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Biomedical subjects

K M Knigge

Publications and source records attributed to K M Knigge.

At least 19 recordsLinked to original sources
PubMed1991

Neuronal NADPH diaphorase is a nitric oxide synthase.

NADPH diaphorase histochemistry selectively labels a number of discrete populations of neurons throughout the nervous system. This simple and robust technique has been used in a great many experimental and neuropathological studies; however, the function of this enzyme has remained a matter of speculation. We, therefore, undertook to characterize this enzyme biochemically. With biochemical and immunochemical assays, NADPH diaphorase was purified to apparent homogeneity from rat brain by affinity chromatography and anion-exchange HPLC. Western (immunoblot) transfer and immunostaining with an antibody specific for NADPH diaphorase labeled a single protein of 150 kDa. Nitric oxide synthase was recently shown to be a 150-kDa, NADPH-dependent enzyme in brain. It is responsible for the calcium/calmodulin-dependent synthesis of the guanylyl cyclase activator nitric oxide from L-arginine. We have found that nitric oxide synthase activity and NADPH diaphorase copurify to homogeneity and that both activities could be immunoprecipitated with an antibody recognizing neuronal NADPH diaphorase. Furthermore, nitric oxide synthase was competitively inhibited by the NADPH diaphorase substrate, nitro blue tetrazolium. Thus, neuronal NADPH diaphorase is a nitric oxide synthase, and NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide.

Amino Acid Oxidoreductases↗
PubMed1991

Monitoring of specific antibodies to human immunodeficiency virus structural proteins: clinical significance.

Levels of antibodies to six major structural proteins of human immunodeficiency virus type 1 (gp120, gp41, p66, p31, p24, and p17) were assessed in serial samples from 22 persons with severe hemophilia (16 asymptomatic and 6 who developed acquired immunodeficiency syndrome [AIDS] or AIDS-related complex) with an automated dot blot assay using purified recombinant antigens. High and sustained levels of antibody to gp120, gp41, and p31 were found in all patients irrespective of their clinical condition for 4 to 6 years after seroconversion. In contrast, immune response to p66 and p17 was significantly lower in symptomatic patients. Over time, the levels of these two antibodies, as well as anti-p24, decreased and tended to become undetectable. Abnormal immune response and low levels of antibody to p66 and p17 are early indications of rapid clinical progression.

Adult↗
PubMed1989

Topography of oxytocin and vasopressin neurons in the forebrain of Equus caballus: further support of proposed evolutionary relationships for proopiomelanocortin, oxytocin and vasopressin neurons.

The present study describes the topography of immunoreactive (ir) oxytocin (OXY) and vasopressin (AVP) neurons in the forebrain of Equus caballus and the coexistence of ir proopiomelanocortin (POMC)-derived peptides in the same cells. These data are compared to those for other mammalian species and the possible significance of species variations is considered. As expected, magnocellular neurons of the equine hypothalamus, which contain ir OXY or AVP, have prominent discernible projections to the neurohypophysis. Further, as in other mammalian species, the field of ir OXY perikarya generally extends rostral and dorsal to groups of ir AVP cell bodies, and caudal projections from OXY neurons appear to be more numerous than ir AVP projections to the brainstem and/or spinal cord. Interestingly, however, the brain of E. caballus also contains: (1) perikarya staining for OXY in the arcuate nucleus, (2) ir AVP and OXY cell bodies in the suprachiasmatic nucleus, and (3) neurons in the supraoptic and paraventricular nuclei that stained for beta-endorphin but not for other posttranslational products of POMC or dynorphin. These results give further credence to the proposal that there is an evolutionary relationship between OXY-, AVP- and POMC-producing hypothalamic neurons. Whether or not species differences in peptide coexistence reflect functional differences in neuronal populations or species differences in residual genomic expression by these neuroendocrine cells warrants further investigation.

Animals↗
PubMed1989

Interaction of putative vasopressin receptor proteins of rat brain and bovine pituitary gland with an antibody against a nanopeptide encoded by the reverse message of the complementary mRNA to vasopressin.

