Biomedical subjects
H H Schmidt
Publications and source records attributed to H H Schmidt.
Expression and purification of recombinant human apolipoprotein A-I in Chinese hamster ovary cells.
The expression of apolipoprotein A-I (apoA-I) has been shown to be very difficult due to its amphiphilic character, autoaggregation, and degradation. We have expressed apoA-I using CHO cells, Baculovirus, and Escherichia coli [Schmidt et al., J. Biol. Chem. (1995) 270, 469-475]. Here we report about optimized conditions for the expression of proapoA-I in CHO cells, testing various serum-free media. We were able to yield apoA-I expression up to 80 micrograms/ml, by far the highest ever reported. However, immunoblot analysis revealed degraded apoA-I. The best apoA-I expression testing various conditions was about 20-30 micrograms/ml without any evidence of degradation. Interestingly, the apoA-I expression resulted in reproducible apoA-I fragments of 26 and 14 kDa. These fragments are consistent with already reported in vivo findings, in which carboxy-terminal proteolysis was suggested. The use of the protease inhibitors pepstatin and chymostatin, both carboxy-peptidase inhibitors, did result in contrast to other studied protease inhibitors in increased apoA-I yield. Therefore, limited carboxy-terminal proteolysis contributes to the degradation of CHO cell-secreted apoA-I. In addition, we evaluated various purification methods for the preparative isolation of recombinant apoA-I. In our hands we obtained the best recovery and no degradation with reversed-phase chromatography using a FPLC system.
[Formation and effects of nitrogen monoxide (NO) in the vascular system].
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Erdheim-Chester disease: low low-density lipoprotein levels due to rapid catabolism.
We have identified a 44-year-old patient with symmetrically excessive xanthomatosis, called Erdheim-Chester disease (ECD), and simultaneously decreased levels of low-density lipoprotein (LDL) cholesterol. Clinically, this patient presents lipoidgranulomatosis of numerous long and flat bones with involvement of the liver, spleen, pericardium, pleura, thyroid, skin, conjunctiva, and gingiva. However, the patient does not have any signs of atherosclerosis. So far, the underlying defect has not been elucidated. We performed a LDL-apolipoprotein B (apoB) kinetic study in the ECD patient and a normal control to determine the etiology of the low LDL level in ECD. LDL was isolated from both subjects, radioiodinated with either 131I or 125I, and injected simultaneously into the ECD patient and the normal control. Normal and ECD LDL was catabolized at the same rate after injection into the control subject (fractional catabolic rate [FCR], 0.43/d and 0.46/d, respectively). Therefore, LDL isolated from an ECD subject is metabolically normal. In contrast, autologous LDL injected into the ECD subject showed a markedly increased catabolism (FCR, 0.69/d) compared with that in the control subject (FCR, 0.43/d). This is the first report about increased catabolism of LDL cholesterol in a patient.
Preparative electrophoresis: an improved method for the isolation of human recombinant apolipoprotein A-I.
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Neurological complications in celiac disease.
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The spleen as a storage pool in lipid metabolism.
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No .NO from NO synthase.
The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of L-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form L-citrulline (Cit) and nitric oxide (.NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify .NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable .NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal .NO standards were only partially recovered and the dismutation of superoxide (O2-.), which otherwise scavenges. .NO to yield ONOO-, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO- resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8-1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to .NO or that .NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal .NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO-) to .NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from .NO decomposition, was consistent with formation of an .NO precursor molecule such as NO-. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that .NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.
[Liver cell transplantation as somatic gene therapy].
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Identification of uterine-related sympathetic neurons in the rat inferior mesenteric ganglion: neurotransmitter content and afferent input.
The rat uterus is innervated by sensory and autonomic nerves. Sensory and sympathetic fibers travel in the hypogastric nerves and are associated with the thoracolumbar spinal cord levels T13-L3. The inferior mesenteric ganglion (IMG) contains the somata of sympathetic postganglionic neurons and some of these may project axons to the uterus. Sensory and parasympathetic fibers travel in the pelvic nerve and are associated with the lumbosacral cord levels L6-S1 and pelvic ganglion (PG). We previously reported data concerning the neurochemical anatomy of the PG with regard to the uterine innervation; the present study was undertaken to characterize the neurochemical anatomy of the IMG with regard to it involvement in uterine innervation. A retrograde axonal tracer was used to verify projections of axons of IMG neurons to the uterus. Immunostaining of cryostat sections of the IMG revealed neurons immunoreactive for neuropeptide Y (NPY) and for tyrosine hydroxylase (TH). Immunostaining for the synaptic terminal protein synapsin I (SYN) revealed numerous fine terminals immediately surrounding the principal neurons and in the interneuronal spaces. Varicosities immunoreactive for calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), enkephalin (ENK), substance P (SP) and galanin (GAL) appear to be associated with principal neurons. Additional varicosities stained for nicotinamide adenine dinucleotide phosphate (reduced)-diaphorase (NADPH-d) and nitric oxide synthase (NOS), thus indicating sites of neuronal nitric oxide synthesis. This study revealed that the IMG contains uterine-related neurons and that some of the retrogradely labeled uterine-related neurons contain NPY, TH or both NPY/TH. In addition, uterine-related neurons received abundant afferent inputs indicated by SYN-immunoreactive (-ir) terminals and some of these varicosities labeled for GAL, CGRP, VIP, ENK, or NADPH-d/NOS.
