Reversible dementia with psychosis: Hashimoto's encephalopathy.

A case of presumed Hashimoto's encephalopathy (HE) is presented. The presentation included memory loss, delusions, functional decline and culminated in a generalized seizure. Anti-thyroid antibodies were detected and symptoms resolved with prednisolone. Patients with HE may present with prominent neuropsychiatric symptoms, attract psychiatric diagnoses and present to psychiatric services. Primarily a diagnosis of exclusion, HE should be considered in cases of encephalopathy in which standard investigations are negative.

The cytochrome c fold can be attained from a compact apo state by occupancy of a nascent heme binding site.

NMR techniques and 8-anilino-1-napthalenesulphonate (ANS) binding studies have been used to characterize the apo state of a variant of cytochrome c(552) from Hydrogenobacter thermophilus. In this variant the two cysteines that form covalent thioether linkages to the heme group have been replaced by alanine residues (C11A/C14A). CD studies show that the apo state contains approximately 14% helical secondary structure, and measurements of hydrodynamic radii using pulse field gradient NMR methods show that it is compact (R(h), 16.6 A). The apo state binds 1 mol of ANS/mol of protein, and a linear reduction in fluorescence enhancement is observed on adding aliquots of hemin to a solution of apo C11A/C14A cytochrome c(552) with ANS bound. These results suggest that the bound ANS is located in the heme binding pocket, which would therefore be at least partially formed in the apo state. Consistent with these characteristics, the formation of the holo state of the variant cytochrome c(552) from the apo state on the addition of heme has been demonstrated using NMR techniques. The properties of the apo state of C11A/C14A cytochrome c(552) reported here contrast strongly with those of mitochondrial cytochrome c whose apo state resembles a random coil under similar conditions.

A further clue to understanding the mobility of mitochondrial yeast cytochrome c: a (15)N T1rho investigation of the oxidized and reduced species.

A new approach was developed to overproduce 15N-enriched yeast iso-1-cytochrome c in the periplasm of Escherichia coli in order to perform a study of the motions in the ms-micros time scale on the oxidized and reduced forms through rotating frame 15N relaxation rates and proton/deuterium exchange studies. It is confirmed that the reduced protein is rather rigid whereas the oxidized species is more flexible. The regions of the protein that display increased internal mobility upon oxidation are easily identified by the number of residues experiencing conformational equilibria and by their exchange rates. These data complement the information already available in the literature and provide a comprehensive picture of the mobility in the protein. In particular, oxidation mobilizes the loop containing Met80 and, through specific contacts, affects the mobility of helix 3 and possibly of helix 5, and of a section of protein connecting the heme propionates to helix 2. The relevance of internal motions to molecular recognition and to the early steps of the unfolding process of the oxidized species is also discussed. In agreement with the reported data, subnanosecond mobility is found to be less informative than the ms-micros with respect to redox dependent properties.

Amyloid fibril formation by a helical cytochrome.

The substitution of alanines for the two cysteines which form thioether linkages to the haem group in cytochrome c(552) from Hydogenobacter thermophilus destabilises the native protein fold. The holo form of this variant slowly converts into a partially folded apo state that over prolonged periods of time aggregates into fibrillar structures. Characterisation of these structures by electron microscopy and thioflavin-T binding assays shows that they are amyloid fibrils. The data demonstrate that when the native state of this cytochrome is destabilised by loss of haem, even this highly alpha-helical protein can form beta-sheet structures of the type most commonly associated with protein deposition diseases.

Loss of either of the two heme-binding cysteines from a class I c-type cytochrome has a surprisingly small effect on physicochemical properties.

Almost without exception, c-type cytochromes have heme covalently attached via two thioether linkages to the cysteine residues of a CXXCH motif. The reasons for the covalent attachment are not understood. Reported here is cytoplasmic expression in Escherichia coli of AXXCH and CXXAH variants of cytochrome c(552) from Hydrogenobacter thermophilus; remarkably, the single thioether bond proteins have, apart from an altered visible absorption spectrum, almost identical properties, including thermal stability and reduction potential, to the wild type CXXCH protein. In combination with previous work showing that an AXXAH variant of cytochrome c(552) is much less stable than the CXXCH form, it can be concluded that covalent attachment of heme via either of thioether bonds is sufficient to confer considerable stability and that these bonds contribute little to the setting of the reduction potential. The absence of AXXCH or CXXAH heme-binding motifs from bacterial cytochromes c may relate to the coexistence of the assembly pathway with that for formation of disulfide bonds in the bacterial periplasm.

