. Our results indicate that metformin induces 5-HT3 receptor-independent release of 5-HT from human duodenal mucosa via neuronal and non-neuronal mechanisms. Part of the gastrointestinal side effects observed during treatment with metformin could, thus, be produced by the release of 5-HT and other neurotransmitter substances within the duodenal mucosa.
Animals↗
Bioanalytical method validation--a revisit with a decade of progress.
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Biological Assay↗
Functional characterization of the P2X(4) receptor orthologues.
1. The aim of this study was to functionally characterize the recombinant mouse P2X(4) receptor and to compare its pharmacological properties with those of the human and rat orthologues. 2. Whole cell recordings were made from rafts of HEK-293 cells stably expressing recombinant mouse, rat or human P2X(4) receptors, using Cs-aspartate containing electrodes (3 - 8 MOmega) in a HEPES-buffered extracellular medium. 3. The agonist potency of ATP at the three species orthologues was similar, with mean EC(50) values of 2.3 microM, 1.4 microM and 5.5 microM, respectively. 4. Adenosine-5'-tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X(4) receptors (EC(50)=2.6 and 3.0 microM), but was significantly less potent at the rat orthologue (EC(50)=20.0 microM). alpha,beta-methylene adenosine-5'-triphosphate (alpha,beta-meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X(4) and 24% at the human P2X(4) receptor. 5. In contrast to the other species orthologues, alpha,beta-meATP failed to elicit a significant agonist response at rat P2X(4) receptors, and was found to act as an antagonist, with an IC(50) of 4.6 microM, against 10 microM ATP. 6. Mouse P2X(4) receptors were found to be sensitive to the antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (IC(50)=10.5 microM), as were human P2X(4) receptors (IC(50)=9.6 microM). The rat receptor however, showed a low sensitivity to PPADS (IC(50)>100 microM). 7. All three orthologues were relatively suramin-insensitive (IC(50)>100 microM) and insensitive to 1-[N, O-Bis(5-isoquinoline sulphonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinoline sulphonamide (KN-62; IC(50)>3 microM). 8. Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X(4) receptor, and differ markedly from the rat receptor.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Hepatocyte growth factor affects satellite cell activation and differentiation in regenerating skeletal muscle.
Hepatocyte growth factor (HGF) is the only known growth factor that activates quiescent satellite cells in skeletal muscle. We hypothesized that local delivery of HGF may enhance regeneration after trauma by increasing the number of myoblasts available for restoring normal tissue architecture. Injection of HGF into muscle at the time of injury increases myoblast number but does not enhance tissue repair as determined using quantitative histological analyses. Rather, depending on the dose and the timing of HGF administration relative to the injury, regeneration can be inhibited. The greatest inhibitory effect is observed when HGF is administered on the day of injury and continued for 3 days, corresponding to the time when satellite cell activation, proliferation, and early differentiation normally occur. To establish a mechanism for this inhibition, we show that HGF can act directly on primary muscle cells to block differentiation. These results demonstrate that 1) exogenous HGF synergizes with factors in damaged muscle to increase myoblast number, 2) regeneration is not regulated solely by myoblast number, and 3) HGF inhibits muscle differentiation both in vitro and in vivo.
Animals↗
Welcome to the real world: reflections on teaching and administration.
The author compares his former position as an assistant professor in a program preparing future teachers of deaf and hard of hearing students with his present position as an administrator of a public school program serving these students. He maintains that in some ways, teacher training programs in deafness and the public school settings hiring these graduates are separate worlds. The emphasis in teacher training programs appears to be on preparing graduates to work with deaf students in self-contained or residential school settings even though most teaching positions are with hard of hearing students mainstreamed in public schools. Other important areas, such as collaboration with general education teachers, litigation, parental relationships, and individualized education programs, seem to be overlooked by teacher training programs. The author employs the mockingbird metaphor from the novel To Kill A Mockingbird (Lee, 1960) to highlight differences between teacher training programs and public school settings, while making recommendations for strengthening connections between the two.