Relation of cholesterol-year score to severity of calcific atherosclerosis and tissue deposition in homozygous familial hypercholesterolemia.
The high concentrations of low-density lipoprotein cholesterol in plasma lead to accelerated atherosclerosis in patients homozygous for familial hypercholesterolemia (FH). We addressed the hypothesis that lipid deposition in the arterial vasculature and in nonvascular tissues in these patients correlates with both the duration and severity of their hypercholesterolemia. The severity of calcific atherosclerosis was defined by calcification scores and a calcified volume determined by electron beam tomography. The extent of tendinous xanthomatosis was quantitated by computed tomography. A cholesterol-year score was calculated based on the age and the yearly mean serum cholesterol concentration of each patient. Seventeen patients homozygous for FH were followed up. The average total cholesterol concentration in the study group was 780 +/- 231 mg/dl (20.2 mmol/L), and the cholesterol-year scores ranged from 2,172 mg-year/dl (56 mmol-year/L) to 32,260 mg-year/dl (834 mmol-year/L). Achilles tendon width (r=0.86) and cross-sectional area (r=0.81; both p <0.001) were best correlated with the cholesterol-year score. In addition, the coronary (r=0.61; p<0.05), ostial (r=0.45; p<0.05), and total (r= 0.77; p<0.001) calcification atherosclerosis scores all were best correlated with the cholesterol-year score. Calcific atherosclerosis was not observed in these patients until the cholesterol-year score exceeded 10,000 mg-year/dl (260 mmol-year/L). These findings establish a direct association of cholesterol-year with extravascular lipid deposition in tissues of patients with FH. The cholesterol-year score may be useful in defining the risk of atherosclerosis in patients with more common forms of hypercholesterolemia.
NADPH-diaphorase activity in brain macrophages during postnatal development in the rat.
NADPH-diaphorase histochemistry, that allows the visualization of cells producing the gaseous intercellular messenger nitric oxide, was used in the study of the forebrain during the first three postnatal weeks in the rat. Subpopulations of NADPH-diaphorase positive neurons were observed at all ages studied. In addition, non-neuronal NADPH-diaphorase-stained cells were detected in the subcortical white matter, and were very numerous in the supraventricular portion of the corpus callosum, and in the internal and external capsules. These cells were present during the first two postnatal weeks, and were especially prominent at the end of the first postnatal week. They were round-shaped and morphologically similar to the brain macrophages, whose phagocytic activity has been shown in previous studies to play a role in naturally occurring cell death and elimination of exhuberant axons. Series of sections adjacent to those stained with NADPH-diaphorase were processed with immunohistochemistry, using two different antibodies (OX-42 and ED-1) that detect macrophagic and microglial markers, and antibodies that recognize the neuronal form of nitric oxide synthase. Furthermore, brain sections from rats at postnatal day 7 were sequentially processed for either OX-42 or nitric oxide synthase immunohistochemistry followed by NADPH-diaphorase histochemistry. The morphological features and distribution of the non-neuronal NADPH-diaphorase-positive cells were superimposable to those obtained with OX-42 and ED-1 immunohistochemistry. In addition, these cells did not display nitric oxide synthase immunoreactivity. Double-labelled NADPH-diaphorase-positive and OX-42-immunoreactive cells were detected at postnatal day 7. The present results show that brain macrophages express NADPH-diaphorase activity during the early stages of the normal postnatal maturation and suggest that nitric oxide produced by brain macrophages could be involved in the development reshaping of the central nervous system.
Bone marrow granulomas in hairy cell leukaemia.
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Quantification of lipoprotein(a): comparison of an automated latex-enhanced nephelometric assay with an immunoenzymometric method.