An antibody directed against the reverse message of the complementary mRNA for arginine vasopressin was demonstrated to be immunoreactive with 62 and 55 kdalton proteins, obtained by affinity chromatography of rat brain and bovine posterior pituitary extracts and believed to be associated with the vasopressin binding site. A similar pattern of immunoreactivity was seen with an anti-idiotypic antibody for arginine vasopressin.

Animals↗
PubMed1988

An alternate method utilizing small quantities of ligand for affinity purification of monospecific antibodies.

An alternate method was designed to couple a limited quantity of protein to an affinity support when a conventional technique was unsuccessful. This was achieved through the introduction of a small number of sulfhydryl groups to the ligand by reaction with 2-iminothiolane which resulted in a limited number of reactive sites on the protein. Amino groups on an AH-Sepharose 4B matrix were linked to sulfhydryl groups on the ligand using the heterobifunctional agent m-maleimidobenzoyl sulfosuccinimide ester (sulfo-MBS). This method was employed to prepare an affinity support using a cytosolic protein that activates glyceraldehyde-3-phosphate dehydrogenase as a ligand. Monospecific antibody purified from the affinity column recognized only this protein on a Western blot of a cytosolic extract of kidney epithelial cells.

Animals↗
PubMed1988

Immunocytochemistry of a vasopressin (AVP) receptor with anti-idiotype antibody: inhibition of staining with a peptide (PVA) encoded by an RNA that is complementary to AVP mRNA.

Immunocytochemical staining of putative presynaptic (auto-) receptors associated with vasopressin (AVP) neurons by anti-idiotype antibody can be markedly reduced or abolished by preincubation of the antibody with peptide PVA. This peptide, Ser-Ser-Trp-Ala-Val-Leu-Glu-Val-Ala, represents amino acids encoded by a nucleotide sequence complementary to the mRNA code of AVP. These results suggest that PVA may have some binding characteristics similar to the AVP autoreceptor.

Animals↗
PubMed1988

Kinetics of a novel cytosolic protein during the onset of renal epithelial cell growth.

Exposure of monkey kidney epithelial cells (BSC-1 line) to medium with a reduced K concentration (3.2 mM) stimulated growth and transiently activated glyceraldehyde-3-phosphate dehydrogenase (G3PD). The increase in enzyme activity was mediated by a cytosolic modifier protein that was purified using affinity and size-exclusion chromatography, and anion-exchange high-performance liquid chromatography. The apparent molecular mass of the protein was 62 kDa. A monospecific antibody to the protein was prepared from rabbit antiserum and used as an immunoprobe. Immunocytochemical staining and Western blotting revealed that the protein was a normal constituent of the cytosol and that it accumulated in cells exposed to low-K medium. A quantitative enzyme-linked immunosorbent assay showed that the amount of modifier protein increased progressively for up to 2 h in cells exposed to low-K medium, and then returned to the control value, a kinetic profile similar to that observed for G3PD activity. These results indicate that the modifier protein is a constituent of renal epithelial cells and accumulates transiently in the cytosol where it could regulate G3PD activity during the onset of growth induced by the low-K mitogenic signal.

Animals↗
PubMed1988

Comparative topography of the immunoreactive alpha-melanocyte-stimulating hormone neuronal systems in the brains of horses and rats.