Deafness↗
Effect of low water activity on staphylococcal enterotoxin A and B biosynthesis.
Staphylococcus aureus strains FDA 743 (staphylococcal enterotoxin A [SEA]-producing), FDA 778 (staphylococcal enterotoxin B [SEB]-producing), and S6 (SEA- and SEB-producing) were used to examine the effect of low water activity (a(w)) on SEA and SEB biosynthesis. In this report, we show that SEB production is more sensitive to low a(w) than SEA production. We also show that when proline is available as a compatible solute for S. aureus, SEB production is significantly stimulated at low a(w). This stimulatory effect was not observed when other compatible solutes (i.e., glycine betaine or carnitine) were added to low a(w) growth media. Finally, Northern blot analysis revealed that the stimulation of SEB production at low a(w) by added proline occurs at the level of transcription.
Blotting, Northern↗
Effects of growth at low water activity on the thermal tolerance of Staphylococcus aureus.
Staphylococcus aureus is the most osmotolerant foodborne pathogen, and outbreaks of staphylococcal food poisoning are often linked to foods of reduced water activity (a(w)) values. While it is generally known that the thermal tolerance of microorganisms increases as the a(w) of the heating menstruum is decreased, surprisingly little research has examined the influence of growth medium a(w) on microbial thermal tolerance. In the present study, we show that growth of S. aureus at an a(w) value of 0.94 leads to the development of dramatically enhanced thermal tolerance (i.e., less than 1 log reduction after heating for 20 min at 60 degrees C). We further show that the identity of the accumulated compatible solute within cells grown at low a(w) can also influence the overall level of thermal tolerance of S. aureus. Finally, we provide evidence that the synthesis of general stress and/or osmotic stress proteins is required for the development of enhanced thermal tolerance of S. aureus at low a(w).
Heat-Shock Proteins↗
Liquid ventilation: a case study.
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Acute Disease↗
Cloning, sequencing, and characterization of the cgmB gene of Sinorhizobium meliloti involved in cyclic beta-glucan biosynthesis.
Periplasmic cyclic beta-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation. In Sinorhizobium meliloti (also known as Rhizobium meliloti), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an S. meliloti Tn5 insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic beta-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346-6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn5 flanking sequences (amplified by inverse PCR) as a probe, an S. meliloti genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb HindIII-EcoRI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transform Rhizobium leguminosarum bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic beta-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an S. meliloti phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of S. meliloti is designated the cgm (cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB.
Base Sequence↗
Systemic administration of the NF-kappaB inhibitor curcumin stimulates muscle regeneration after traumatic injury.
Skeletal muscle is often the site of tissue injury due to trauma, disease, developmental defects or surgery. Yet, to date, no effective treatment is available to stimulate the repair of skeletal muscle. We show that the kinetics and extent of muscle regeneration in vivo after trauma are greatly enhanced following systemic administration of curcumin, a pharmacological inhibitor of the transcription factor NF-kappaB. Biochemical and histological analyses indicate an effect of curcumin after only 4 days of daily intraperitoneal injection compared with controls that require >2 wk to restore normal tissue architecture. Curcumin can act directly on cultured muscle precursor cells to stimulate both cell proliferation and differentiation under appropriate conditions. Other pharmacological and genetic inhibitors of NF-kappaB also stimulate muscle differentiation in vitro. Inhibition of NF-kappaB-mediated transcription was confirmed using reporter gene assays. We conclude that NF-kappaB exerts a role in regulating myogenesis and that modulation of NF-kappaB activity within muscle tissue is beneficial for muscle repair. The striking effects of curcumin on myogenesis suggest therapeutic applications for treating muscle injuries.
Animals↗
Serotonin 5-HT2A receptor activation inhibits cytokine-stimulated inducible nitric oxide synthase in C6 glioma cells.