Conversion of a c type cytochrome to a b type that spontaneously forms in vitro from apo protein and heme: implications for c type cytochrome biogenesis and folding.

Cytochrome c(552) from Hydrogenobacter thermophilus, a thermophilic bacterium, has been converted into a b type cytochrome, after mutagenesis of both heme-binding cysteines to alanine and expression in the cytoplasm of Escherichia coli. The b type variant is less stable, with the guanidine hydrochloride unfolding midpoint occurring at a concentration 2 M lower than for the wild-type protein. The reduction potential is 75 mV lower than that of the recombinant wild-type protein. The heme can be removed from the b type variant, thus generating an apo protein that has, according to circular dichroism spectroscopy, an alpha-helical content different from that of the holo b type protein. The latter is readily reformed in vitro by addition of heme to the apo protein. This reforming suggests that previously observed assembly of cytochrome c(552), which has the typical class I cytochrome c fold, in the E. coli cytoplasm is a consequence of spontaneous thioether bond formation after binding of heme to a prefolded polypeptide. These observations have implications for the general problem of c type cytochrome biogenesis.

Quantitative expression analysis of genes regulated by both obesity and leptin reveals a regulatory loop between leptin and pituitary-derived ACTH.

Absence of the hormone leptin leads to dramatic increases in appetite, food intake, and adiposity. The primary site of action, at least with respect to appetite, is the hypothalamus. Leptin also has significant effects on the function(s) of peripheral organs involved in maintaining body composition. Some of these effects are mediated through direct interaction of leptin with its receptor on the target tissue, and some effects are indirectly mediated through secondary hormonal and neural pathways. Few of the genes that are responsible for regulating body composition and the peripheral effects of leptin are known. We have used a new gene profiling technology to characterize gene expression changes that occur in the pituitary, hypothalamus, fat, muscle, and liver in response to both obesity and treatment with exogenous leptin. These differences were then overlaid to allow the identification of genes that are regulated by obesity and at least partially normalized by leptin treatment. By using this process we have identified five genes (POMC, PC2, prolactin, HSGP25L2G, and one novel) that are both abnormally expressed in the pituitaries of obese mice and are sensitive to the effects of leptin. We also show that adrenocorticotropic hormone appears to be involved in a regulatory loop involving leptin.

Controlled plasmid delivery and gene expression : applications for nucleic Acid-based vaccines.

After the concept of genetic immunization was first demonstrated by Johnston's group in 1992 (1), numerous studies have reported the potential prophylactic and therapeutic use of nucleic acid-based vaccines for combating various infectious diseases (2-4). Vaccines of this composition appear to be both efficacious in the short term, and able to elicit a prolonged anamnestic response capable of preventing or resolving infection when challenged at up to one year after vaccination (5). Nucleic acid-based vaccines elicit a broader immune response than do subunit vaccines, inducing both cellular and humoral responses that are reminiscent of attenuated and whole-killed viral vaccines. Further, nucleic acid-based vaccines can be prepared with relative ease of synthesis and production. Expression plasmids can be generated quickly once the antigen's coding sequence is known and small- and large-scale purification methods are well established. Nucleic acid-based vaccines also avoid some of the safety concerns of conventional vaccines in that there is no chance of disease due to co-purification of contaminating virus or reversion of the attenuated strain in the patient. This is not to claim that the safety issues surrounding nucleic acid-based vaccines are minimal. The major theoretical concerns surrounding the safety of this technology include plasmid integration into the host genome, transformation of somatic or stem cells, and tolerability. However, there is no published evidence that administration of unformulated or 'naked' plasmid produces a severe short or long term deleterious effect (6).

Randomized phase II study of high-dose paclitaxel with or without amifostine in patients with metastatic breast cancer.