The present study describes and compares the topography of alpha-melanocyte-stimulating hormone (alpha-MSH)-immunoreactive neurons in the forebrain of rats and horses. Two groups of immunoreactive cells were present in both species. These groups were distinguished by their cross-immunoreactivity and topography. The topography of cell bodies with cross-immunoreactivity for peptides derived from pro-opiomelanocortin (POMC) including adrenocorticotropin, 16K, alpha-MSH and beta-endorphin was similar in both species. However, the topography of perikarya which stained only for alpha-MSH, and not for other POMC-derived neuropeptides, was subject to pronounced species differences. In rats, perikarya which stained only for alpha-MSH (alpha 2-system) have a specific distribution in the dorsal and lateral hypothalamic regions from the level of the midoptic chiasm to the premamillary region of the posterior hypothalamus. At anterior levels of the rat brain, the cells are bipolar and oriented horizontally. Posteriorly, all alpha 2-group perikarya are stellate in rats. In the equine species, alpha 2-group cell bodies occupy a shorter anterioposterior field extending from the posterior level of the paraventricular nucleus to the supramamillary nucleus. Stellate cells are mixed with bipolar cells at all levels of the alpha 2-field in the equine hypothalamus. Further, in contrast to the continuous cell group found in rats, the alpha 2-group divides into perifornical and periventricular subpopulations at posterior levels of the equine hypothalamus. The results from this study suggest that the topography of the arcuate opiocortin cell pool, which contains peptides derived from POMC, is similar in phylogenetically diverse mammalian species whereas the topography of the cell pool staining only for alpha-MSH is subject to species differences. In both species, the major terminus of alpha 2-neurons appears to be the cerebral cortex and hippocampus. It has been proposed that alpha-MSH is one of several neuropeptides concerned with memory and behavior. Thus, species differences in the topography of the alpha 2-pool may reflect evolutionary changes in the importance of this peptide to these physiological processes.

Animals↗
PubMed1987

The effects of nitrous oxide on the central endogenous pro-opiomelanocortin system in the rat.

The hypothesis that nitrous oxide stimulates the central pro-opiomelanocortin system in vivo was explored in this study. A concentration-dependent stimulation of central pro-opiomelanocortin neuropeptides was demonstrated after exposures to variable concentrations of nitrous oxide with oxygen. Rats exposed to 60% and 80% nitrous oxide with oxygen demonstrated an elevation of beta-endorphin concentration along the neuraxis involved with analgesia; no similar effect was observed in alpha-MSH concentration, neither duration of exposure nor acclimation to the enclosed environment altered this stimulation. The discontinuation of nitrous oxide exposure resulted in the diminution of beta-endorphin concentration to pre-exposure levels in 15-30 min. With an ACTH1-39 antisera, a semiquantitative increase in opiocortin immunoreactivity after exposures to nitrous oxide was demonstrated. In conclusion, the increase in beta-endorphin concentration and immunoreactive ACTH1-39 staining in the cells of origin, areas of fiber projection and terminal fields suggest that nitrous oxide stimulates the central pro-opiomelanocortin system in vivo in the rat.

Administration, Inhalation↗
PubMed1987

The effects of nitrous oxide on the secretory activity of pro-opiomelanocortin peptides from basal hypothalamic cells attached to cytodex beads in a superfusion in vitro system.

Dispersed cells from adult rat basal hypothalami, attached to Cytodex-3 microcarrier beads, were placed in a column and superfused with aerated high glucose media or media enriched with variable concentrations of nitrous oxide with oxygen. beta-Endorphin and alpha-MSH content was measured in the effluent collected during superfusion and demonstrated a near constant baseline release. Nitrous oxide, 60% (P less than 0.025) and 80% (P less than 0.02), caused significant increases in release of beta-endorphin. Potassium chloride (50 mM) caused a significant increase in release (P less than 0.007) of beta-endorphin whereas saline and 30% nitrous oxide did not. Neither nitrous oxide-enriched media nor potassium chloride had any statistically significant effect on alpha-MSH release. The increase in beta-endorphin secretory activity during exposure to nitrous oxide demonstrates that nitrous oxide may have a stimulatory effect on central pro-opiomelanocortin neurons.

Animals↗
PubMed1987

Staining of magnocellular neurons of the supraoptic and paraventricular nuclei with vasopressin anti-idiotype antibody: a potential method for receptor immunocytochemistry.