Several studies indicate the relevance of lipoprotein(a) (Lp(a)) in the genesis of premature coronary artery disease. A simple method for determining the concentration of Lp(a) is therefore of great interest for assessing the risk of coronary artery disease in patients. We compared a new latex-enhanced immunonephelometric assay (Behringwerke AG, Marburg, Germany), using the Behring Nephelometer System 100, with an established immunoenzymometric assay (Immuno, Heidelberg, Germany). A total of 163 patients was studied. Intra- and inter-assay coefficients of variation were between 2.2% and 7.1%, and between 3.4% and 8.6%, depending on the concentration of Lp(a). The correlation between the studied assays was excellent (r = 0.93, y = 0.98x -1.57, Spearman rank, Passing & Bablok). When values above 1000 mg/l for Lp(a) were excluded, the correlation was even higher. Increased light scattering with particle size, which hitherto has been a disadvantage of the nephelometric technique, seems to be negligible using the improved latex-enhanced approach. In patients with triacylglycerol values above 4.5 mmol/l (n = 19) there was no interference with the Behring system, i.e. the results of the nephelometric method were not increasing, and they agreed with those of the immunoenzymometric assay. In conclusion, this new latex-enhanced nephelometric immunoassay represents a rapid and precise method for the quantification of Lp(a).
Should we treat cholestatic liver disease with cholesterol synthesis inhibitors?
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[Reverse cholesterol transport].
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Thiol dependence of nitric oxide synthase.
Nitric oxide synthases (NOS) require NADPH and tetrahydrobiopterin (H4biopterin) to convert L-arginine to L-citrulline. The additional requirement and effects of thiols during purification and activity assays of NOS are unclear; for example, glutathione (GSH) has been reported to stimulate or, in the presence of catalase, to inhibit enzyme activity. We therefore studied the effects of different thiols, thiol reagents, antioxidants, and H4biopterin-regenerating systems on purified porcine cerebellum NOS. GSH in the presence of catalase did not inhibit NOS. In contrast, GSH and, to a lesser degree, several other thiols consistently stimulated total L-arginine turnover up to 4-fold. In the presence of GSH, Vmax of NOS was increased, the usually observed loss of activity during the 15 min assay was less dramatic, and the apparent S0.5 value for H4biopterin decreased. Stabilization of NOS activity by GSH was augmented by protein disulfide isomerase (PDI), indicating that, at least in part, GSH acted by reductive protection of NOS protein thiols. Consistent with this, four different protein thiol reagents abolished NOS activity. In other experiments, specific allosteric binding was excluded as a potential mechanism of GSH regulation of NOS. In addition, GSH may affect NOS kinetics by recycling or preventing the autoxidation of H4biopterin. In support of this, the non-thiol reductant ascorbate and dihydropteridine reductase mimicked the effects of GSH on NOS kinetics, but not on NOS stability. Thus, NOS activity depends on both H4biopterin and the reduced state of essential protein thiols.
Carboxyl-terminal domain truncation alters apolipoprotein A-I in vivo catabolism.
Apolipoprotein A-I (apoA-I), the major protein of high density lipoproteins, facilitates reverse cholesterol transport from peripheral tissue to liver. To determine the structural motifs important for modulating the in vivo catabolism of human apoA-I (h-apoA-I), we generated carboxyl-terminal truncation mutants at residues 201 (apoA-I201), 217 (apoA-I217), and 226 (apoA-I226) by site-directed mutagenesis. ApoA-I was expressed in Escherichia coli as a fusion protein with the maltose binding protein, which was removed by factor Xa cleavage. The in vivo kinetic analysis of the radioiodinated apoA-I in normolipemic rabbits revealed a markedly increased rate of catabolism for the truncated forms of apoA-I. The fractional catabolic rates (FCR) of 9.10 +/- 1.28/day (+/- S.D.) for apoA-I201, 6.34 +/- 0.81/day for apoA-I217, and 4.42 +/- 0.51/day for apoA-I226 were much faster than the FCR of recombinant intact apoA-I (r-apoA-I, 0.93 +/- 0.07/day) and h-apoA-I (0.91 +/- 0.34/day). All the truncated forms of apoA-I were associated with very high density lipoproteins, whereas the intact recombinant apoA-I (r-apoA-I) and h-apoA-I associated with HDL2 and HDL3. Gel filtration chromatography revealed that in contrast to r-apoA-I, the mutant apoA-I201 associated with a phospholipid-rich rabbit apoA-I containing particle. Analysis by agarose gel electrophoresis demonstrated that the same mutant migrated in the pre-beta position, but not within the alpha position as did r-apoA-I. These results indicate that the carboxyl-terminal region (residue 227-243) of apoA-I is critical in modulating the association of apoA-I with lipoproteins and in vivo metabolism of apoA-I.