Compound heterozygosity for metachromatic leukodystrophy and arylsulfatase A pseudodeficiency alleles is not associated with progressive neurological disease

Several allelic mutations at the arylsulfatase A (ASA) locus cause substantial deficiencies of this lysosomal enzyme. Depending on the genetically determined degree of the deficiency, the clinical outcome may be very different—either metachromatic leukodystrophy (MLD), a lethal lysosomal storage disorder affecting the nervous system, or, more frequently, the so-called pseudodeficiency (PD), which has no apparent clinical consequence. Because of compound heterozygosity for MLD and PD, 1/1,000 individuals in the population have low residual enzyme activities, which are intermediate between those of MLD patients and those of PD homozygous normal individuals. In order to assess whether PD/MLD compound heterozygotes bear a health risk, we examined clinically and biochemically 16 individuals with this genotype. Of these subjects, two had neurological symptoms and two showed lesions, without clinical symptoms, in magnetic resonance imaging of the brain. None of these symptoms was progressive, nor did they resemble those of MLD. Nerve conduction velocities were normal in these probands, and they secreted only low amounts of sulfatide in the urine. We conclude that the observed neurological symptoms are unrelated to the ASA genotype and that PD/MLD compound heterozygotes are not at an increased risk for developing progressive nervous system diseases.     

