Metallic taste and retronasal smell

A series of experiments investigated the nature of metallic taste reports and whether they can be attributed to the development of a retronasal smell. Two studies showed that the metallic sensation reports following oral stimulation with solutions of FeSO4 were reduced to baseline when the nose was occluded. No such reduction was seen for CuSO4 or ZnSO4, which were more bitter and astringent, respectively, and less metallic. A discrimination test based on weak but equi-intense levels of FeSO4 and CuSO4 showed that FeSO4 could be discriminated from water with the nose open but not when occluded, but that discrimination of CuSO4 from water was not impaired by nasal occlusion. A discrimination test demonstrated that the headspace over solutions of FeSO4 was not different from water, although some subjects could discriminate FeSO4 solutions from water in the mouth when the nose was occluded, perhaps by tactile or astringent cues. These results confirm that metallic taste reports following oral stimulation with FeSO4 are likely due to development of a retronasal smell, possibly following a lipid oxidation reaction in the mouth. However, metallic taste reports may arise from different mechanisms with copper and zinc salts.     

Human Immunodeficiency Virus Inhibits Multilineage Hematopoiesis In Vivo

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4⁺ lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34⁺ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.     

Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis

The polysaccharide chains of enterobacterial common antigen (ECA) consist of linear trisaccharide repeat units with the structure -->3)- alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1--> 4)-alpha-d-GlcNAc-(1-->, where Fuc4NAc is 4-acetamido-4, 6-dideoxy-d-galactose, ManNAcA is N - acetyl-d- mannosaminuronic acid, and GlcNAc is N -acetyl-d-glucosamine. The major form of ECA (ECAPG) consists of polysaccharide chains that are believed to be covalently linked to diacylglycerol through phosphodiester linkage; the phospholipid moiety functions to anchor molecules in the outer membrane. The ECA trisaccharide repeat unit is assembled as a polyisoprenyl-linked intermediate which has been tentatively identified as Fuc4NAc-ManNAcA-GlcNAc- pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongation presumably occurs by a block-polymerization mechanism. However, the identity of the polyisoprenoid carrier-lipid has not been established. Accordingly, the current studies were conducted in an effort to structurally characterize the polyisoprenyl lipid-carrier involved in ECA synthesis. Isolation and characterization of the lipid carrier was facilitated by the accumulation of a ManNAcA-GlcNAc- pyrophosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonella typhimurium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA synthesis. Analyses of lipid II preparations by fast atom bombardment tandem mass spectroscopy (FAB-MS/MS) resulted in the identification of the lipid-carrier as the 55-carbon polyisoprenyl alcohol, undecaprenol. These analyses also resulted in the identification of a novel glycolipid which copurified with lipid II. FAB-MS/MS analyses of this glycolipid revealed its structure to be 1,2-diacyl- sn -glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP- disaccharide). An examination of purified ECAPGby phosphorus-31 nuclear magnetic resonance spectroscopy confirmed that the polysaccharide chains are linked to diacylglycerol through phosphodiester linkage. Thus, DGP-disaccharide does not appear to be an intermediate in ECAPGsynthesis. Nevertheless, although the available evidence clearly indicate that lipid II is a precursor of DGP-disaccharide, the function of this novel glycolipid is not yet known, and it may be an intermediate in the biosynthesis of a molecule other than ECAPG.      

Further freeze-fracture studies on the uterine epithelium of Salamandra salamandra (L.) (Amphibia,Urodela) using the antibiotic filipin

Summary The apical portion of the uterine lining of the ovoviviparous fire salamander, Salamandra salamandra, was studied by the freeze-fracture technique in conjunction with the polyene antibiotic filipin. Filipin-sterol complexes were found in the luminal plasmalemma and in the membranes limiting the mucous secretory granules typical of this epithelium. In all females, but particularly in non-pregnant females, more or less discrete clusters of filipinsterol complexes were occasionally found overlying heavily affected secretory granules. The findings are discussed with regard to comparable results (Orci et al. 1980) based on the examination of collapsed and stretched urinary bladders of toads.We are indebted to Mrs. K. Ott for excellent technical assistance, to Miss E.S. MacLure for linguistic corrections and to Dr. J.E. Grady of the Upjohn Co., Kalamazoo, Michigan USA, for kindly providing the filipin     