PURPOSE: To determine whether the neurotoxicity of paclitaxel 250 mg/m(2) given over 3 hours every 3 weeks could be reduced by pretreatment with amifostine 910 mg/m(2). Secondary objectives included comparing myelosuppression, myalgias, and response rates of the two groups. PATIENTS AND METHODS: Forty women with metastatic breast cancer were randomized to receive either paclitaxel alone (arm 1) or paclitaxel preceded by amifostine (arm 2). All were assessable for toxicity, and 37 were assessable for response. At baseline and after each cycle, all patients completed questionnaires for neurologic symptoms and had standardized neurologic examinations, including objective assessments of power and vibration sense. In addition, standard follow-up assessments for other toxicities and tumor response were undertaken. Changes from baseline after courses 1, 2, and 3 were assessed. The sample size was sufficient to detect a 50% improvement in the expected determination in sensory change. RESULTS: There were no differences observed in any of the measures of neurotoxicity. Other toxicity was similar in arms 1 and 2, including hair loss (95% v 90%), neurosensory changes (100% v 100%), fatigue/lethargy (85% v 90%), myalgia (95% v 90%), and grade 4 neutropenia (47% v 60%). Nausea, vomiting, dizziness, hypotension, and sneezing were more common in the amifostine arm. Response rates (22.2% v 36.8%) and paclitaxel pharmacokinetics were not significantly different. CONCLUSION: There was no protection from paclitaxel-related neurotoxicity or hematologic toxicity in this study. These results suggest that the mechanism of action of paclitaxel-related toxic effects is not amenable to the cytoprotective action of amifostine.

A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy.

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).

Molecular mimics of insulin-like growth factor 1 (IGF-1) for inhibiting IGF-1: IGF-binding protein interactions.

IGF-1 (insulin-like growth factor 1) is a 70-residue protein hormone which has both metabolic and mitogenic activities mediated through IGF-1 binding to cell surface receptors. However, an unrelated class of proteins, the IGF-binding proteins (IGFBPs) also bind IGF-1 in the serum and tissues and block or modulate its activity in vivo. Therefore, inhibitors of the IGFBPs can alter the distribution between free and bound IGF-1 [Loddick, S. A., Liu, X.-J., Lu, Z.-X., Liu, C., Behan, D. P., Chalmers, D. C., Foster, A. C., Vale, W. W., Ling, N., and De Souza, E. B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1894-1898] and potentially affect the distribution of IGF-1 among body tissues. We report here that phage-displayed peptide libraries have yielded a peptide that binds IGFBP-1 and produces IGF-like activity at sub-micromolar concentrations. The 14-residue peptide has an extremely well-defined solution conformation that can aid in the design of smaller, orally active compounds. Interestingly, the peptide structure contains a helix, as does one region of IGF-1 previously implicated in IGFBP binding, yet displays side chains different from those of the IGF-1 helix I. Furthermore, an IGF-1 variant lacking receptor-signaling activity in vitro is shown here to produce IGF-like mitogenic and metabolic activity in vivo. These results suggest that small antagonist mimetics of protein ligands, identified by binding selection to otherwise inhibitory factors, may be useful as indirect agonists for a variety of therapeutic applications.

Growth hormone secretagogues stimulate the hypothalamic-pituitary-adrenal axis and are diabetogenic in the Zucker diabetic fatty rat.