Chimeric Nitrogenase-like Enzymes of (Bacterio)chlorophyll Biosynthesis

Nitrogenase-like light-independent protochlorophyllide oxidoreductase (DPOR) is involved in chlorophyll biosynthesis. Bacteriochlorophyll formation additionally requires the structurally related chlorophyllide oxidoreductase (COR). During catalysis, homodimeric subunit BchL₂ or ChlL₂ of DPOR transfers electrons to the corresponding heterotetrameric catalytic subunit, (BchNB)₂ or (ChlNB)₂. Analogously, subunit BchX₂ of the COR enzymes delivers electrons to subunit (BchYZ)₂. Various chimeric DPOR enzymes formed between recombinant subunits (BchNB)₂ and BchL₂ from Chlorobaculum tepidum or (ChlNB)₂ and ChlL₂ from Prochlorococcus marinus and Thermosynechococcus elongatus were found to be enzymatically active, indicating a conserved docking surface for the interaction of both DPOR protein subunits. Biotin label transfer experiments revealed the interaction of P. marinus ChlL₂ with both subunits, ChlN and ChlB, of the (ChlNB)₂ tetramer. Based on these findings and on structural information from the homologous nitrogenase system, a site-directed mutagenesis approach yielded 10 DPOR mutants for the characterization of amino acid residues involved in protein-protein interaction. Surface-exposed residues Tyr¹²⁷ of subunit ChlL, Leu⁷⁰ and Val¹⁰⁷ of subunit ChlN, and Gly⁶⁶ of subunit ChlB were found essential for P. marinus DPOR activity. Next, the BchL₂ or ChlL₂ part of DPOR was exchanged with electron-transferring BchX₂ subunits of COR and NifH₂ of nitrogenase. Active chimeric DPOR was generated via a combination of BchX₂ from C. tepidum or Roseobacter denitrificans with (BchNB)₂ from C. tepidum. No DPOR activity was observed for the chimeric enzyme consisting of NifH₂ from Azotobacter vinelandii in combination with (BchNB)₂ from C. tepidum or (ChlNB)₂ from P. marinus and T. elongatus, respectively.Chlorophyll and bacteriochlorophyll biosynthesis, as well as nitrogen fixation, are essential biochemical processes developed early in the evolution of life (1). During biological fixation of nitrogen, nitrogenase catalyzes the reduction of atmospheric dinitrogen to ammonia (2). Enzyme systems homologous to nitrogenase play a crucial role in the formation of the chlorin and bacteriochlorin ring system of chlorophylls (Chl)2 and bacteriochlorophylls (Bchl) (3, 4) (Fig. 1a). For the synthesis of both Chl and Bchl, the stereospecific reduction of the C-17-C-18 double bond of ring D of protochlorophyllide (Pchlide) catalyzed by the nitrogenase-like enzyme light-independent (dark-operative) protochlorophyllide oxidoreductase (DPOR) results in the formation of chlorophyllide (Chlide) (Fig. 1a, left) (5, 6). DPOR enzymes consist of three protein subunits which are designated BchN, BchB and BchL in Bchl-synthesizing organisms and ChlN, ChlB and ChlL in Chl-synthesizing organisms. A second reduction step at ring B (C-7-C-8) unique to the synthesis of Bchl converts the chlorin Chlide into a bacteriochlorin ring structure to form bacteriochlorophyllide (Bchlide) (Fig. 1a, right, Bchlide). This reaction is catalyzed by another nitrogenase-like enzyme, termed chlorophyllide oxidoreductase (COR) (7). COR enzymes are composed of subunits BchY, BchZ, and BchX.Open in a separate windowFIGURE 1.Comparison of the three subunit enzymes DPOR, COR, and nitrogenase. a, during Chl and Bchl biosynthesis, ring D is stereospecifically reduced by the nitrogenase-like enzyme DPOR (subunit composition BchL₂/(BchNB)₂ or ChlL₂/(ChlNB)₂) leading to the chlorin Chlide. Subunits N, B, and L are named ChlN, ChlB, and ChlL in Chl-synthesizing organisms and BchN, BchB, and BchL in Bchl-synthesizing organisms. The synthesis of Bchl additionally requires the stereospecific B ring reduction by a second nitrogenase-like enzyme called COR, with the subunit composition BchX₂/(BchYZ)₂. COR catalyzes the formation of the bacteriochlorin Bchlide. Subunits Y, Z, and X of the COR enzyme are named BchY, BchZ, and BchX. b, the homologous nitrogenase complex has the subunit composition NifH₂/(NifD/NifK)₂. Rings A–E and the carbon atoms are designated according to IUPAC nomenclature (41). R is either a vinyl or an ethyl moiety. The position marked by an asterisk indicates either a vinyl or a hydroxyethyl moiety (42).All subunits share significant amino acid sequence homology to the corresponding subunits of nitrogenase, which are designated NifD, NifK, and NifH, respectively (1) (compare Fig. 1, a and b). Whereas subunits BchL or ChlL, BchX and NifH exhibit a sequence identity at the amino acid level of ∼33%, subunits BchN or ChlN, BchY, NifD, and BchB or ChlB, BchZ, and NifK, respectively, show lower sequence identities of ∼15% (1). For all enzymes a common oligomeric protein architecture has been proposed consisting of the heterotetrameric complexes (BchNB)₂ or (ChlNB)₂, (BchYZ)₂, and (NifD/NifK)₂, which are completed by a homodimeric protein subunit BchL₂ or ChlL₂, BchX₂, and NifH₂, respectively (compare Fig. 1, a and b) (3, 7, 8).Nitrogenase is a well characterized protein complex that catalyzes the reduction of nitrogen to ammonia in a reaction that requires at least 16 molecules of MgATP (2, 9, 10). During nitrogenase catalysis, subunit NifH₂ (Fe protein) associates with and dissociates from the (NifD/NifK)₂ complex (MoFe protein). Binding, hydrolysis of MgATP and structural rearrangements are coupled to sequential intersubunit electron transfer. For this purpose, NifH₂ contains an ATP-binding motif and an intersubunit [4Fe-4S] cluster coordinated by two cysteine residues from each NifH monomer (1, 11). Electrons from this [4Fe-4S] cluster are transferred via a [8Fe-7S] cluster (P-cluster) onto the [1Mo-7Fe-9S-X-homocitrate] cluster (MoFe cofactor). Both of the latter clusters are located on (NifD/NifK)₂, where dinitrogen is reduced to ammonia (10). Three-dimensional structures of NifH₂ in complex with (NifD/NifK)₂ revealed a detailed picture of the dynamic interaction of both subcomplexes (8, 12).Based on biochemical and bioinformatic approaches, it has been proposed that the initial steps of DPOR reaction strongly resemble nitrogenase catalysis. Key amino acid residues essential for DPOR function have been identified by mutagenesis of the enzyme from Chlorobaculum tepidum (formerly denoted as Chlorobium tepidum) (3). The catalytic mechanism of DPOR includes the electron transfer from a “plant-type” [2Fe-2S] ferredoxin onto the dimeric DPOR subunit, BchL₂, carrying an intersubunit [4Fe-4S] redox center coordinated by Cys⁹⁷ and Cys¹³¹ in C. tepidum. Analogous to nitrogenase, Lys¹⁰ in the phosphate-binding loop (P-loop) and Leu¹²⁶ in the switch II region of DPOR were found essential for DPOR catalysis. Moreover, it was shown that the BchL₂ protein from C. tepidum does not form a stable complex with the catalytic (BchNB)₂ subcomplex. Therefore, a transient interaction responsible for the electron transfer onto protein subunit (BchNB)₂ has been proposed (3).The subsequent [Fe-S] cluster-dependent catalysis and the specific substrate recognition at the active site located on subunit (BchNB)₂ are unrelated to nitrogenase. The (BchNB)₂ subcomplex was shown to carry a second [4Fe-4S] cluster, which was proposed to be ligated by Cys²¹, Cys⁴⁶, and Cys¹⁰³ of the BchN subunit and Cys⁹⁴ of subunit BchB (C. tepidum numbering) (3). No evidence for any type of additional cofactor was obtained from biochemical and EPR spectroscopic analyses (5, 13). Thus, despite the same common oligomeric architecture, the catalytic subunits (BchNB)₂ and (ChlNB)₂ clearly differ from the corresponding nitrogenase complex, as no molybdenum-containing cofactor or P-cluster equivalent is employed (5, 14). From these results it was concluded that electrons from the [4Fe-4S] cluster of (BchNB)₂ or (ChlNB)₂ are transferred directly onto the Pchlide substrate at the active site of DPOR.The second nitrogenase-like enzyme, COR, catalyzes the reduction of ring B of Chlide during the biosynthesis of Bchl (7). Therefore, an accurate discrimination of the ring systems of the individual substrates is required. COR subunits share an overall amino acid sequence identity of 15–22% for BchY and BchZ and 31–35% for subunit BchX when compared with the corresponding DPOR subunits (supplemental Figures S2–S4). In amino acid sequence alignments of BchX proteins with the closely related BchL or ChlL subunits of DPOR, both cysteinyl ligands responsible for [4Fe-4S] cluster formation and residues for ATP binding are conserved (1). Furthermore, all cysteinyl residues characterized as ligands for a catalytic [4Fe-4S] cluster in (BchNB)₂ or (ChlNB)₂ are conserved in the sequences of subunits BchY and BchZ of COR (7). These findings correspond to a recent EPR study in which a characteristic signal for a [4Fe-4S] cluster was obtained for the COR subunit BchX₂ as well as for subunit (BchYZ)₂ (15). These results indicate that the catalytic mechanism of COR strongly resembles DPOR catalysis. In vitro assays for nitrogenase as well as for DPOR and COR make use of the artificial electron donor dithionite in the presence of high concentrations of ATP (7, 16, 17).