C6-glioma cells endogenously express both 5-HT2A receptors and inducible nitric oxide synthase (iNOS). iNOS can be induced by transcriptional activation to produce nitric oxide (NO) in response to a challenge with the pro-inflammatory cytokines TNF-alpha and IFN-gamma. Experiments were conducted to determine whether 5-HT2A receptor activation could modify the production of NO in response to the inducing agents. 1 muM DOI produced a dose-dependent inhibition of the cytokine-inducted nitrite levels of 40% which was inhibited by spiperone and ritanserin. In addition, the DOI-mediated decrease was prevented by the PKC inhibitor chelerythrine (100 nM). The effectiveness of DOI was lost when added more than two hours after the addition of inducing agent, suggesting that DOI was regulating iNOS at the level of transcription rather than post-translationally. We suggest that there is a link between the serotonergic system and NO-mediated immune responses in the brain.
Amphetamines↗
Prevention of DOI-mediated wet dog shakes by inhibitors of nitric oxide synthase.
The purpose of this study was to determine if (+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced wet dog shakes (WDS) required the involvement of nitric oxide synthases (NOS). Systemic administration of the general NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), but not its d-isomer, and the neuronal NOS (nNOS) inhibitor 7-nitroindazole completely blocked DOI-mediated WDS in a dose dependent manner. The data provides evidence that serotonin 5HT2 receptors are coupled to nNOS activation in the rat brain.
Amphetamines↗
Cibacron blue allosterically modulates the rat P2X4 receptor.
We have used whole-cell patch clamp electrophysiology to characterise the actions of the P2 antagonist, cibacron blue, on the rat recombinant P2X4 receptor, stably expressed in human embryonic kidney 293 (HEK293) cells. In single cells, adenosine triphosphate (ATP) evoked inward currents, but the response was subject to considerable run down which precluded obtaining quantitative data. However, when recordings were made from cells that were part of a group of 20-40 electrically coupled cells (cell rafts), run-down of current was not observed and reproducible responses could be obtained. When studied using cell rafts, cibacron blue was a weak antagonist of the rat P2X4 receptor (IC50 > 300 microM) when co-applied with ATP. However, when cell rafts were preincubated with low concentrations of cibacron blue (3-30 microM) for 5 min prior to ATP addition, cibacron blue increased responses to ATP by increasing its potency (up to 4-fold) without affecting the maximum current. Potentiation of ATP-evoked currents was also observed following washout of high, inhibitory concentrations of cibacron blue (300 microM). In contrast to these effects on P2X4 receptors, cibacron blue inhibited the ATP-induced response in both single cells and rafts of HEK293 cells expressing the P2X2 receptor (IC50 approximately 600-800 nM). The effects of cibacron blue on the P2X4 receptor were quantitatively similar to those of Zn2+ which also increased ATP-evoked currents by decreasing the EC50 of ATP (up to 3.5-fold). These data are consistent with the concept that cibacron blue, like zinc, allosterically regulates the function of the P2X4 receptor.
Adenosine Triphosphate↗
Evidence for P2X3 receptors in the developing rat brain.
P2X receptor-mediated responses to the ATP analogue, alpha,beta-methylene ATP, in rat brain cannot be accounted for by the receptor proteins known to be present. Such experiments are often performed on cells from neonates and, since differential developmental regulation of P2X1 and P2X2 receptor messenger RNAs has already been demonstrated, this is likely to be the case for other P2X receptors. This study was designed to address the possible existence of alpha,beta-methylene ATP-sensitive P2X3 receptors in rat brains of various ages using a P2X3 receptor-selective antibody. P2X3 receptor protein was found in discrete regions of the embryonic (E16) and neonatal rat brain (P7 and P14) but was not detectable in adult animals. This is the first demonstration of the presence of these receptors in brains from various ages of rat and the differential expression of these receptors in neonates may account for some reported electrophysiological responses to alpha,beta-methylene ATP.
Age Factors↗