Non-perturbative calculation of molecular magnetic properties within current-density functional theory.

We present a novel implementation of Kohn-Sham density-functional theory utilizing London atomic orbitals as basis functions. External magnetic fields are treated non-perturbatively, which enable the study of both magnetic response properties and the effects of strong fields, using either standard density functionals or current-density functionals-the implementation is the first fully self-consistent implementation of the latter for molecules. Pilot applications are presented for the finite-field calculation of molecular magnetizabilities, hypermagnetizabilities, and nuclear magnetic resonance shielding constants, focusing on the impact of current-density functionals on the accuracy of the results. Existing current-density functionals based on the gauge-invariant vorticity are tested and found to be sensitive to numerical details of their implementation. Furthermore, when appropriately regularized, the resulting magnetic properties show no improvement over standard density-functional results. An advantage of the present implementation is the ability to apply density-functional theory to molecules in very strong magnetic fields, where the perturbative approach breaks down. Comparison with high accuracy full-configuration-interaction results show that the inadequacies of current-density approximations are exacerbated with increasing magnetic field strength. Standard density-functionals remain well behaved but fail to deliver high accuracy. The need for improved current-dependent density-functionals, and how they may be tested using the presented implementation, is discussed in light of our findings.

The umbrella motion of core-excited CH3 and CD3 methyl radicals.

An accurate experimental and theoretical study of the lowest core excitation of CH(3) and CD(3) methyl radicals is presented. The complex vibrational structure of the lowest band of the x-ray absorption spectrum (XAS) is due to the large variation of the molecular geometry, which is planar in the ground state and pyramidal in the core-excited state. The XAS spectra of the two radicals were recorded at high resolution and assigned by theoretical simulations of the spectra, taking into account the coupling of symmetrical stretching and symmetrical bending (umbrellalike) deformations of the radicals. An excellent agreement between experimental and theoretical spectral profiles allowed us to accurately characterize the vibrational structure of the electronic transition. The similarities, as well as the differences, of the peculiar vibrational progression observed for the two radicals are explained by the strong anharmonicity along the umbrella coordinate and by the isotopic variation, leading to a different probing of the double-well potential energy surface of the core excited state during the nuclear motion.

Adrenocorticotrophic hormone retarded metabolism of low-density lipoprotein in rats.

In humans, treatment with adrenocorticotrophic hormone (ACTH) has well-documented plasma cholesterol lowering and low-density lipoprotein (LDL) lowering effects. Moreover, it has recently been demonstrated that ACTH directly inhibits apolipoprotein B expression and secretion in the HepG2 cell line. The aim of the present study was to demonstrate the effects of ACTH on lipid metabolism in the rat, and particularly on LDL metabolism. Rats were treated with porcine ACTH for 3 days and plasma lipid parameters were determined. Surprisingly, the total cholesterol level and LDL-cholesterol level were increased in plasma after ACTH administration, displaying an opposite effect of ACTH in humans. Furthermore, clearance and distribution of radiolabeled human LDL in different tissues were investigated in the rat after ACTH treatment. The clearance of radiolabeled LDL was slightly decreased after ACTH treatment suggesting that ACTH can inhibit LDL catabolism in the rat. Unlike previous observations performed in human hepatic cell cultures, there was no change in apoB expression in rat liver, or in apoE and apoM expression, after treatment with ACTH. This study clearly demonstrates that ACTH has species specificity differences in humans and in the rat.

Different clinical expressions in two families with Stargardt's macular dystrophy (STGD1).

PURPOSE: To describe the clinical expressions, with emphasis on electrophysiological examinations, in two Swedish families with Stargardt's macular dystrophy (STGD1). METHODS: Two pairs of siblings with STGD1, for whom diagnosis had been confirmed by genetic linkage to the ABCA4 gene region, were examined regarding visual acuity, kinetic perimetry, fundus photography, full-field ERG and multifocal ERG (MERG). Possible disease-causing mutations were screened for by DNA sequencing of selected regions of the ABCA4 gene. RESULTS: All STGD1 patients had visual acuity 0.07-0.1. The two families presented different fundus appearances, MERGs and implicit times on 30 Hz flicker white light full-field ERGs. Genetic analysis revealed one unique sequence variation in exon 19 of the ABCA4 gene, in one allele from the patients of one of the families. This point mutation causes the amino acid substitution T972N in the ABCR protein. CONCLUSION: Two pairs of siblings with STGD1 presented two different expressions of the disease regarding the distribution of the retinal dysfunction. One possible molecular explanation to the different clinical expressions may be the T972N substitution present in the ABCR protein in one of the STGD1 families investigated.