Besides stimulating GH release, some GH secretagogues also release ACTH and adrenal steroids. Several novel classes of potent GH secretagogues have recently been described, and we have now tested their ability to release corticosterone in conscious normal rats. All analogs that released GH also stimulated corticosterone release to some degree, though the relative effects on GH and corticosterone varied somewhat. The corticosterone responses for some analogs were in the range of those obtained with CRF (2 microg, iv), whereas closely related analogs inactive for GH release failed to release corticosterone. Activation of the hypothalamic-pituitary-adrenal axis with GH release by GHRPs could be a highly diabetogenic combination in susceptible individuals. Therefore, a potent GHRP pentapeptide analog (G7039, 100 microg/day, sc, bid) was given to young obese male Zucker diabetic fatty rats (ZDF, n = 8/group) for 24 days. Other groups received hGH (500 microg/day, sc, bid), recombinant human insulin-like growth factor (rhIGF)-1 (750 microg/day, sc, infusion) or excipient, alone or in combination. Both G7039 and hGH increased weight gain, markedly raised serum glucose (G7039, 542 +/- 37; hGH, 725 +/- 30; excipient, 330 +/- 57 mg/dl) and doubled insulin levels but had opposite effects on serum triglycerides (G7039, 1412 +/- 44; hGH 501 +/- 46; excipient 1058 +/- 73 mg/dl) and fat depot weights. In contrast, treatment with IGF-1, alone or in combination with hGH or G7039, improved the diabetic state and stimulated growth. Thus, both G7039 and hGH treatment stimulated growth in ZDF rats, but greatly worsened diabetes, unless IGF-1 was coadministered. Some of the effects ofG7039 could be explained by GH release, but the effects on blood lipids and body fat were not seen with hGH and may reflect the additional activation of the hypothalamic-pituitary-adrenal axis by the secretagogue. The magnitude of these adverse effects in the ZDF animals suggest that chronic administration of GHRP analogs with cortisol-releasing activity to obese or diabetes-prone individuals warrants careful evaluation.

In vitro metabolism of dexamethasone (DEX) in human liver and kidney: the involvement of CYP3A4 and CYP17 (17,20 LYASE) and molecular modelling studies.

Dexamethasone (DEX) has previously been shown to be extensively metabolised to 6-hydroxylated and side-chain cleaved metabolites in human liver in vitro. CYP3A4 is responsible for 6alpha- and 6beta-hydroxylation of DEX and CYP17 is thought to mediate side-chain cleavage to generate 9alphafluoro-androsta-1,4-diene-11beta-hydroxy-16alpha-methyl-3,17-dione (9alphaF-A). Although 9alphaF-A has not previously been isolated as a metabolite in its unhydroxylated form in human liver incubations, it is formed as an intermediate metabolite, which is subsequently rapidly hydroxylated to OH-9alphaF-A. A main part of this study has been to conclusively show that DEX undergoes extensive side-chain cleavage to form 9alphaF-A in human kidney fractions, which is in contrast to profiles obtained for DEX metabolism in parallel human liver microsomal incubations where 6-hydroxylation is the predominant pathway. Furthermore, molecular models of CYP3A4 and CYP17 (17,20 lyase) have been used to model the enzyme fits of DEX. From these modelling studies it has been shown that DEX complements both putative enzyme active sites in orientations likely to lead to the formation of the metabolites identified in vitro. We have also been able to rationalise the preferential formation of the 6betaOH-DEX isomer.

Cationic lipid-based gene delivery systems: pharmaceutical perspectives.

Gene delivery systems are designed to control the location of administered therapeutic genes within a patient's body. Successful in vivo gene transfer may require (i) the condensation of plasmid and its protection from nuclease degradation, (ii) cellular interaction and internalization of condensed plasmid, (iii) escape of plasmid from endosomes (if endocytosis is involved), and (iv) plasmid entry into cell nuclei. Expression plasmids encoding a therapeutic protein can be, for instance, complexed with cationic liposomes or micelles in order to achieve effective in vivo gene transfer. A thorough knowledge of pharmaceutics and drug delivery, bio-engineering, as well as cell and molecular biology is required to design optimal systems for gene therapy. This mini-review provides a critical discussion on cationic lipid-based gene delivery systems and their possible uses as pharmaceuticals.

Dexamethasone metabolism in vitro: species differences.