Abnormal serum concentrations of proteins in Parkinson’s disease

Blood serum was used to identify protein biomarkers for diagnosis of Parkinson’s disease (PD) using analytically validated quantitative 2D-gel electrophoresis, and single variable and multivariate statistics. Using banked samples from a first medical center, we identified 57 specific protein spot biomarkers with disease-specific abnormal levels in serum of patients with PD, Alzheimer’s disease, amyotrophic lateral sclerosis and similar neurodegenerative conditions (337 samples), when compared to age-matched normal controls (132 samples). To further assess their clinical usefulness in Parkinson’s disease, we obtained prospective newly drawn blood serum samples from a second (56 PD, 30 controls) and third (6 PD, 48 controls) medical center. The protein concentrations of the 57 biomarkers were assessed by 2D-gel electrophoresis. Stepwise linear discriminant analysis selected a combination of 21 of the 57 as optimal to distinguish PD patients from controls. When applied to the samples from the second site, the 21 proteins had sensitivity of 93.3% (52 of 56 PD correctly classified), specificity of 92.9% (28 of 30 controls correctly classified); 15 of 15 patients with mild, 28 of 30 with moderate to severe symptoms, and all of the 6 PD patients from the third site were correctly classified. Eleven of the 21 proteins showed statistically significant abnormal concentrations in patients with mild symptoms, and 14 in patients with moderate-severe symptoms. The protein identities reflect the heterogeneity of Parkinson’s disease, and thus may provide the capability of monitoring the blood for a diverse range of PD pathophysiological mechanisms: cellular degeneration, oxidative stress, inflammation, and transport.     

Klinik und Genetik des Joubert-Syndroms

Joubert syndrome generally represents an autosomal recessive and rarely X-linked disorder characterized by hypotonia, an irregular breathing pattern, abnormal eye movements, ataxia, developmental delay and a complex mid-hindbrain malformation causing the molar tooth sign on magnetic resonance imaging (MRI). Many patients have additional features, with nephronophthisis, retinal dystrophy, coloboma and hepatic fibrosis representing the most frequent features. Due to its clinical variability and overlap with other syndromes, the term “Joubert syndrome and related disorders” (JSRD) was proposed. To date 10 genes are known to cause JSRD. The encoded proteins are localized to cilia, linking JSRD to other human ciliopathies.     

Decrypting the biochemical function of an essential gene from Streptococcus pneumoniae using ThermoFluor technology

The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.     

Impact of cytokines on replication in the thymus of primary human immunodeficiency virus type 1 isolates from infants

Early infection of the thymus with the human immunodeficiency virus (HIV) may explain the more rapid disease progression among children infected in utero than in children infected intrapartum. Therefore, we analyzed infection of thymocytes in vitro by HIV type 1 primary isolates, obtained at or near birth, from 10 children with different disease outcomes. HIV isolates able to replicate in the thymus and impact thymopoiesis were present in all infants, regardless of the timing of viral transmission and the rate of disease progression. Isolates from newborns utilized CCR5, CXCR4, or both chemokine receptors to enter thymocytes. Viral expression was observed in discrete thymocyte subsets postinfection with HIV isolates using CXCR4 (X4) and isolates using CCR5 (R5), despite the wider distribution of CXCR4 in the thymus. In contrast to previous findings, the X4 primary isolates were not more cytopathic for thymocytes than were the R5 isolates. The cytokines interleukin-2 (IL-2), IL-4, and IL-7 increased HIV replication in the thymus by inducing differentiation and expansion of mature CD27(+) thymocytes expressing CXCR4 or CCR5. IL-2 and IL-4 together increased expression of CXCR4 and CCR5 in this population, whereas IL-4 and IL-7 increased CXCR4 but not CCR5 expression. IL-2 plus IL-4 increased the viral production of all pediatric isolates, but IL-4 and IL-7 had a significantly higher impact on the replication of X4 isolates compared to R5 isolates. Our studies suggest that coreceptor use by HIV primary isolates is important but is not the sole determinant of HIV pathogenesis in the thymus.     

Structure and dynamics of lipid-associated states of apocytochrome c.

Apocytochrome c (apocyt c), which in aqueous solution is largely unstructured, acquires an alpha-helical conformation upon association with lipid membranes. The extent of alpha-helix induced in apocyt c is lipid-dependent and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The structural and dynamic properties of apocyt c in lipid membranes were investigated by attenuated total reflection Fourier transform infrared spectroscopy combined with amide H-D exchange kinetics. Apocyt c acquires a higher content of alpha-helical structure with negatively charged membranes than with zwitterionic ones. For all membranes studied here, the helices of these partially folded states of apocyt c have a preferential orientation perpendicular to the plane of the lipid membrane. The H-D exchange revealed that a small fraction of amide protons of apocyt c, possibly associated with a stable folded domain protected by the lipid, remained protected from exchange over 20 min. However, a large fraction of amide protons exchanged in less than 20 min, indicating that the helical states of apocyt c in lipid membranes are very dynamic.