Percutaneous cutting needle biopsies for histopathological assessment and sperm retrieval in men with azoospermia.

BACKGROUND: Twenty-three men (45 testes) with azoospermia underwent percutaneous testicular biopsy under local anaesthesia. METHODS: In all but one of the 45 testes two biopsies were taken close to each other, one with a 16 gauge (n = 44) and another with a 14 gauge (n = 45) cutting needle, both with a 19 mm notch. Three quarters of the tissue was used for histopathological assessment and one quarter for direct microscopy. RESULTS: The histopathological findings were similar between the two needles. The observations with direct microscopy corresponded with the histopathological assessments concerning the presence of mature spermatids in 41 of 45 (91%) biopsies using the 14 gauge and in 40 of 44 (91%) biopsies using the 16 gauge needle. There were no post-operative complications except for minimal pain and minor local swelling. CONCLUSIONS: Percutaneous material retrieved using 16 gauge and 14 gauge needles is sufficient for histopathological assessment, and the two needles are equally reliable for testicular sperm retrieval. However, needle biopsy with one puncture may not be representative of the entire testis.

ACTH decreases the expression and secretion of apolipoprotein B in HepG2 cell cultures.

Administration of adrenocorticotropic hormone (ACTH) has been shown to decrease plasma concentrations of apolipoprotein B (apoB) containing lipoproteins, including lipoprotein(a), in man. However, the mechanism behind this hypolipidemic effect is unknown. This study aimed at distinguishing between the main possibilities (increased elimination or decreased production of lipoproteins) using HepG2 cell cultures. Addition of ACTH to the cell culture medium selectively down-regulated apoB mRNA expression and apoB secretion in a dose-dependent manner. At 100 pmol/liter ACTH, the apoB mRNA level was about 40% lower than in the untreated cells, and the secretion of apoB into the medium was decreased to a similar extent. The expression and secretion of other apolipoproteins (apoA-I, apoE, and apoM), however, were not affected by ACTH. Under normal culture conditions the level of secretion of apoB from HepG2 cells is quite low. In the presence of 0.4 mmol/liter oleic acid secretion of apoB increased 3-fold, but this phenomenon was not seen in ACTH-treated cells. Binding and internalization of radiolabeled low density lipoprotein (LDL) by HepG2 cell, as well as LDL-receptor mRNA and scavenger receptor B-I mRNA levels, were not influenced by ACTH. In conclusion, ACTH directly and selectively down-regulated the production and secretion of apoB in HepG2 cell cultures, suggesting that a principal mechanism behind the cholesterol-lowering effect of ACTH in vivo may be a decreased production rate of apoB-containing lipoproteins from the liver.

The molecular mechanism for the genetic disorder familial defective apolipoprotein B100.

Familial defective apolipoprotein B100 (FDB) is a genetic disorder in which low density lipoproteins (LDL) bind defectively to the LDL receptor, resulting in hypercholesterolemia and premature atherosclerosis. FDB is caused by a mutation (R3500Q) that changes the conformation of apolipoprotein (apo) B100 near the receptor-binding site. We previously showed that arginine, not simply a positive charge, at residue 3500 is essential for normal receptor binding and that the carboxyl terminus of apoB100 is necessary for mutations affecting arginine 3500 to disrupt LDL receptor binding. Thus, normal receptor binding involves an interaction between arginine 3500 and tryptophan 4369 in the carboxyl tail of apoB100. W4369Y LDL and R3500Q LDL isolated from transgenic mice had identically defective LDL binding and a higher affinity for the monoclonal antibody MB47, which has an epitope flanking residue 3500. We conclude that arginine 3500 interacts with tryptophan 4369 and facilitates the conformation of apoB100 required for normal receptor binding of LDL. From our findings, we developed a model that explains how the carboxyl terminus of apoB100 interacts with the backbone of apoB100 that enwraps the LDL particle. Our model also explains how all known ligand-defective mutations in apoB100, including a newly discovered R3480W mutation in apoB100, cause defective receptor binding.

Expression of an LDL receptor allele with two different mutations (E256K and I402T).