TABLE 1

Amino acid sequence identities of the individual subunits of DPOR, COR, and nitrogenaseAmino acid sequences of the individual subunits of DPOR, COR, and nitrogenase employed in the present study (compareFig. 3A) were aligned by using the ClustalW method in MegAlign (DNASTAR), and sequence identities were calculated.

Enhanced microglial pro‐inflammatory response to lipopolysaccharide correlates with brain infiltration and blood–brain barrier dysregulation in a mouse model of telomere shortening

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self‐renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as ‘priming’. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first‐generation G1 mTerc^?/?)‐ and late‐generation (third‐generation G3 and G4 mTerc^?/?) telomerase‐deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late‐generation mTerc^?/? microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc^?/? microglia are comparable with microglia derived from G1 mTerc^?/? mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc^?/? microglia mice show an enhanced pro‐inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age‐associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood–brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening.     

Attitudes of families affected by adrenoleukodystrophy toward prenatal diagnosis, presymptomatic and carrier testing, and newborn screening

Families affected by adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN) were surveyed to elicit attitudes toward prenatal, presymptomatic and carrier testing, and newborn screening in order to determine the level of support that these families have for current and future genetic testing protocols. Identifying attitudes toward genetic testing, including newborn screening, is especially important because of new data regarding therapeutic options and the possible addition of ALD to newborn screening regimens. The Kennedy Krieger Institute (KKI) database identified 327 prospective participants. Families that were willing to participate in the study received an anonymous questionnaire for completion. Frequencies were generated using SPSS software for Windows. Questionnaires were returned from 128 families for a response rate of 39%. Sons who were at risk for inheriting the ALD gene would be tested by 93% of respondents, and 89.3% would ideally have this testing performed prenatally or in the newborn period. Eighty-nine percent would test an at-risk daughter and 51.2% would ideally have this testing performed prenatally or shortly after birth. ALD newborn screening for males and females was supported by 90% of respondents. If newborn screening for ALD/AMN commences, or there is a new diagnosis of ALD, genetic professionals need to be prepared to have extensive conversations with families regarding the benefits and limitations of current therapeutic and genetic testing options.     

Effect of tumor necrosis factor administration in vivo on lipoprotein lipase activity in various tissues of the rat

When added to murine adipocytes in culture, tumor necrosis factor (TNF) decreases the levels of lipoprotein lipase (LPL). Semb et al (1987. J. Biol Chem. 262: 8390-8394) have shown that administration of murine TNF to rats decreases lipoprotein lipase (LPL) in the epididymal fat pad with maximal inhibition requiring several hours. We have now tested the effects of treatment of rats with TNF on LPL activity in a variety of tissues and find that few show decreases in LPL under conditions that acutely increase serum triglycerides. Ninety minutes after treatment of male rats with human TNF (25 micrograms/200 g, i.v.), serum triglycerides rose 2.2-fold but there was no decrease in LPL activity in epididymal fat. Sixteen hours after TNF treatment LPL activity had decreased by 44% in epididymal fat, consistent with the previously reported data. In contrast, in female rats, no significant decrease was seen in LPL activity in parametrial adipose tissue at either 90 min or 16 hr after TNF administration despite increases in serum triglycerides (1.8-fold and 1.5-fold, respectively). There was little change in LPL activity in most other adipose tissue sites of male or female rats at either time after TNF treatment. No effect of TNF was seen on heart or diaphragm muscle LPL at any time. TNF treatment of both male and female rats produces consistent increases in de novo hepatic lipogenesis in vivo under conditions that increase serum triglycerides. It is unlikely that the limited effects of TNF on LPL in vivo can account for the rapid and sustained increase in serum triglycerides.     