A vasopressin anti-idiotype antibody was generated by immunization with a primary anti-vasopressin IgG. This antibody was capable of immunostaining vasopressinergic neurons in the supraoptic and paraventricular nuclei of the hypothalami of normal and Brattleboro rats. Staining was eliminated by preabsorption or coincubation of the antibody with a vasopressin binding protein prepared from rat neural membranes. The anti-idiotype also inhibited binding of [3H]vasopressin to this neural membrane protein in a dose-dependent manner. These experiments suggest that the anti-idiotype antibody recognizes a receptor associated with vasopressinergic neurons.

Animals↗
PubMed1986

Immunocytochemistry of magnocellular neurons of supraoptic and paraventricular nuclei of normal and Brattleboro rats with vasopressin anti-idiotype antibody.

A vasopressin anti-idiotype antibody was generated by immunization with purified IgG of a primary vasopressin antiserum. The anti-idiotype antibody immunostained neurons in the supraoptic and paraventricular nuclei of the hypothalamus of normal and Brattleboro rats. The distribution of immunostained perikarya in these hypothalamic nuclei together with the staining of fibers in median eminence and neural lobe was similar to that observed in normal rats with anti-vasopressin and suggests strongly that vasopressinergic neurons are being stained. Absorption studies with vasopressin and a vasopressin-binding receptor protein further indicate that a receptor associated with vasopressinergic neurons is recognized by the anti-idiotype antibody.

Animals↗
PubMed1985

Anatomy of the corticotropin-releasing factor and opiomelanocortin systems of the brain.

Using antiserums generated against the neuropeptides corticotropin-releasing factor (CRF), beta-endorphin, and adrenocorticotropin, we have immunocytochemically mapped the organization of these hormones throughout the brain. The distribution of CRF perikarya is widespread throughout the forebrain, brain stem, and spinal cord. The central opiomelanocortin system is represented by two separate pools of neuronal perikarya: the hypothalamic arcuate pool, and the medullary (nucleus tractus solitarius) pool. From these groups fibers distribute widely throughout forebrain and brain stem. A conspicuous codistribution pattern exists between the CRF perikarya and opiomelanocortin fibers. Analysis of these systems within the brain stem and spinal cord reveals innervation and distribution patterns within regions containing catecholamine systems, areas associated with regulation of sympathetic and parasympathetic outflow, cardiovascular and respiratory homeostatic centers, and regions associated with stress, autonomic homeostasis, and pain modulation. By virtue of these extensive brain-stem distributions, it is suggested that an anatomical substrate exists for participation of these peptide systems in the integration of certain autonomic, visceral, and homeostatic mechanisms.

Adrenocorticotropic Hormone↗
PubMed1985

Pulmonary platelet trapping induced by beta-endorphin injection in the cerebrospinal fluid in dogs.

The effect of beta-endorphin injected into either the lateral ventricle or the cisterna magna on blood pressure, heart rate, peripheral platelet and leukocyte counts, hematocrit levels, catecholamines, and pulmonary platelet trapping was studied. The effect of endotoxin on the endogenous opiocortin system was also investigated. Injection of beta-endorphin caused a significant decrease in blood pressure, bradycardia, and pulmonary platelet trapping. beta-Endorphin had no effect on peripheral platelet and leukocyte counts, catecholamines, or hematocrit levels. Endotoxin shock caused a marked rise in circulating beta-endorphin and a decrease in cerebrospinal fluid beta-endorphin. Our results confirm that endotoxin shock activates the opiocortin system, and we suggest that the endorphins may participate in the evolution of the lung injury seen in septic shock.

Animals↗
PubMed1982

Relationship of the central ACTH-immunoreactive opiocortin system to the supraoptic and paraventricular nuclei of the hypothalamus of the rat.

ACTH-immunoreactive (ir) fibers of the central opiocortin system are present in high density in the mid-portion of the paraventricular nucleus (PVN). ACTH-ir fibers, however, do not appear to directly contact oxytocin-ir vasopressin-ir, containing perikarya or axons of vasopressinergic neurons. Magnocellular neurons located more anteriorly and posteriorly in the PVN as well as those located laterally have few ACTH-ir fibers associated with them. No ACTH-ir fibers are present in the supraoptic nucleus.

Adrenocorticotropic Hormone↗