AIMS: To investigate the disease causing event in patients with familial hypercholesterolaemia, carrying two mutations each, E256K in exon 6 and I402T in exon 9, of the gene encoding the low density lipoprotein (LDL) receptor. It was not known whether the mutations were positioned in cis or trans, or if they were each pathogenic separately or only when present together. METHODS: Polymerase chain reaction, denaturing gradient gel electrophoresis and sequencing were used to characterise the LDL receptor locus of the patients and family members. The different LDL receptor mutants, constructed in vitro by oligonucleotide directed mutagenesis, were expressed in LDL receptor deficient Chinese hamster ovary (CHO1d1A7) cells, to determine the effects of the mutations on LDL receptor function. RESULTS: The two mutations were located on the same allele of the LDL receptor gene. All mutant constructs resulted in the production of a detectable protein in CHO cells. The cells expressing only the I402T mutation, or the combination of I402T and E256K mutations, were seriously affected in mediating uptake and degradation of LDL. Contrary to initial predictions, the cells expressing only the E256K mutation showed essentially the same binding, uptake, and degradation of 125I labelled LDL as cells transfected with normal LDL receptor cDNA. These results suggest that the pathogenic mutation in the patients heterozygous for the E256K/I402T allele is the I402T mutation, and that E256K alone is a rare sequence variation, which does not affect LDL receptor protein function. E256K was not detected either in DNA from a healthy population or in DNA from other hypercholesterolaemic patients studied. CONCLUSIONS: Despite the information available on the structure-function relations between the LDL receptor and LDL receptor like proteins, predictions about the disease causing potential of a mutation are not reliable. These results suggest that the I402T mutation is pathogenic and that the substitution of E256K alone is a rare sequence variation, without a detectable phenotype modulating effect.

An individual with a healthy phenotype in spite of a pathogenic LDL receptor mutation (C240F).

Familial hypercholesterolemia (FH) is caused by a defect in the function of the low density lipoprotein (LDL) receptor and inherited in an autosomal, codominant way. In this study we present a 13-year-old girl, compound heterozygote for the LDL receptor mutations C240F and Y167X. Fibroblasts from the patient showed very low cholesterol esterification rate, LDL uptake, and degradation compared to normal fibroblasts (< 2%, 8%, and < 2%, respectively). The C240F mutant was expressed in LDL receptor deficient CHOMldlA7 cells. Analysis of cell extracts by immunoblotting demonstrated delayed processing of the mutated LDL receptor, which was accumulated as a precursor protein of normal size. A high molecular weight form of the receptor was also detectable in these cells, which probably reflects cross-linking through the unpaired cysteine residue in the binding domain. Cells expressing the C240F mutant protein were unable to mediate uptake and degradation of LDL. The two siblings of the index case also carried the C240F mutation, but surprisingly one of them (a 17-year-old brother) showed no signs of hypercholesterolemia. This observation is consistent with the view that there may be cholesterol lowering mechanisms that can be activated, perhaps by mutations in known or hitherto unknown genes.

A Swedish family with a mutation in the peripherin/RDS gene (Arg-172-Trp) associated with a progressive retinal degeneration.

PURPOSE: To clinically characterize a Swedish family with autosomal dominant retinitis pigmentosa due to a mutation, Arg-172-Trp, in the peripherin/RDS gene. METHODS: Full clinical evaluation including kinetic visual field testing, measurement of dark-adaptation threshold, and full-field electroretinography in seven patients with autosomal dominant retinitis pigmentosa and three healthy family members. Denaturing gradient gel electrophoresis (DGGE) was used for mutation screening in seven patients and six healthy members of the family. RESULTS: Three of four siblings from the middle generation and four of the younger generation were heterozygous for the peripherin /RDS Arg-172-Trp mutation. The mutation segregated with the disease. Visual acuity decreased progressively with age and visual fields were moderately constricted in young patients, while central scotoma and constriction of the fields were detected in the family members above 50 years of age. The results from full-field electrography were comparable with a widespread retinal degeneration. CONCLUSIONS: Earlier, the peripherin/RDS Arg-172-Trp mutation was associated primarily with a macular degeneration phenotype. One previous study indicated that this mutation also can give rise to a degeneration of the more peripheral parts of the retina. In the present study, a widespread retinal degeneration is seen in the patients above 50 years of age, carrying the Arg-172-Trp mutation.

Mutations in the low-density lipoprotein receptor gene in Swedish familial hypercholesterolaemia patients: clinical expression and treatment response.

BACKGROUND: Familial hypercholesterolaemia, an autosomal co-dominant disorder caused by defects in the low-density lipoprotein receptor gene, is strongly associated with premature development of cardiovascular disease. METHODS: In this study, we have applied a gene screening method in a population of familial hypercholesterolaemia patients in order to describe the genetic background of the disease in southern Sweden. These patients were studied with the aim of relating the presence of the different mutations to the clinical expression of the disease and to the response to pharmacological treatment. RESULTS: In 16 out of 21 patients, potentially disease-causing low-density lipoprotein receptor gene defects were found, including five not previously described alterations (C240-->F, C122-->stop, C356-->Y, 785insG, 165delG). No defects in apolipoprotein B were found. One group of patients (n = 4) carried the mutation C122-->stop and another group of patients (n = 4) a mutation causing the substitution W66-->G. Patients in the two genotype subgroups were very similar with respect to lipid levels before treatment. CONCLUSION: A tendency towards differential susceptibility to treatment with statins was observed for the patient groups, encouraging further comparative studies of heterozygous FH patients.