Antigen-experienced T cells undergo a transient phase of unresponsiveness following optimal stimulation

Interaction of the Ag-specific receptor of T lymphocytes with its Ag/MHC ligand can lead either to cell activation or to a state of unresponsiveness often referred to as anergy. It has been generally assumed that anergy develops as a consequence of inadequate stimulation, such as in response to altered peptide ligands or to agonists presented by costimulatory-deficient accessory cells. The present study uncovers an alternative way of inducing an unresponsive state in T cells. Indeed, we demonstrate herein that Ag-stimulation of murine CD4+ Th clones induces cellular activation, characterized by cytokine production and cell proliferation, followed by a state of transient (lasting up to 6 days) unresponsiveness to further antigenic stimulation. This state of activation-induced unresponsiveness 1) is not a consequence of inadequate costimulation, as it occurs when cells are stimulated in the presence of dendritic cells or anti-CD28 Abs; 2) develops after an optimal response to Ag; 3) is not due to cell death/apoptosis or CTLA-4 engagement; 4) down-regulates the proliferation and cytokine production of both Th1- and Th2-like clones; and 5) does not affect the early steps of signal transduction. Finally, naive T cells are not sensitive to this novel form of unresponsiveness, but become gradually susceptible to activation-induced unresponsiveness upon Ag stimulation. Collectively, these data suggest that activation-induced T cell unresponsiveness may represent a regulatory mechanism limiting the clonal expansion and effector cell function of Ag-experienced T cells, thus contributing to the homeostasis of an immune response.     

Differentiation of isolated murine embryonic palatal epithelium in culture: exogenous transforming growth factor alpha modulates matrix biosynthesis in defined experimental conditions

Summary A novel culture technique, which supports the growth and differentiation of mouse embryonic palatal epithelial cells in the absence of either an extracellular matrix substratum or feeder layers, has been developed. Using this technique we have investigated the effects of exogenous transforming growth factor alpha (TGFα) and serum on extracellular matrix biosynthesis by primary cultures of mouse embryonic epithelial sheets under defined experimental conditions. In all culture treatments (chemically defined medium with and without TGFα or serum) the palatal epithelial sheets differentiated into three regionally distinct cell phenotypes after 36 h. Nasal and oral cells differentiated into pseudostratified, cilliated columnar, and stratified squamous keratinizing epithelium, respectively. In addition, basal medial edge epithelial (MEE) cells at the oral/nasal regional interface assumed an elongated cobblestoned phenotype. In serum-free medium, collagen types IV and V, laminin, fibronectin, and heparan sulphate proteoglycan were detected immunocytochemically throughout the entire epithelial sheet. Tenascin and collagen IX were present almost exclusively in MEE cells. Types I, II, and III collagen were completely absent. Addition of TGFα or serum universally increased the intensity of staining, most notably that for tenascin and collagen IX in MEE cells. These results indicate that mouse embryonic palatal epithelial sheets can be maintained under defined culture conditions during which they exhibit patterns of differentiation similar to those observed in vivo. TGFα, known to localize to the MEE in vivo, can modulate palatal extracellular matrix biosynthesis, particularly by the MEE, suggesting a regulatory role for this factor. The culture system is suitable for further investigating the effects of exogenous factors on mouse embryonic palatal epithelial cell bioactivity and differentiation.     

Volatile constituents and fatty acid composition of lipids in Durio zibethinus

The predominating flavour compounds in the fruit pulp of Durio zibethinus were hydrogen sulfide, ethyl hydrodisulfide and several dialkyl polysulfides, particularly (C₂H₅)₂S_n, where n = 2 or 3. Ethyl acetate, 1,1-diethoxyethane and ethyl 2-methylbutanoate contribute to an additional fruity odour note. Hydrodisulfides are probably the precursors of the dialkyl sulfides. In the pericarp and seed no volatile sulfur compounds could be detected. The fatty acid composition of the lipids in pericarp, pulp and seed depended on the origin and/or harvest season of the fruit. The main components were oleic and palmitic acids or arachidic acid together with appreciable quantities of palmitoleic, stearic, linoleic and linolenic acids.