Dexamethasone (DEX) is extensively metabolized to 6-hydroxyDEX (6OH-DEX) and side-chain cleaved metabolites in human liver both in vitro and in vivo with CYP3A4 responsible for the formation of 6-hydroxylated products. In the present study, the metabolism of [3H]DEX has been examined in the liver fractions from various mammalian species and metabolite profiles compared with those obtained with human liver microsomes. Metabolites were quantified by radiometric high-pressure liquid chromatography (HPLC) and characterized by liquid chromatography-mass spectrometry (LC-MS) and co-chromatography with chemical standards, where available. 6OH-DEX formation was quantified for each species and the inhibitory potency of ketoconazole at 1 and 20 microM determined. Glycyrrhetinic acid, a specific inhibitor of 11-dehydrogenase, was also used to determine the extent of reductive DEX metabolism. Species differences in metabolite profiles obtained from microsomal incubations were both quantitative and qualitative. 6-Hydroxylation was variable (highest in the hamster) and was not always the major route of metabolism, and formation was sex-specific in the rat (male >> female). The inhibition of 6-hydroxylation (CYP3A) by ketoconazole was variable, and indicates that ketoconazole cannot be regarded as a selective inhibitor of CYP3A proteins in all species. Cytosolic incubations produced similar profiles in different species with the formation of a metabolite (M5) which was inhibited by glycyrrhetinic acid and tentatively identified in this study as 11-dehydro-side-chain cleaved DEX (11DH-9alphaF-A). In conclusion, the male rat gave a metabolite profile which was closest to that seen in the human. However, 6-hydroxylation was most extensive in the hamster which may therefore be a suitable model to use for further studies on DEX metabolism by CYP3A.

Dexamethasone metabolism by human liver in vitro. Metabolite identification and inhibition of 6-hydroxylation.

The metabolism of the synthetic glucocorticoid dexamethasone in human liver microsomal incubations has been studied. Metabolites were analyzed by radiometric high-performance liquid chromatography and were identified by liquid-chromatography-mass spectrometry; in addition, the major metabolite 6beta-hydroxydexamethasone was identified by cochromatography with a chemically synthesized standard. A total of 17 human livers were used in this study and the following metabolites were identified: 6beta-hydroxydexamethasone, 6 alpha-hydroxydexamethasone, 6-hydroxy-9 alpha-fluoro-androsta-1,4-diene-11 beta-hydroxy-16 alpha-methyl-3,17-dione (6-hydroxy-9 alpha-F-A) and 9 alpha-fluoro-androsta-1,4-diene-11 beta-hydroxy-16 alpha-methyl-3,17-dione (9 alpha-F-A). Dexamethasone underwent side-chain cleavage to form 9 alpha-F-A. This metabolite was then a substrate for 6-hydroxylation. There was considerable interindividual variability in metabolic profiles. Mean (+/-S.D.) K(m) values for 6 beta- and 6 alpha-hydroxydexamethasone formation were 23.2 +/- 3.8 and 25.6 +/- 1.6 microM (n = 4), respectively. The corresponding V max values were 14.3 +/- 9.9 and 4.6 +/- 3.1 pmol x min(-1) mg protein (-1). Ketoconazole (3 microM) completely inhibited 6 alpha- and 6 beta-hydroxylation, indicating that formation of both metabolites was catalyzed by CYP3A4. This was confirmed in studies of correlations between the rate of metabolite formation and the relative expression of CYP3A4: r = 0.74 for 6 beta-hydroxydexamethasone, P = .003; r = 0.70 for 6 alpha-hydroxydexamethasone, P = .006. In addition to ketoconazole, both ellipticine and gestodene caused marked inhibition of 6-hydroxylation. Ellipticine is clearly not a selective CYP1A inhibitor as has been stated previously. However, furafylline (CYP1A inhibitor), tolbutamide (CYP2C substrate), and sulfaphenazole (CYP2C inhibitor) were essentially noninhibitory. The relatively simple metabolic profile of dexamethasone compared to other steroids may point to this being a potentially useful in vivo probe for CYP3A4 in humans.

Investigation of the uptake of drugs by individual cells using a scanning proton microprobe (SPM).

In this paper we demonstrate the use of micro-PIXE (proton induced X-ray emission) for measuring the quantitative uptake of anti-AIDS drugs, containing metal atoms, by individual Vero cells (African green monkey kidney cell line). Hetero-polytungstates, which are assessed to present an activity against the HIV virus, were studied using Vero cells. It was found that unlike other techniques, SPM offers both the sensitivity and the spatial resolution to carry out these programs of investigations. The use of elemental analysis in single cells of cultured cell lines has shown to have distinct advantages over peripheral blood lymphocytes.