Phenotypic expression of autosomal dominant retinitis pigmentosa in a Swedish family expressing a Phe-211-Leu variant of peripherin/RDS.

PURPOSE: To characterize the clinical phenotype, with emphasis on electrophysiology, of members of a Swedish family with autosomal dominant retinitis pigmentosa due to a novel mutation, F211L, in the peripherin/RDS gene. METHODS: Nine patients with autosomal dominant retinitis pigmentosa and two healthy family members underwent a full clinical evaluation including kinetic visual field testing, measurement of dark adaptation threshold, and full-field electroretinography. Blood samples were collected and DNA analysis was performed using denaturing gradient gel electrophoresis (DGGE). RESULTS: The grandfather, six of seven siblings from the middle generation, and two young boys carried the mutation F211L in the peripherin/RDS gene. The mutation segregated with the clinical presentation of disease. Fundus examination revealed mainly macular atrophy. All assessed parameters of retinal function (visual acuity, dark adaptation threshold, visual fields, and full-field electroretinograms) demonstrated a successive reduction with increasing age. Full-field electroretinograms showed a diminished rod response in all affected individuals and a reduction of the cone b-wave amplitudes with increasing age, indicating retinitis pigmentosa. In the affected family members, the disease seems to progress at a similar rate with increasing age. CONCLUSIONS: The peripherin/RDS gene mutation F211L is associated with a clinical phenotype and includes early loss of rod function and successive reduction of cone function with increasing age, but impressively well-preserved visual acuity and visual fields in young and middle-aged patients and moderately reduced vision in the old patient. Compared to previously described phenotypes segregating with mutations in the peripherin/RDS gene, the present family demonstrates a more benign clinical phenotype, which is concordant within the family.

Detection of alterations in all three exons of the peripherin/RDS gene in Swedish patients with retinitis pigmentosa using an efficient DGGE system.

AIMS: To develop a sensitive mutation screening procedure suitable for routine analysis of the peripherin/RDS gene, and to estimate the nature and prevalence of peripherin/RDS gene mutations in Swedish patients with autosomal dominant retinitis pigmentosa. METHODS: To make the method as sensitive as possible, as many as eight segments, covering the three exons and the flanking intron sequences of the peripherin/RDS gene, were analysed by denaturing gradient gel electrophoresis. A group of 38 Swedish patients with a clinical diagnosis of autosomal dominant retinitis pigmentosa were screened for mutations in the peripherin/RDS gene. RESULTS: Three point mutations were found in four of the patients and five polymorphisms were defined. One mutation in exon 1, R172W, has been described previously in other ethnic groups as causing a macular degeneration. Another mutation, in exon 2 and causing the substitution F211L, was found in two unrelated patients. A third mutation, resulting in the likely non-pathogenic substitution S289L, as well as a polymorphism not reported previously, was found in exon 3. CONCLUSIONS: The screening procedure described allows detection of mutations in all of the exons, including the polymorphic 5' and 3' ends of the gene, and is therefore suitable for routine screening of peripherin/RDS gene defects in patients with autosomal dominant retinitis pigmentosa. The frequency of mutations found in the Swedish patient group indicates that defects in the peripherin/RDS gene might be a more common cause of autosomal dominant retinitis pigmentosa than was thought previously.

Effects of heparin and low molecular weight heparin on lipid transport during parenteral feeding in the rat.

BACKGROUND: Treatment with heparin has been reported to interfere with lipid metabolism by release of Lipoprotein Lipase (LPL) into the circulation. The purpose of the present study was to determine the effects on LPL activity by anticoagulants in combination with total parenteral nutrition (TPN) in the rat. In an earlier investigation we could show that TPN, per se, caused a three-fold increase of triglyceride content in liver tissue, retention of lipids in the circulation and disturbed cholesterol metabolism with accumulation of cholesterol in the non High Density Lipoprotein (HDL) fraction of lipoproteins. The activity of Hepatic Lipase (HL) was decreased, while the activities of LPL in adipose tissue and heart were up-regulated. METHODS: Effects on lipid metabolism by TPN for seven days with or without simultaneous administration of heparin or Low Molecular Weight Heparin (LMWH) were studied in 52 healthy male Sprague-Dawley rats. Combinations of Heparin or LMWH and discontinuous or continuous administration of TPN solutions (including approximately 8 g triglycerides/kg body weight daily) were investigated. RESULTS: Addition of LMWH, but not heparin, to treatment with TPN resulted in significant up-regulation of LPL activity in the heart. Combination of heparin and continuous administration of TPN solutions was followed by modest, but significant, increases of S-Triglycerides and HDL-Triglycerides. No differences between the TPN groups were observed concerning liver steatosis, cholesterol metabolism, phospholipid metabolism or HL activity. CONCLUSION: Treatment with LMWH during TPN resulted in up-regulated LPL activity in the heart, which might represent a compensatory mechanism for enzyme release from the capillary walls induced by anticoagulants. Administration of heparin, a more effective lipase-releasing agent, was not associated with increased LPL activity. Heparin treatment in combination with continuous TPN administration was followed by increased levels of triglycerides in blood and HDL particles, suggesting that treatment with heparin might have impaired the capacity for LPL up-regulation, resulting in the development of hyperlipidemia. Further investigations are necessary for evaluation of the mechanisms. Depletion of LPL activity could not be demonstrated by this study in healthy rats.

An efficient screening procedure detecting six novel mutations in the LDL receptor gene in Swedish children with hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal semi-dominant disorder caused by defects in the low density lipoprotein receptor (LDLR) gene and is a well-documented risk factor for developing cardiovascular disease. The LDLR genes of five Swedish children with FH were examined in this study. Initial mutation screening was performed by denaturing gradient gel electrophoresis (DGGE) with enzymatically amplified exon-sized fragments, each containing a tailing GC-rich requence. The GC-clamped fragments had been synthesized with a restriction site adjacent to the intron-corresponding sequence to allow detachment of the clamps, thereby rendering the fragments suitable for subsequent analysis by single-strand conformation polymorphism (SSCP) analysis of samples from patients with no DGGE-detectable mutations. In addition, all the LDLR genes of the patients were screened for large alterations by restriction fragment length polymorphism analysis. Following this strategy, seven different, potentially disease-causing mutations were detected in the five children with FH. Six of the alterations, five single-base substitutions and one dinucleotide deletion, have not previously been described. DGGE detected six of the mutations and SSCP the seventh.

Effects of total parenteral nutrition on lipid metabolism in rats.

BACKGROUND: The pathophysiologic mechanisms behind the development of liver steatosis during total parenteral nutrition (TPN) and the possible relationship to alterations of lipoprotein lipase activities in different tissues are not fully known. It is also unknown whether continuous and discontinuous administration of TPN affect lipid metabolism differently. METHODS: TPN, including 8.4 g of triglycerides per kilogram per day, was given for 10 days to two groups of male Sprague-Dawley rats that received the infusions discontinuously and continuously, respectively. Freely fed rats were used as controls. RESULTS: TPN led to hyperlipidemia and accumulation of triglycerides in the liver. High-density lipoproteins were enriched in triglycerides, whereas high-density lipoprotein cholesterol and phospholipid levels were low. The activities of hepatic lipase were markedly decreased, and lipoprotein lipase activities in adipose tissue and in cardiac muscle were both up-regulated. The increased levels of cholesterol and phospholipids in the serum of TPN animals were more pronounced after discontinuous administration. CONCLUSIONS: TPN including lipids interferes with the normal regulation of lipid metabolism. Although the mechanisms remain obscure, the elevation of lipoprotein lipase activities seems functionally important to accommodate the increased input of triglycerides during TPN. Possibly, the observed alterations in lipase activities may be attributed to a state of hypothyroidism.

Hormone release from thyrotrophs and lactotrophs during hypo- and hyperglycemia.

To test whether changes in carbohydrate metabolism influence anterior pituitary function, iv TRH tests (25 micrograms TRH) were carried out on three different occasions in 6 normal subjects. On one of these occasions TRH was administered during normoglycemia (blood glucose level 4.5 mmol/l - on the other, during hyperglycemia (10 mmol/l) - and on the third, during hypoglycemia (3 mmol/l). Hypoglycemia reduced the TRH-elicited TSH response significantly (19 +/- 6%), but failed to affect the corresponding PRL response. Hyperglycemia left both the TSH and PRL responses to TRH unaffected. These results imply that thyrotrophs and lactotrophs react differently to changes in carbohydrate metabolism. Thyrotrophs - in contrast to lactotrophs - seem to require a certain minimal glucose delivery to function normally. Glucose excess does not change the reactivity of these pituitary cells significantly.